(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2015/038960 Al 19 March 2015 (19.03.2015) P O P C T
(51) International Patent Classification: (74) Agents: CANADY, Karen S. et al; canady + lortz LLP, C12Q 1/68 (2006.01) A61K 31/7105 (2006.01) 3701 Wilshire Blvd. Suite 508, Los Angeles, California C12N 15/113 (2010.01) A61P 35/00 (2006.01) 90010 (US). (21) International Application Number: (81) Designated States (unless otherwise indicated, for every PCT/US20 14/055493 kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, (22) Date: International Filing BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, 12 September 2014 (12.09.2014) DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (25) Filing Language: English HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, (26) Publication Language: English MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, (30) Priority Data: PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, 61/877,902 13 September 2013 (13.09.2013) US SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (71) Applicant: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA [US/US]; 1111 Franklin Street, Oak (84) Designated States (unless otherwise indicated, for every land, California 94607-5200 (US). kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, (72) Inventors: POTHOULAKIS, Charalabos; UCLA Med- TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, div Of Digestive Diseases, Box 957019, 15 19 Mrl, Los TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, Angeles, California 90095-7019 (US). ILIOPOULOS, Di- DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, mitrios; UCLA Med-div Of Digestive Diseases, Box LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, 956949, 44-133 Chs, Los Angeles, California 90095-6949 SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, (US). LAW, Ka Man; UCLA Med-div Of Digestive Dis GW, KM, ML, MR, NE, SN, TD, TG). eases, Box 957019, 1519 Mrl, Los Angeles, California 90095-7019 (US). Declarations under Rule 4.17 : — of inventorship (Rule 4.17(iv))
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(54) Title: MIR-133a AS A MARKER AND THERAPEUTIC TARGET FOR COLITIS AND INFLAMMATORY BOWEL DIS EASE (57) Abstract: The invention provides a method for detection and monitoring of in flammatory bowel disease (IBD) in a subject that comprises assaying a specimen from the subject for miR-133a, AFTPH, and, option ally NTSR1. An elevated amount of miR- n f 133a and/or NTSRl, and/or a decreased amount of AFTPH, present in the specimen compared to control sample is indicative of inflammation. The invention further provides a method of treating inflammatory disease, such as IBD or colon cancer, in a subject by administering an inhibitor of miR-133a. Published: — with sequence listing part of description (Rule 5.2(a)) — with international search report (Art. 21(3)) — before the expiration of the time limit for amending the claims and to be republished in the event of receipt of amendments (Rule 48.2(h)) MIR-133a AS A MARKER AND THERAPEUTIC TARGET FOR COLITIS AND INFLAMMATORY BOWEL DISEASE
[0001] This application claims priority to United States provisional patent application number 61/877,902, filed September 13, 2013, the entire contents of which are incorporated herein by reference.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
This invention was made with Government support under DK060729, awarded by the National Institutes of Health. The Government has certain rights in the invention.
TECHNICAL FIELD OF THE INVENTION
The present invention relates generally to detection, diagnosis, and monitoring of inflammatory bowel disease. The invention more specifically pertains to use of miR-133a as a marker and treatment target for inflammatory bowel disease.
BACKGROUND OF THE INVENTION
Inflammatory bowel disease (IBD), which includes ulcerative colitis (UC) and Crohn's disease (CD), is a chronic inflammatory disease of the gastrointestinal (Gl) tract. Monoclonal antibodies against Tumor Necrosis Factor-alpha (TNF-a) remain one of the most effective treatments against IBD. In addition aminosalicylates, corticosteroids and other immunomodulators/immunosuppresants are also used as treatment modalities. However, remissions are common. IBD is a multifactorial disease, and all of the currently employed treatment modalities are associated with several and some times debilitating side effects.
There is a need to identify improved methods for the detection and treatment of inflammatory bowel disease.
SUMMARY OF THE INVENTION
The invention provides a method for detection of inflammatory disease, such as inflammatory bowel disease, in a subject. In a typical embodiment, the method comprises contacting a specimen obtained from the subject with reagents for assaying for miR-133a; measuring the amount of miR-133a present in the specimen as compared to a control sample; and determining the presence of inflammatory bowel disease when an elevated amount of miR-133a is present in the specimen compared to the control sample. The invention additionally provides a method of detecting or monitoring inflammatory disease by assaying for levels of miR-133a, aftiphilin (AFTPH) and/or neurotensin receptor 1 (NTSR1). The method can be used to distinguish between ulcerative colitis and Crohn's disease.
In one embodiment, the specimen is intestinal biopsy tissue, such as, for example, colon tissue, or intestinal fluid. Representative examples of inflammatory bowel disease include, but are not limited to, ulcerative colitis, Crohn's disease or Clostridium difficile colitis. In a typical embodiment, the measuring comprises polymerase chain reaction (PCR) assay, such as rea l time PCR. In another embodiment, the measuring comprises an immunoassay (e.g., for AFTPH or NTSR1). In one embodiment, the immunoassay detects cytokines deregulated during inflammatory bowel disease, including, but not limited to, ulcerative colitis, Crohn's disease or Clostridium difficile colitis.
The method for monitoring the efficacy of treatment of inflammatory disease, such as inflammatory bowel disease, in a subject typically comprises contacting a specimen obtained from the subject at a first time point with reagents for assaying for miR-133a, AFTPH and/or NTSR1 ; contacting a specimen obtained from the subject at a second time point with reagents for assaying for miR-1 33a, AFTPH and/or NTSR1 , wherein the subject has been treated for inflammatory bowel disease prior to the second time point. The method further comprises measuring the amount of miR-1 33a, AFTPH and/or NTSR1 present in the specimens obtained at the first and second time points; and determining whether an increased or decreased amount of miR-1 33a, AFTPH and/or NTSR1 is present in the specimen obtained at the second time point compared to the specimen obtained at the first time point, which decreased amount of miR- 133a or NTSR1 , or increased amount of AFTPH, is indicative of effective amelioration of the inflammatory bowel disease. In one embodiment, the above method is modified to monitor the progression of inflammatory bowel disease in a subject, optionally performed in the absence of treatment, by comparing measurements obtained at the two time points. An increase in the amount of miR-1 33a and/or NTSR1 (or decrease in the amount of AFTPH) at the second time point compared to the first time point is indicative of disease progression.
The specimen can be blood or other bodily fluid, such as peritoneal fluid, or a tissue specimen. Typically, the specimen is intestinal fluid or tissue. Examples of specimens include intestinal biopsy tissue, such as colon biopsy. Examples of inflammatory bowel disease include, but are not limited to, ulcerative colitis, Crohn's disease or Clostridium difficile colitis. The measuring typically comprises PCR or immunoassay. The invention additionally provides a method of treating inflammatory disease, such as inflammatory bowel disease, or cancer, in a subject. In one embodiment, the method comprises administering to the subject a therapeutically effective amount of an inhibitor of miR-1 33a. In one embodiment, the administering is intracolonic or intravenous. Examples of inhibitors of miR- 133a include an antisense miR-1 33a oligonucleotide. The antisense oligonucleotide can be provided in a more stabilized form, such as, in one example, a locked-nucleic acid-based antisense miR-1 33a oligonucleotide. The method can comprise administering the antisense miR-1 33a oligonucleotide either directly, or via a lentiviral vector. The inflammatory bowel disease can be, for example, ulcerative colitis, Crohn's disease or Clostridium difficile colitis.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1A-1C. MiR-1 33a down-regulation inhibits receptor recycling. (1A) NTSR1 localization in NCM460-NTSR1 cells transfected with control or antisense-miR-133a. Cells were incubated with vehicle or NTS (100 nM) for 1 h , washed and recovered in NTS-free medium for 3 h . (arrows, intracellular NTSR1; arrowheads, membrane-associated NTSR1). Scale, 10 µηη . (1B) Mean Fluorescence Intensity (MFI) was measured in the intracellular and cell surface- associated NTSR1 labeling and the surface and intracellular values were expressed as a ratio.
(1C) Membrane-associated NTSR1 in biotinylation assay after vehicle, NTS (100 nM) treatment and recovery for 3 h were analyzed in an NTSR1 -specific ELISA. * P<0.05 when compared to antisense miR-control group.
Figures 2A-2F. AFTPH is the binding target of miR-1 33a. (2A) Diagram showing complementary binding site of miR-1 33a in AFTPH 3' UTR in different species. (2B) RT-PCR analysis of miR-1 33a levels in NCM460-NTSR1 cells transfected with control and antisense- miR-133a 2 days prior to NTS treatment (100 nM) for 30 min.(2C) RT-PCR analysis of AFTPH levels in NCM460-NTSR1 cells with the above mentioned treatment. (2D) Luciferase activity assay of NCM460-NTSR1 cells with the above mentioned treatment and exposed to 100 nM NTS for 1 h . (2E) Luciferase activity assay of cells transfected with plasmids of AFTPH 3' UTR with or without miR-1 33a binding site 2 days prior to NTS (100 nM) treatment for 1 h . (2F) Luciferase activity assay of HEK293 cells transfected with control or miR-1 33a precursors 2 days in prior. * P<0.05 when compared to vehicle treatment or control group.
Figures 3A-3D. AFTPH regulates NTS-induced NTSR1 recycling. (3A) Localization of AFTPH and TGOLN2 in untreated NCM460-NTSR1 cells. (3B) Localization of NTSR1 and AFTPH in untreated NCM460-NTSR1 cells. (3C) Localization of NTSR1 in NCM460-NTSR1 cells transfected with control si-RNAs and si-RNAs against AFTPH, and followed by treatment with vehicle control , 100 nM NTS treatment, 3 h in NTS-free medium after NTS treatment. (Arrows, intracellular NTSR1 ; arrowheads, membrane-associated NTSR1). Scale, 10 µηη . (3D) Membrane-associated NTSR1 in biontinylation assay after NTS (100 nM) treatment and recovery for 3 h analyzed in an NTSR1 -specific ELISA. * P<0.05 when compared to si-control- transfected cells.
Figures 4A-4B. Inhibition of TGN functioning inhibits NTS-induced NTSR1 recycling. (4A) NCM460-NTSR1 cells were treated with vehicle control, 100 nM NTS treatment and 3 h in NTS- free medium after NTS treatment in the presence or absence of 10 nM Brefeldin A (Arrows, intracellular NTSR1 ; arrowheads, membrane-associated NTSR1). Scale, 10 m. (4B) Membrane-associated NTSR1 in biontinylation assay after NTS (100 nM) treatment and
recovery for 3 h analyzed in an NTSR1 -specific ELISA. * P<0.05, * * P<0.01 when compared to vehicle control-treated cells.
Figures 5A-5G. ZEB1 is the negative transcription regulator of miR-133a. (5A) Diagram showing the complementary ZEB1 binding site in miR-133a promoter. (5B) RT-PCR analysis of miR-133a levels in NCM460-NTSR1 cells transfected with control si-RNAs or si-RNAs against
ZEB1 2 days prior to 100 nM NTS exposure for 1 h . (5C) Luciferase activity assay of NCM460- NTSR1 cells transfected with AFTPH 3' UTR luciferase and control si-RNAs or si-RNAs against ZEB1 (5D) RT-PCR analysis of AFTPH levels in cells with the above mentioned treatment. (5E) Chromatin-immunoprecipitation assay (ChIP) of ZEB1 binding sites from NCM460-NTSR1 cells incubated with vehicle control or 100 nM NTS for 1 h (5F) MiR-133a promoter-driven luciferase activity assay of cells transfected with control si-RNAs and si-RNAs against ZEB1 2 days prior
to 100 nM NTS exposure for 1 h . (5G) Luciferase activity assay of NCM460-NTSR1 cells transfected with miR-1 33a promoter with or without ZEB1 binding site 2 days prior to 100 nM
NTS treatment, 1 h . * P<0.05 when compared to vehicle treatment in control group.
Figures 6A-6E. MiR-1 33a and AFTPH regulate tumor growth in vitro and in vivo. (6A) Tumor volume measured from mice xenografts induced by injection of HCT-1 16 and SW480 cells and treated with NTS and antisense-control miR (as-miR-control) or antisense-miR-133a (as-miR- 133a) at day 10, 15, 20, 25 and 30. (6B) RT-PCR analysis of miR-1 33a, AFTPH and IL-8 mRNA levels in tumors from the above mentioned treatment. (6C) Anchorage-independent colony formation of HCT-1 16 and SW480 cells transfected with control si-RNAs (si-control) and si- RNAs against AFTPH (si-AFTPH). (6D) Tumor invasion assay of HCT-1 16 and SW480 of the above mentioned treatment. (6E) Tumor volume measured from mice xenografts induced by injection of HCT-1 16 and SW480 cells and treated with control si-RNAs (si-control) and si-RNAs against AFTPH (si-AFTPH) at day 10, 15, 20, 25 and 30. * P<0.05 when compared to untreated cells or mice.
Figures 7A-7B. Levels of miR-133a were upregulated after NT exposure in human colonic
epithelial cells. (7A) Expression of miR-133a in human colonic epithelial NCM460 cells after NT
exposure was analyzed by qPCR analysis. **P <0.01 , compared to vehicle control. (7B) miR- 133a levels in human colon cancer HCT-1 16 cells after NT exposure were analyzed by qPCR
analysis. **P <0.01 , compared to vehicle control.
Figures 8A-8G. Downregulation of miR-133a attenuated NT-associated proinflammatory signaling in NCM460-NTSR1 cells. (8A) Levels of miR-133a in NCM460-NTSR1 cells upon NT stimulation after transfection of as-miR-133a and its control were analyzed by qPCR analysis.
*P <0.05, compared to cells transfected with as-miR-control. (8B) Levels of MAP kinase phosphorylation, including p38, ERK1/2 and c-Jun and (8C) NF- κΒ phosphorylation in NCM460- NTSR1 cells transfected with as-miR-133a and its control were analyzed as stated in Methods.
*P <0.05, compared to cells transfected with as-miR-control; P <0.01 , compared to cells treated with NT. (8D) IL-6 and (8E) IL-8 levels in NCM460-NTSR1 cells transfected with as-miR-133a
and its control was analyzed by qPCR analysis. **P <0.01 , compared to cells transfected with
as-miR-control and P <0.05, compared with NT-treated cells. (8F) IL-8 production in NCM460- NTSR1 cells transfected with as-miR-133a and its control was measured using ELISA.
**P <0.01 , compared to cells treated with vehicle control; P <0.05, compared with NT-treated
cells. (8G) IL-1 β and IL-1ra production in NCM460-NTSR1 cells transfected with as-miR-133a
and its control was measured as stated in Methods. *P <0.05, compared to cells treated with vehicle control.
Figures 9A-9F. Overexpression of miR-133a promoted proinflammatory signaling in NCM460- NTSR1 cells. (9A) Levels of MAP kinase phosphorylation, including ERK1/2 and c-Jun and (9B)
N F-KB p65 phosphorylation in NCM460-NTSR1 cells transfected with miR-133a and its control was analyzed as stated in Methods. *P<0.05, compared to cells transfected with control miRNA precursor. (9C) Translocation of NF- κΒ p65 in NCM460-NTSR1 cells transfected with miR-133a
and its control was analyzed by ELISA. *P <0.05, compared to cells transfected with control miRNA precursor. (9D) Expression of IL-8, IL-1 β and TNF-a in NCM460-NTSR1 cells
transfected with miR-133a and its control was analyzed by qPCR analysis. *P <0.05, compared to cells transfected with control miRNA precursor. (9E) IL-8 production in NCM460-NTSR1 cells transfected with miR-133a and its control was measured using IL-8 ELISA.*P<0.05, compared to cells transfected with control miRNA precursor. (9F) IL-1 β and IL-1 ra production in NCM460- NTSR1 cells transfected with miR-133a and its control was measured as stated in Methods. * P<0.05, compared to cells treated with vehicle control; P<0.05, compared to cells transfected with miR control precursor.
Figures 10A-10D. Levels of miR-133a were upregulated during experimental colitis in vivo. (10A) miR-133a levels in colon tissues from C57BL6/J mice intracolonically administered with NT (300 g g) was analyzed by qPCR analysis. * P<0.05, compared to mice receiving vehicle
control. ( 10B) Expression of miR-133a in colon tissues from C57BL6/J mice collected 2 and 7 days after 5 mg/kg TNBS administration and 5 days after 5% DSS feeding was analyzed by
qPCR analysis. * P<0.05, compared to treatment control. (10C) Representative images of in situ hybridization of miR-133a of colon tissues from TNBS-treated C57BL6/J mice and their control counterparts (arrows, epithelial cells; arrow heads, lamina propria cells infiltration). Scale: 50 µιη . (10D) Expression of miR-133a in colon tissues from NTSR1KO and its control counterparts collected 5 days after 5% DSS feeding was analyzed by qPCR analysis. * P<0. 05, compared to wild type mice with same treatment.
Figures 11A-1 1E. Intracolonic administration of as-miR-133a attenuated TNBS-induced colitis
development in wild type mice. ( 1 1A) Representative images from H&E staining of colon tissues from C57BL6/J mice intracolonically administered with as-miR-133a and its control with or without TNBS treatment. Scale: 50 µιη . ( 1 1B) Levels of miR-133a in colon tissues from C57BL6/J mice intracolonically administered with as-miR-133a and its control were analyzed
using qPCR analysis. * P<0.05, compared to mice administered with control as-miRNA. ( 1C) Colon weight from C57BL6/J mice intracolonically administered with as-miR-133a and its control with or without TNBS treatment were measured and normalized by colon length. * P<0.05,
compared to mice administered with control as-miRNA. ( 1 1D) Total histological score, neutrophil infiltration and mucosal integrity of colon tissues from C57BL6/J mice intracolonically administered with as-miR-133a and its control with TNBS treatment were scored. * P<0.05,
* * P<0.01 , *** P<0.005 compared to mice administered with control as-miRNA. ( 1E) Levels of
Icn2, TNF-a and cxcH in colon tissues from C57BL6/J mice intracolonically administered with as-miR-133a and its control with or without TNBS treatment were analyzed using quantitative PCR analysis. * P<0.05, compared to mice administered with control as-miRNA.
Figures 12A-12H. AFTPH was the downstream target of NTS-induced miR-133a upregulation. (12A) Conservative miR-133a binding sites were identified in 3'UTR region of aftiphilin across different species. (12B) Levels of miR-133a in NCM460-NTSR1 cells transfected with as-miR- 133α and its control was analyzed by qPCR analysis. * P<0.05, compared to cells stimulated by vehicle. (12C) AFTPH 3'UTR-driven luciferase activities from NCM460-NTSR1 cells transfected with as-miR-133a and its control were measured after NTS stimulation. * P<0.05, compared to cells stimulated by vehicle. (12D) Luciferase activity driven by AFTPH 3'UTR with or without miR-133a binding sequence was measured after NTS stimulation. * P<0.05, compared to cells stimulated by vehicle. (12E) AFTPH 3'UTR-driven luciferase activities from HEK293 cells transfected with miR-133a precursor (miR-133a) and its control were measured. * P<0.05, compared to cells transfected with control miRNA precursor. (12F) Immunoblot analysis of AFTPH expression in NCM460-NTR1 cells 30 min and 6 h after NT exposure in the presence or absence of SR48962 (10 nM). (12G) AFTPH levels in colon tissues from C57BL6/J mice intracolonically administered with NT and its control and (12H) from C57BL6/J mice intracolonically administered with as-miR-133a and its control with or without TNBS treatment were analyzed using qPCR analysis. * P<0.05, compared to mice administered with control as- miRNA.
Figures 13A-13C. Gene silencing of AFTPH promoted NTS-associated proinflammatory response in vitro. Levels of (13A) c-Jun and (13B) NF-κΒ phosphorylation in NCM460-NTSR1 cells transfected with si-AFTPH and its control was measured as stated in Methods. * P<0.05, compared to cells transfected with control siRNA; P<0.05, compared to NTS-treated cells. (13C) IL-1 β and IL-1 ra production of NCM460-NTSR1 cells transfected with si-AFTPH and its control in the presence or absence of NTS stimulation was analyzed as stated in Methods. * P<0.05, compared to cells transfected with control siRNA.
Figures 14A-14B. Levels of miR-133a and AFTPH were significantly dysregulated in UC patients. (14A) Levels of miR-133a and AFTPH in colon tissues from normal and UC patients were analyzed using qPCR analysis. (14B) Representative images from immunohistochemistry against AFTPH and villin of colon tissues from normal and UC patients. Scale: 50 µηη .
Figures 15A-15C. Intracolonic administration of as-miR-133a attenuated DSS-induced colitis development in wild type mice. (15A) Percentage body weight loss in C57BL6/J mice intracolonically administered with as-miR-133a and its control treated with DSS. * P<0.05,
compared to mice administered with control as-miRNA (15B) Levels of TNF-a and (15C) IL- β in colon tissues from C57BL6/J mice intracolonically administered with as-miR-133a and its control were analyzed using qPCR analysis. * P<0.05, compared to mice administered with control as-miRNA. DETAILED DESCRIPTION OF THE INVENTION
While both genetic and environmental factors contribute to IBD pathogenesis, epigenetic regulators, such as microRNAs have not previously been known to have a role in IBD. MicroRNAs (miRs) are short (19-25 nucleotides), single-stranded RNA molecules, acting as negative transcriptional regulators. They bind to the 3' untranslated regions (UTRs) of transcripts and lead to messenger RNA (mRNA) degradation, or inhibition of translation into
protein (McKenna LB, et al., Gastroenterology 2010;139:1654-64, 1664 e 1; Bartel DP, Cell 2009;136:215-33).
The invention described herein is based on the unexpected expression of miR-133a in biopsy material from inflammatory bowel disease. The invention is further based on the demonstration that, in an experimental colitis model, intracolonic administration of anti-sense miR-133a reduces intestinal inflammation, while overexpression of this microRNA in human colonic epithelial cells activates the global mediator of inflammation NF-kB and increases expression of the potent IBD-related human chemokine interleukin-8. The data show that miR-133a is an important mediator in IBD and a target for IBD treatment.
Definitions
All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified. As used in this application, the following words or phrases have the meanings specified.
As used herein, "inflammatory disease" means a clinical disorder in which activation of the innate or adaptive immune response is a prominent contributor to the clinical condition.
As used herein, a "specimen" from a subject means a specimen obtained from the subject that contains blood or blood-derived cells, other bodily fluid, such as intestinal fluid, or biopsy tissue. Examples of biopsy tissue include intestinal tissue, such as colon biopsy tissue.
As used herein, a "control sample" means a specimen that represents a normal, healthy condition. The specimen may be blood, serum, or other fluid or tissue understood by those skilled in the art to serve as a suitable control. A sample of normal colon tissue or intestinal fluid obtained from a healthy patient is a typical example of a control sample.
As used herein, the term "subject" includes any human or non-human animal. The term "non- human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, horses, sheep, dogs, cows, pigs, chickens, and other veterinary subjects. As used herein, "a" or "an" means at least one, unless clearly indicated otherwise.
Methods of Detecting and Treating Inflammatory Disease
The invention provides a method for detection of inflammatory disease activity, such as inflammatory bowel disease, in a subject. In one embodiment, the method comprises assaying a specimen obtained from the subject for miR-133a, an oligonucleotide having the sequence: GGTTGAAGGGGACCAA (SEQ ID NO: 1). In another embodiment, the method comprises assaying a specimen obtained from the subject for aftiphilin (AFTPH), encoded by the sequence GGGTATGTTA GAGCCCACCA AGGAACCACT GAAACCACTT TCTGCTGCAG AAAAAATAGC TTCCATCGGT (SEQ ID NO: 2). In a further embodiment, the method comprises assaying the specimen for two, three, or more markers of IBD. The markers of IBD are selected from the group consisting of miR133a, AFTPH, and neurotensin receptor 1
(NTSR1). In some embodiments, one, two, three or more additional markers of IBD are used in addition to those named here. The assaying typically comprises contacting the specimen with reagents specific for miR-133a, AFTPH, and/or NTSR1, and measuring the amount of miR- 133a, AFTPH, and/or NTSR1 present in the specimen. In one embodiment, the NTSR1 is encoded by the sequence TCCGTTCCTCT ATGACTTCTA CCACTACTTC TACATGGTGA CCAACGCACT CTTCTACGTC AGCTCCACCA TCAACCCCAT CCTGTA (SEQ ID NO: 3).
The inflammatory disease detected in this manner includes both colon cancer and inflammatory
bowel disease. In particular, this method can be used to detect ulcerative colitis (UC), and to distinguish UC from Crohn's disease. Thus, the invention additionally provides a method of specifically detecting UC.
The amount of miR-133a, AFTPH, and/or NTSR1 present in the specimen is then compared to that present in a control sample. An elevated amount of miR-133a or NTSR1 present in the specimen compared to the control sample is indicative of inflammatory disease. A reduced amount of AFTPH present in the specimen compared to the control sample is indicative of inflammatory bowel disease. Typically, the amount of increase or decrease in the presence of the marker (miR-133a, AFTPH, and/or NTSR1) in the specimen obtained from a subject who has inflammatory disease is a statistically significant difference compared to a normal control sample.
In one embodiment, the amount of miR-133a and/or NTSR1 in a specimen obtained from an IBD subject is elevated 2-fold compared to a normal control, and the amount of AFTPH is
decreased to about half that of a normal control. In some embodiments, the difference is an increase (in the case of miR-133a and NTSR1) or decrease (in the case of AFTPH) 10%, 20%, 30%, 40%, 50%, 75%, 100% relative to normal control.
The measuring or assay for miR-133a can involve isolating microRNA from the specimen and/or performing a polymerase chain reaction (PCR) assay, such as real-time PCR, or other suitable PCR assay known in the art. Alternatively, the assay can be an in situ hybridization assay. The assay for AFTPH and for NTSR1 can be PCR or an immunoassay, such as enzyme-linked immunosorbent assay (ELISA), immunoblotting, radioimmunoassay, or other immunoassays known in the art.
Probes for detection of miR-133a, AFTPH, and/or NTSR1 can be detectably labeled. In one embodiment, the probe is labeled with a radioisotope, an enzyme, a fluorescent substance, a luminescent substance or biotin. In another embodiment, the 5' end of an oligonucleotide probe is labeled with a reporter fluorescent dye and the 3' end of the probe is labeled with a quencher dye. In some embodiments, the probe is an antibody and the detectable label is an antibody that binds AFTPH or NTSR1 , or a secondary antibody that binds a primary antibody. Representative antibodies are described in the Examples below. Reagents and kits, including antibodies, probes, PCR primers and related materials are commercially available.
The specimen is typically intestinal fluid or intestinal tissue, such as biopsy tissue. Other specimens may be obtained in accordance with the judgment of the treating physician. Specimens can be obtained from subjects using conventional means.
The inflammatory disease can be inflammatory bowel disease, including ulcerative colitis, Crohn's disease, or sub-clinical inflammation. Inflammatory bowel disease (IBD) refers to a group of disorders that cause the intestines to become inflamed (red and swollen). The two most common forms of IBD are ulcerative colitis and Crohn's disease. The inflammatory disease may also be an autoimmune disease (such as rheumatoid arthritis, systemic lupus erythematosis, or multiple sclerosis), or a chronic inflammatory disease (such as pseudomembranous colitis (positive or negative for C. difficile toxin), chronic diverticulitis, or chronic obstructive pulmonary disease).
Those skilled in the art will appreciate additional variations suitable for the method of detecting inflammation through detection of miR-133a in a specimen, as it provides a means of monitoring to assess disease activity and response to treatment. This method can also be used to monitor levels of miR-133a, AFTPH and/or NTSR1 in a sample from a patient undergoing treatment. The suitability of a therapeutic regimen for initial or continued treatment can be determined by monitoring miR-133a, AFTPH and/or NTSR1 levels using this method. The extent of miR-133a, AFTPH and/or NTSR1 present in a given patient or specimen can provide a prognostic indicator to guide treatment strategy. Accordingly, one can use information about the level of miR-133a, AFTPH and/or NTSR1 present in a subject to assist in selecting an appropriate treatment protocol. For example, mesalamine treatment of ulcerative colitis could be monitored by miR- 133a, AFTPH and/or NTSR1 as a surrogate biomarker to quantitatively measure the level of persisting disease activity. If disease activity persists above an acceptable level, the clinician would consider increasing the treatment dose, or changing to a different therapeutic agent.
The invention additionally provides a method of treating inflammatory disease, such as inflammatory bowel disease, or cancer, in a subject. In one embodiment, the method comprises administering to the subject a therapeutically effective amount of an inhibitor of miR-1 33a. A therapeutically effective amount is an amount sufficient to ameliorate disease symptoms, including, but not limited to, inflammation, and IL-8 expression. In one embodiment, the
administering is intracolonic or intravenous. In another embodiment, the administering is intratumoral. Examples of inhibitors of miR-1 33a include an antisense miR-1 33a oligonucleotide, such as an antisense oligonucleotide directed against SEQ ID NO: 1. The
antisense oligonucleotide can be provided in a more stabilized form, such as, in one example, a locked-nucleic acid-based antisense miR-1 33a oligonucleotide. The method can comprise administering the antisense miR-1 33a oligonucleotide directly, or via a lentiviral vector. Use of a suitable delivery vector, such as a lentiviral or adenoviral vector, for example, may be selected to enhance the efficacy of intravenous administration.
Treatment of colon cancer in a subject comprises administering a therapeutically effective amount of an inhibitor of miR-1 33a, such as an antisense miR-1 33a oligonucleotide, to the subject. A therapeutically effective amount is an amount sufficient to ameliorate symptoms of disease, such as tumor growth and/or size, and IL-8 expression. The administering can be intravenous, intracolonic, and/or intratumoral. In one embodiment, the inhibitor of miR-1 33a is administered intraoperatively at the time of biopsy and/or tumor resection.
Treatment of inflammatory disease or cancer can be administered in a single dose or as a series of doses administered over time. Dosage and treatment regimens can be determined by the treating physician, taking into account disease severity, patient condition, and other factors. Kits
For use in the diagnostic applications described herein, kits are also within the scope of the invention. Such kits can comprise a carrier, package or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in the method. The antibodies, probes, primers, and other reagents of the kit may be provided in any suitable form, including frozen, lyophilized, or in a pharmaceutically acceptable buffer such as TBS or PBS. The kit may also include other reagents required for utilization of the reagents in vitro or in vivo such as buffers (i.e., TBS, PBS), blocking agents (solutions including nonfat dry milk, normal sera, Tween-20 Detergent, BSA, or casein), and / or detection reagents (i.e., goat anti-mouse IgG biotin, streptavidin-HRP conjugates, allophycocyanin, B-phycoerythrin, R- phycoerythrin, peroxidase, fluors (i.e., DyLight, Cy3, Cy5, FITC, HiLyte Fluor 555, HiLyte Fluor 647), and / or staining kits (i.e., ABC Staining Kit, Pierce)). The kits may also include other reagents and / or instructions for using antibodies and other reagents in commonly utilized assays described above such as, for example, flow cytometric analysis, ELISA, immunoblotting (i.e., western blot), in situ detection, immunocytochemistry, immunohistochemistry.
In one embodiment, the kit provides the reagent in purified form. In another embodiment, the reagents are immunoreagents that are provided in biotinylated form either alone or along with an avidin-conjugated detection reagent (i.e., antibody). In another embodiment, the kit includes a fluorescently labeled immunoreagent which may be used to directly detect antigen. Buffers and the like required for using any of these systems are well-known in the art and may be prepared by the end-user or provided as a component of the kit. The kit may also include a solid support containing positive- and negative-control protein and / or tissue samples. For example, kits for performing spotting or western blot-type assays may include control cell or tissue lysates for use in SDS- PAGE or nylon or other membranes containing pre-fixed control samples with additional space for experimental samples.
The kit of the invention will typically comprise the container described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In addition, a label can be provided on the container to indicate that the composition is used for a specific application, and can also indicate directions for use, such as those described above. Directions and or other information can also be included on an insert, which is included with the kit. EXAMPLES
The following examples are presented to illustrate the present invention and to assist one of ordinary skill in making and using the same. The examples are not intended in any way to otherwise limit the scope of the invention.
Example 1: Neurotensin-induced tumor formation is regulated by neurotensin receptor 1 (NTSR1)/microRNA-133a-associated NTSR1 recycling involving the negative regulator zinc finger E-box-binding homeobox 1 (ZEB1) We recently identified miR-1 33a and its downstream target, aftiphilin (AFTPH), localized in the
trans-golgi network (TGN), as regulators of NTSR1 recycling in human colonocytes (DDW2012: Tu1823). This Example describes the mechanism by which NTS regulates miR-1 33a, and the
correlation of miR-1 33a and AFTPH expression with colon cancer development in mouse xenografts and human colon tissue samples.
MiR-1 33a transcriptional regulation was verified by quantitative PCR, promoter-driven luciferase, promoter site-directed mutagenesis, and chromatin immunoprecipitation (ChIP) assays in human colonic epithelial cells overexpressing NTSR1 (NCM460-NTSR1). The association of miR-1 33a and AFTPH with tumor growth was examined by tumor colony formation assays and mouse xenografts using SW480 and HCT1 16 colon cancer cells.
The genomic sequence of 2000bp upstream to the start of miR-1 33a was analyzed by transcription binding site prediction software and identified a binding site for zinc finger E-box homeobox 1 (ZEB1 , a negative transcriptional regulator). ZEB1 gene silencing in non- stimulated NCM460-NTSR1 cells increased miR-1 33a (3.3±0.6 fold, p<0.05) and reduced AFTPH mRNA (27.2±0.1%, p<0.01). ChIP analysis showed that upon NTS exposure ZEB1 was
dissociated from the miR-1 33a promoter (41 .0±1.7%, p<0.05 compared to non-stimulated cells).
MiR-1 33a overexpression increased cyclin D 1 expression (5.2±0.7 fold, p<0.001) in SW480 colon cancer cells. Blocking NTSR1 recycling through AFTPH-localized TGN by Brefeldin A
reduced NTS-induced tumor colony formation (44.1 ± . 1 % , p<0.05). MiR-1 33a overexpression
and AFTPH gene silencing also promoted tumor growth in vitro (-2.0 fold and ~ 1.7 fold
respectively, p<0.05) and in mouse cancer xenografts (~1 .25 fold and ~ 1.6 fold respectively, p<0.05), while miR-1 33a knock-down attenuated NTS-induced tumor growth (31 .6%, p<0.05).
MiR-1 33a mRNA was negatively correlated with AFTPH mRNA in human tumor samples (r= - 0.8979, n=42), and well correlated with tumor stage (p<0.01). Conclusions: NTSR1 signaling modulates miR-1 33a/AFTPH expression through dissociation of ZEB1 from the miR-1 33a
promoter, which promotes NTSR1 recycling. NTSR1/miR-1 33a/AFTPH interactions regulate colonic tumor growth. This is the first study providing evidence for an important role of microRNAs in regulation of GPCR recycling linked to development of colon cancer.
Example 2 : Neurotensin/miR-133a interactions modulate neurotensin-induced pro- inflammatory response during acute experimental colitis We have recently shown that NTS exposure to NTSR1 -expressing human colonic NCM460 epithelial cells (NCM460-NTSR1) induces differential expression of miRNAs (Gastroenterology 201 1;141 :1749-61) and NTS-modulated miR-133a expression regulates NTSR1 recycling and NTS-related tumor growth by inhibiting expression of its downstream target aftiphilin (AFPTH); (DOM Research Day 2012). This Example elucidates the mechanism by which miR-133a regulates NTS signaling in vitro, and describes the functional consequences of this response in the development of colitis in vivo.
NTS/miR-133a -regulated proinflammatory cytokine transcription in NCM460-NTR1 cells and colon tissues was examined by qPCR. MiR-133a and AFTPH -regulated NTSR1 intracellular trafficking was examined in immunocytohistochemistry of NCM460-NTSR1 cells transfected with antisense (as) miR-133a and si-AFTPH. Acute colonic inflammation was induced by intracolonic administration of TNBS (250 mg/kg, 48 h). Expression of miR-133a in the colon was inhibited by intracolonic administration of as-miR-133a (3 times every two days) before TNBS treatment. The degree of inflammation was evaluated on distal colon segments stained with H&E.
MiR-133a knock-down attenuated NTS-induced mRNA expression of IL-8 and IL-6 in vitro (P<0.01). Immunocytochemistry showed miR-133a knock-down decreased recycling of NTSR1 to the plasma membrane. miR-133a knock-down resulted in NTSR1 localization in the trans- golgi network, while AFTPH gene silencing promoted trafficking of NTSR1 to early endosomes. This result suggested that NTS-induced miR-133a promotes the rapid recycling of NTSR1 in human epithelial cells, thereby enhancing NTS-regulated proinflammatory signaling in vitro. MiR-133a knock-down in mouse colon reduced mucosal TNF-a expression (P<0.05) and
neutrophil infiltration (P<0.05) in colonic tissues and improved mucosal integrity (P<0.05) and overall histological score (P<0.05) in wild type mice with TNBS-induced colitis.
The results indicate that miR-133a regulates NTSR1 recycling in human colonocytes by targeting AFTPH, a novel miR-133a target. Silencing of miR-133a also reduces cytokine expression and histologic damage and inflammation in a mouse model of experimental colitis. These results suggest that miR-133a/AFTPH interactions promote NTS-induced proinflammatory responses in the colonic mucosa by enhancing NTSR1 recycling. The results also support targeting of miR-133a in the colon for treatment in Inflammatory Bowel Disease.
Example 3 : Neurotensin-induced tumor formation is regulated by microRNA-133a- aftiphilin- dependent receptor recycling
Recycling of the G protein-coupled receptor (GPCR) neurotensin receptor 1 (NTSR1 ; sometimes referred to as NTR1) regulates proliferative and pro-inflammatory signaling in colonocytes. MicroRNAs (miRs) are negative regulators of gene expression affecting multiple signaling pathways, but their involvement in GPCR recycling is unknown. This Example describes the role of miRNA(s)-regulated NTSR1 trafficking in colonic tumor development regulated by NTS/NTSR1 interactions.
Immunocytochemistry and a biotinylation assay were used to study NTSR1 trafficking. Potential transcriptional regulator(s) and binding target(s) of miRs were identified by bioinformatic analysis, real-time polymerase chain reaction (RT-PCR) and chromatin immunoprecipitation. Tumorigenesis was assessed in soft agar assays and human colon cancer xenografts.
RESULTS: In human colonocytes, NTS increased miR-133a expression. Antisense-miR-133a inhibited NTSR1 recycling, but not endocytosis, following NTS exposure. Bioinformatic analysis and RT-PCR identified aftiphilin (AFTPH), a protein associated with protein trafficking, as a miR- 133a binding target that is down-regulated during NTS exposure. AFTPH silencing enhanced NTSR1 recycling. We also found that NTS induces miR-133a expression by reducing binding of the transcription factor Zinc finger E-box binding homeobox 1 (ZEB1) to the miR-133a promoter. NTS injection to mice increased miR-133a and interleukin-8 (IL-8) and reduced AFTPH mRNA levels in HCT-1 16 and SW480 colon cancer xenografts and stimulated tumor growth. All these responses were reversed by intra-tumoral injection of as-MiR-133a.
This Example shows that NTS-regulated miR-133a expression modulates NTSR1 recycling and promotes tumorigenesis by down-regulating the previously unknown miR-133a target AFTPH in colonocytes. The results show that miRs modulate colonic tumorigenesis by regulating trafficking of GPCRs in colonocytes.
Based on these considerations, we hypothesized that some of the NTS-upregulated miRs may modify NTSR1 trafficking, thereby altering signaling pathways important for tumorigenesis. We show that among 8 miRs stimulated by NTS, only miR-133a regulates NTSR1 recycling to the plasma membrane without affecting receptor internalization. We demonstrate that the mechanism by which NTS increases miR-133a expression involves the transcription factor ZEB1. Importantly, we present evidence that miR-133a modulates NTSR1 recycling by downregulating aftiphilin (AFTPH), a previously unrecognized miR-133a downstream target that regulates NTSR1 recycling as well as colonic tumor growth in vitro and in vivo.
Materials and Methods
Cell culture. NCM460-NTSR1 cells were generated from human colonic epithelial NCM460 cells (INCELL) transduced with lentivirus particles expressing human NTSR1 as previously described 3 . HCT-1 16 and SW480 colon cancer cells and HEK293 human embryonic kidney fibroblasts were maintained in McCoy's 5a Medium Modified, Roswell Park Memorial Institute (RPMI) 1640 medium and Eagle's Minimum Essential Medium (MEM), respectively, and supplemented with 10% fetal bovine serum.
Immunofluorescence. NCM460-NTSR1 cells were transfected with as-miR-133a or si-AFTPH and their corresponding controls. Cells were serum-fasted overnight 2 days post transfection
and exposed to 100 nM NTS for 1 h . For NTSR1 recovery studies, cells were washed with Phosphate Buffered Saline (PBS) twice and replenished with NTS-free medium supplemented with 10 µg/ml Brefeldin A or vehicle. Cells were fixed in PBS containing 4% (w/v) paraformeldehyde (pH 7.4, 20 min, 4°C) and blocked in PBS containing 3% bovine serum and
0.1% Triton X . Cells were incubated (16 h , 4°C) with the following antibodies: goat anti-NTSR1 , goat anti-AFTPH and rabbit anti-TGOLN2. Cells were washed and incubated (2 h , room temperature) with the appropriate secondary antibodies conjugated with fluorescent probes. After washing with PBS, cells were mounted with ProLong ® Gold Antifade Reagent (P36930; Invitrogen) and imaged with a Leica TCS SP8 laser scanning confocal microscope (North Ryde, NSW, Australia) using a Leica HCX PL APO 63x oil immersion objective (numerical aperture 1.4). Five Z-stack images were captured (1024x1024 pixel resolution) per treatment. For protocol for image analysis, see below.
Biotinylation assay. Biotinylation of surface NTSR1 was achieved by a modification of a previously described method 3. NCM460-NTSR1 cells were washed with PBS and labeled with 1mg/mL biotin (EZ-link® Sulfo-NHS-LC Biotin, Thermo Scientific) for 15 min at 4°C. All procedures, unless otherwise stated, were performed on ice. Unbound biotin was quenched with Tris-buffered saline (TBS) and cells were washed with HBSS, recovered in media (37°C for
30 min) and exposed to 100 nM NTS for 1 h . After washing with HBSS, 50 mM glutathione in HBSS was used to strip biotin from proteins remaining at the cell surface. Endocytosed NTSR1 was allowed to recycle to plasma membrane in culture media for 3 h at 37°C, biotin bound to recycled NTSR1 was stripped, and unbound biotin was quenched by TBS. Cells were then lysed with 1% Triton X-100 in HBSS supplemented with protease inhibitor cocktail. Quantification of NTSR1 recycling was performed by NTSR1 -specific ELISA. Antibodies against NTSR1 (2 µg/mL) were used to coat 96-well plate (Fisher Scientific). The plate was blocked and equal amount of lysates were loaded to each well in triplicates and incubated for 16 h at 4°C. This was followed by washing with PBS containing 0.05% Tween-20 and incubation with
IRDye® 800CW Streptavidin ( 1 h , room temperature). After washing, the fluorescent intensity was read by Odyssey® CLx Infrared Imager (LI-COR Biosciences) and quantified by LI-COR® Image Studio (LI-COR Biosciences). Membrane-associated NTSR1 was calculated by the equation 1/(biotinylated NTSR1 after treatment/Total biotinylated NTSR1) and analyzed by Student's t-Test.
Luciferase assays. Plasmids encoding AFTPH 3'UTR-driven or miR-133a promoter-driven luciferase reporter were transfected to NCM460-NTSR1 cells using lipofectamine 2000 (Invitrogen). For experimental details, see below. Firefly and Renilla luciferase cell activities were detected using Dual-luciferase reporter assay system (Promega). Results were presented as the relative luciferase activity (mean ± SD) from at least three independent sets of experiments, each with five replicated measurements.
Chromatin immunoprecipitation (ChIP). NCM460-NTSR1 cells were cross-linked and fixed after NTS exposure for 1 h using Pierce Agarose ChIP kit (Thermo Scientific) according to the manufacturer's instructions. The ZEB1 binding region was immunoprecipitated by anti-ZEB1 antibody. ZEB1 binding was quantified by real time PCR using a primer complementary to ZEB1 binding site in miR-133a promoter region (Applied Biosystems, assay ID: AJPACV3, Part no. 4441 114).
Anchorage-independent growth assays. Anochorage-independent growth assays were performed as described 3 . HCT-1 16 and SW480 cells were transfected with miR-133a or si-
AFTPH 2 days prior to the assay. In addition, SW480 cells were treated with 2.5, 5 , 10 µg ml brefeldin A in the presence of 100 nM NTS. Triplicates of 5,000 cells were suspended in complete growth media supplemented with 0.4% agarose [mixing 2% agarose with complete growth media in 1:4(v/v)). Cell mixtures were layered on 0.8% agarose and fed with complete growth media supplemented with 0.4% agarose every 6-7 days. The number of colonies was counted after 15 days. Experiments were repeated 3 times. Statistical significance was calculated using the Student's t-Test. Human colon cancer xenografts. 6x1 0s HCT-1 16 or SW480 cells were injected subcutaneously in the right flank of athymic nude (nu/nu) mice (Charles River Laboratories). When the tumors reached a size of -100 mm3 (10 days) mice were randomly distributed in different groups (4 mice/group). In all experiments, tumor growth was monitored every five days for a total period of 35 days and tumor volumes were calculated by the equation V(mm 3)=axb2/2, where a is the largest diameter and b is the perpendicular diameter.
Statistical analysis. All in vitro results (derived from at least three sets of experiments), expressed as means ± SD and analyzed with Student's t-Tests. Results from
immunofluoresence and in vivo studies were analyzed with one-way-ANOVA (Prism 5). In all statistical comparisons, P< 0.05 was used to indicate significant differences.
Antibodies and reagents We used the following antibodies and reagents: AFTPH ( 1 :100, sc-
167055; Santa Cruz Biotech. Inc), phosphor-NF-KB/p65 (Ser536) ( 1 :1000, 3031 ; Cell Signaling,
Inc), NTSR1 ( 1 :100, sc-7596; Santa Cruz Biotech, Inc), TGOLN2 ( 1 :100, sc-33783; Santa Cruz
Biotech, Inc), bovine anti-goat IgG-FITC ( 1 :500, sc-2348; Santa Cruz Biotech, Inc), bovine-anti-
rabbit IgG-Texas Red ( 1 :500, sc-2787; Santa Cruz Biotech. Inc), IRDye ® 680RD Donkey anti-
Goat IgG (H+L) ( 1 :800, 926-68074; LI-COR Biosciences), ZEB1 ( 1 µg/mL, A301-921A; Bethyl
Laboratories), Brefeldin A (sc-200861; Santa Cruz Biotech. Inc ) , glutathione (BP2521-5, Fisher BioReagents), Hank's balanced salt solution (HBSS, 14175-095, Life Technologies, Inc), NTS (H-4435.0005; Bachem Americas, Inc), paraformaldehyde (CAS 30525-89-4; Santa Cruz
Biotech. Inc ) , complete protease inhibitor cocktail ( 1 1697498001 , Roche), sulfosuccinimidyl-2- (biotinamido) ethyl-1 , 3-dithiopropionate (sulfo-NHS-S-S-biotin, 21331 , Thermo Scientific).
Image analysis protocol. Substacks of five optical sections were generated per image and were analyzed using ImageJ with MacBiophotonics plugins. Briefly, a region of interest (ROI) within the cell surface was defined (ROI 1) and then enlarged by 1 µηη to generate a second ROI (ROI 2), representing the intracellular (ROI 1) and total area including plasma membranes of cells (ROI 2). Mean fluorescence intensity was measured for these two regions, representing the intracellular and membrane-associated NTSR1 labeling, respectively. The relative surface and intracellular values from the same cell were expressed as a ratio (ROI 2/ROI 1). Data were presented as either ratios (mean ± SEM). Only cells with a defined nucleus were analyzed. Treatment groups were compared by one-way ANOVA with Dunnet's post-hoc test.
Transfection experiments. NCM460-NTSR1 , HCT-1 16 and SW480 cells were transfected with 60 nmol/L si-RNA against AFTPH (si-AFTPH, sc-94965; Santa Cruz Biotech. Inc) or ZEB1 (sc- 38643; Santa Cruz Biotech. Inc) using LipofectamineTM RNAiMAX (Invitrogen). For miR-133a silencing or overexpression, the cells were transfected with 60 nmol/L of antisense-miR-133a (as-miR-133a, Ambion) or 60 nmol/L of Pre-miR-133a precursor (Ambion) using siPORTNeoFX transfection reagent (Ambion). Transfection with 60 nmol/L of Control siRNA-A (si-control, Santa Cruz Biotech. Inc), 60 nmol/L of antisense-control miR (as-miR-control, AM17010; Ambion) or 60 nmol/L Pre-miR-negative control (miR-control, AM171 10; Ambion) was used as control.
Plasmid construction and site-directed mutagenesis. pGL3-miR-133o construction, The genomic region of 2000 bp upstream to miR-133a was cloned by the primers, i) miR-133a Xho1 F (5'-ccg etc gag ttt caa aga aat tag ttc aaa get taa-3'; SEQ ID NO: 4) and ii) miR-133a Hindi 11 R (5'-ccc aag ctt agt get get agt ttg gaa tcc-3'; SEQ ID NO: 5). The miR-133a promoter-driven luciferase reporter construct (pGL3-miR-133a) was generated by ligating Xho1/Hindlll-digested PCR products and pGL3-Basic (Promega). pGL3-miR-1 33ο-∆ΖΕΒ 1 construction, Site-directed
mutagenesis was done using QuikChange II XL site-directed mutagenesis Kit (Agilent Technologies) according to the manufacturer's instructions. ZEB1 binding site on the miR-133a promoter region (pGL3-miR-133a-AZEB1) was deleted using with primers: iii) miR133a-del216 (5'-gca ctt aag ttt agg cag ttt aac act tct act aga aaa aat gat gaa aaa g-3'; SEQ ID NO: 6) and iv) miR133a-del216antisense (5'-ctt ttt cat cat ttt ttc tag tag aag tgt taa act gec taa act taa gtg c- 3'; SEQ ID NO: 7). AFTPH 3'UTR-AmiR-1 33a construction, AFTPH 3' UTR luciferase reporter plasmid was purchased from Switchgear Genomics and miR-133a binding site was deleted (AFTPH 3'UTR-AmiR-133a) with primers: v) AFTPH-del198: (5'-atc agt atg att cag aga agg aca tta tat gaa tgt ctt aca atg g-3'; SEQ ID NO: 8) and vi) AFTPH-del198-antisense (5'-cca ttg taa gac att cat ata atg tec ttc tct gaa tea tac tga t-3'; SEQ ID NO: 9).
Messenger RNA and miR expression analysis. NCM460-NTSR1 cells were washed once with ice-cold PBS after various treatments. Total RNA were extracted by TRIzol (Life technologies) and reverse-transcribed into first strand cDNAs using random decamers and reverse transcriptase (Invitrogen) for mRNA expression analysis. Complementary DNAs for microRNA expression analysis were prepared with mirVana quantitative reverse-transcription PCR miRNA Detection Kit and quantitative reverse-transcription PCR primer sets according to the manufacturer's instructions (Ambion).
Luciferase assay. AFTPH 3' UTR -associated luciferase activity: AFTPH 3' UTR luciferase reporter plasmid or AFTPH 3' UTR-AmiR-133a luciferase reporter plasmid and R01_3UTR (Switchgear Genomics, control) were transfected to NCM460-NTSR1 cells in the presence of as-miR-133a and its control (Ambion) and in HEK293 cells in the presence of miR-133a precursor and its control (Ambion). Two days after transfection NCM460-NTSR1 cells were exposed to NTS (100 nM, 1 h), while luciferase activities in transfected HEK293 cells were measured without NTS exposure. The relative AFTPH 3' UTR-associated luciferase activities were calculated by normalizing AFTPH 3' UTR-associated luciferase activities with R01_3' UTR luciferase activity. miR-133a promoter activity: pGL3-miR-133a or pGL3-miR-133a-AZEB1 and pRL-TK (Promega, control) were transfected to NCM460-NTSR1 cells using lipofectamine 2000 (Invitrogen). The relative miR-1 33a promoter-driven luciferase activities were calculated by normalizing Firefly luciferase activity with that from Renilla luciferase.
Cell-based enzyme-linked immunosorbent assay (ELISA). An NTSR1 -specific cell-based ELISA modified from a previous report 4 was used for quantification of NTSR1 in unstimulated
or NTS-stimulated NCM460-NTSR1 cells. In brief, NCM460-NTSR1 cells were transfected and seeded in 96-well culture plate. Cells were serum-fasted overnight 2 days post transfection and
exposed to 100 nM NTS for 1 h . Cells were fixed in PBS containing 4% (w/v) paraformeldehyde (pH 7.4, 20 min, 4°C) and followed by blocking and permeabilization with PBS containing 3%
bovine serum and 0.1% Triton X . Cells were then incubated (16 h , 4°C) with goat anti-NTSR1 in the same blocking buffer with 0.1% Triton X , followed by washing with PBS (containing 0.1%
Tween-20) and incubation with IRDye® 680RD Donkey anti-Goat IgG (H+L) ( 1 h , room temperature). After washing, the fluorescent intensity was read by Odyssey® CLx Infrared Imager (LI-COR Biosciences) and quantified by LI-COR® Image Studio (LI-COR Biosciences). Data are presented as NTSR1 (NT exposure) / NTSR1 (unstimulated) (mean ± SD) and analyzed by Student's t-Test.
Immunoblot analysis. NCM460-NTSR1 cells were washed with ice-cold PBS after various treatments and incubated with radiolabeled immunoprecipitation assay buffer containing protease inhibitor cocktail, phenylmethylsulfonyl fluoride and sodium orthovanadate (Santa Cruz) for 5 min. The insoluble debris was removed by centrifugation at 12,000 rpm, 15 min at 4°C and supernatants were analyzed by immunoblot analysis. Equal amount of cell lysates were loaded (~ 35 µg) and transferred to nitrocellulose membrane. The membrane was blocked with 5% non-fat dry milk (w/v) in Tris-buffered saline with 0.1% Tween 20 (TBS-T). Appropriate antibodies were incubated with the membranes overnight at 4°C, washed with TBS-T and incubated with appropriate secondary antibodies conjugated to horseradish peroxidase. Signals from target proteins were detected with SuperSignal chemiluminescent substrate (Pierce). Immunoblot bands were quantified by densitometry using Multi Gauge V3.1 (Fuji).
IL-8 ELISA. NCM460-NTSR1 cells were stimulated with 100 nM NTS for 6 h after various treatments. IL-8 in supernatants was measured by Duo-Set ® ELISA for IL-8 (R&D Systems) according to the manufacturer's instructions.
Human colon cancer xenografts. Effects of miR-133o as a mediator of NTS tumorigenic activity, the mice bearing xenografts were administered intratumorally as follows: i) untreated, ii) NTS (100 nM) every 5 days until day 30, iii) as-miR-control (5 mg/kg) and NTS (100 nM), iv) as- miR-133a (5 mg/kg) and NTS (100 nM). Effects ofAFTPH inhibition on tumor growth, The mice bearing xenografts were divided in the following groups, i) untreated, ii) si-control (10 mg/kg) and iii) si-AFTPH (10 mg/kg) intratumorally. The dosage of each treatment was 5 mg/kg and all treatments were performed every 5 days until day 30.
Results
NTS-induced miR-133a up-regulation is involved in NTSR1 recycling.
Since NTS triggers expression of miRs in human colonocytes within a time period (30 min -6 h) that coincides with NTSR1 trafficking 23 , we investigated whether the NTS - up-regulated miRs 140, 2 1, 210, 155, 133a, 23a, 23β, and 331-5p 3 modulate NTSR1 internalization and recycling. NCM460-NTSR1 cells transfected with antisense (as) oligonucleotides against all NTS-
upregulated miRs were exposed to 100 nM NTS for 1 h , followed by washing and replenishing with NTS-free medium for 3 h to allow NTSR1 recycling. Membrane-associated NTSR1 was detected at 3 and 6 h by confocal microscopy and quantified by image analysis. Results were expressed as the ratio between NTSR1 fluorescent signals present on the cell surface compare to the cytosol. Consistent with our previous findings 23, NTSR1 was internalized after 1 h NTS (Fig 1A). NTSR1 endocytosis was unaffected by antisense treatment against any of the 8 NTS- upregulated miRs (Fig. 1A and B, Suppl. Result 1A). At 3 hrs, the majority of NTSR1 was recycled to the plasma membrane in control as-miR-transfected cells (Fig. 1 and B). However, in cells transfected with as-miRs, only as-miR-133a caused a prolonged retention of NTSR1 within intracellular vesicles (Fig. 1A, lower panel; Fig 1B, and Suppl Result 1A), indicating inhibition of NTSR1 recycling specifically by miR-133a.
To confirm that NTSR1 recycling is only inhibited by as-miR-133a treatment, we used a biotinylation assay followed by NTSR1-specific ELISA to quantify NTSR1 recycling. Compared to control as-miR, as-miR-133a treatment significantly reduced NTSR1 recycling 3 h after NTS exposure (P<0.05) without affecting NTSR1 internalization (Fig. 1C). None of the other as-miR treatments significantly affected this response (Suppl Result 1B).
MiR-133a directly regulates AFTPH expression through binding its 3' UTR in colonic epithelial cells. MiRs act as gene-silencers by inducing the degeneration of mRNA transcripts through binding to the 3' UTRs of target genes . To identify candidate genes involved in the miR-133a-NTSR1 response, we searched for genes with possible miR-133a binding sites in their 3' UTRs. In silico search using 3 online databases [TargetScanHuman (targetscan.org); miRBase (mirbase.org) and PicTar (pictar.mdc-berlin.de)], identified aftiphilin (AFTPH) with a miR-133a binding site at its 3' UTR highly conserved across species (Fig. 2A). This interaction was validated in human colonocytes transfected with as-miR-133a. We first verified that as-miR- 133a blocked the expected increase of miR-133a in NCM460-NTSR1 cells exposed to NTS (Fig. 2B). NTS stimulation down-regulated AFTPH mRNA levels (Fig. 2C) and its 3' UTR- associated luciferase activity (Fig. 2D) compared to control cells, while as-miR-133a treatment reversed these responses (Figs. 2C & 2D). To further confirm the direct interaction between miR-133a and AFTPH, we deleted miR-133a binding sites on the AFTPH 3' UTR. Deletion of this miR-133a binding site blocked NTS-induced down-regulation of AFTPH 3' UTR luciferase activity (Fig. 2E). In addition, miR-133a overexpression in HEK293 cells significantly reduced AFTPH-3' UTR-associated luciferase activity in the absence of NTS stimulation (Fig. 2F). Thus, NTS stimulation suppresses AFTPH mRNA expression through induction of miR-133a, suggesting that AFTPH is a downstream target of miR-133a in colonocytes.
Trans-golgi network (TGN)-localized AFTPH expression modulates NTS-miR133a - regulated NTSR1 recycling. AFTPH is a 936 amino acid protein with binding motifs for clathrin 24 , adaptor protein-1 (AP-1) and AP-2 25, key mediators of endocytosis and exocytosis. Immunohistochemical experiments with an antibody directed against AFTPH in non-stimulated NCM460-NTSR1 cells showed that endogenous AFTPH is colocalized with the TGN marker TGOLN2 (previously known as TGN38) (Fig. 3A), consistent with previous observations in neurons 2S. AFTPH was also partially colocalized with NTSR1 in human NCM460-NTSR1
colonocytes (Fig. 3B). To investigate a potential role of AFTPH in NTS-induced NTSR1 trafficking, AFTPH expression in NCM460-NTSR1 cells was silenced using small interfering RNA (si-RNA) against AFTPH, which has been shown to have no effect on TGN morphology 2 .
Cells were stimulated with NTS for 1 h , and recovered in NTS-free medium for 3 h to assess receptor endocytosis and recycling. Confocal microscopy showed that AFTPH silencing increased NTSR1 -fluorescence at the plasma membrane (Fig. 3C), suggesting enhanced recycling efficiency of internalized NTSR1 following. Quantification of NTSR1 recycling using the
biotinylation assay confirmed increased membrane-associated NTSR1 during NTS exposure in AFTPH-silenced cells at 1 h (P<0.01) and 3 h after incubation of cells in NTS-free medium (P<0.05, Fig. 3D). The physiological importance of the TGN to NTSR1 recycling was tested by treating NTS-stimulated NCM460-NTSR1 cells with 10 nM Brefeldin A , a TGN transport inhibitor , during NTSR1 recovery to the plasma membrane. The majority of NTSR1- fluorescence signal was retained in the cytosol when NTSR1 was allowed to recycle in NTS-free medium containing Brefeldin A (Fig. 4A). The biotinylation assay confirmed that NTSR1 did not recycle to the plasma membrane at 3 h in Brefeldin A-treated cells compared to cells treated with vehicle (PO.01 , Fig. 4B).
In CHO and HEK293 cells, NTSR1 is degraded in lysosomes upon NTS exposure 2' 4 ' 22. To exclude the possibility that miR-133a and AFTPH are involved in NTSR1 degradation, we compared NTSR1 protein expression in AFTPH and miR-133a silenced cells during NTS exposure using a cell-based ELISA. We found that NTSR1 expression was not significantly
different in as-miR-133a - treated (Suppl. Result 2A) or AFTPH-silenced cells (Suppl. Result 2B) compared to control cells, suggesting that miR-133a and AFTPH do not affect NTSR1 degradation. Thus, NTS-induced NTSR1 recycling involves AFTPH and requires normal TGN function.
Zinc finger E-box binding homeobox 1 (ZEB1) is a negative transcription regulator of miR-133a. To examine the molecular mechanism of NTS-induced miR-133a up-regulation, the genomic sequence of 2000 base pair (bp) upstream to the transcription start site of miR-133a was analyzed by the online transcription binding site prediction software, Transcription Element Search System (TESS) (http://www.cbil.upenn.edu/cgi-bin/tess/tess). This analysis revealed a binding site for the transcription factor zinc finger E-box homeobox 1 (ZEB1) at the promoter region of miR-133a (Fig. 5A). We next knocked down ZEB1 expression by si-RNA in NCM460-
NTSR1 cells and exposed them to 100 nM NTS ( 1 h). In ZEB1 knockdown cells, basal miR- 133a levels were significantly higher compared to control si-RNA transfected cells (Fig. 5B, P< 0.05). However, ZEB1 gene-silencing blocked NTS-induced miR-133a up-regulation in cells transfected with control si-RNA (si-control) (Fig. 5B). Moreover, basal AFTPH 3' UTR- associated luciferase activity was significantly reduced in ZEB1 - silenced cells (Fig. 5C,
P<0.05). In addition, we observed reduced basal AFTPH mRNA levels (Fig. 5D, P<0.05) in ZEB1 - silenced cells compared to si-control transfected cells. There was no significant reduction in AFTPH 3' UTR-associated luciferase activity or AFTPH mRNA levels after NTS exposure (Figs. 5C & 5D).
To directly show that ZEB1 binds to the miR-133a promoter, we performed chromatin - immunoprecipitation (ChIP) with nuclear extracts from control and NTS-exposed cells using a ZEB1 antibody. We found reduced ZEB1 binding to the miR-133a promoter after NTS stimulation (Fig. 5E, P<0.05). To explain this response, the NTS-ZEB1-miR-133a pathway was further investigated by examining miR-133a promoter activity. MiR-133a promoter-driven luciferase activity was increased in control, but not in ZEB1 gene-silenced cells during NTS exposure (Fig. 5F), or in cells transfected with a miR-133a promoter with deleted ZEB1 binding site (Fig 5G). These results indicate that ZEB1 acts as a negative transcription regulator in NTS- associated miR-1 33a transcription.
NTS-induced miR-1 33a up-regulation promotes tumor growth in vitro and in vivo. We previously reported that in colonocytes NTS-induced NTSR1 recycling directly regulates NTS- associated proinflammatory signaling 23, which may have oncogenic function 3' 31, 32. To examine the role of miR-1 33a in NTS signaling, we overexpressed miR-1 33a in NCM460-NTSR1 cells and measured NF-κΒ activation and IL-8 expression. We found that miR-1 33a overexpression in both NTS-stimulated and non-stimulated cells increased IL-8 secretion (Suppl. Result 3A) and NF-KB/p65 activation (Suppl. Result 3B), supporting the hypothesis that miR-1 33a participates in NTS-induced cell signaling by regulating NTS-induced NTSR1 receptor recycling.
Since NTS/NTSR1 coupling promotes colon tumor development 13' 33 and induces similar trends of differential microRNAs expression in colonic cancer cell lines compared to NCM460-NTSR1 cells 13 , we examined the importance of miR-1 33a in NTS-induced colon tumor development. Immunodeficient nude (nu/nu) mice bearing HCT-1 16 or SW480 xenografts were treated with NTS in the presence of as-control miR and as-miR-133a every 5 days until day 30, and tumor growth was measured. As in our previous study, NTS treatment significantly promoted tumor
growth in HCT-1 16 and SW480 xenograft models (by 2.2±0.09 fold and 1.9±0.08 fold, respectively) at the end of treatment, compared to that in the untreated mice (Fig. 6A). Infusion of control as-miR did not contribute to NTS-induced tumor growth. Importantly, intratumoral injection of as-miR-133a suppressed NTS-induced tumor growth after the third NTS and as- miR-133a injections. The final tumor volume was significantly reduced in both xenografts compared to untreated mice (Fig. 6A, P<0.05). Furthermore, NTS treatment increased miR- 133a and IL-8 mRNA and reduced AFTPH mRNA levels in tumors, while as-miR-133a treatment reversed the NTS-induced mRNA changes in these tumors (Fig 6B). These results
suggest that miR-1 33a is involved in NTS-associated colon tumorigenesis in mice.
AFTPH acts as a tumor suppressor gene in colon cancer. We also examined the potential function of AFTPH in colon cancer in vitro and in vivo. AFTPH gene-silencing in HCT-1 16 and SW480 colon cancer cells resulted in increased colony formation (P<0.05 for both, Fig. 6C) and increased invasiveness (Fig. 6D, P<0.05 for both), when compared to untreated cells. Consistent with these findings, pharmacological inhibition of TGN function by Brefeldin A inhibited SW480 cell growth (Suppl. Result 4 , P<0.05). Importantly, tumor growth was increased in HCT-1 16 and SW480 xenografts after intratumoral injections of si-AFTPH (Fig. 6E, P<0.05). These data show that AFTPH has a tumor suppressive function in colon cancer.
Supplementary Results
Supplementary Result 1. Demonstrates that down-regulation of miRs: 140, 2 1, 210, 155, 23a, 23β, 331-5p do not affect NTSR1 recycling. (Result 1A) NTSR1 localization in NCM460-NTSR1 cells transfected with antisense-miR control or antisense-miRs against miR-140, miR-21 , miR- 210, miR-155, miR-23a, miR-233 and miR-331-5p in vehicle control, 100 nM NTS treatment for 1 h and 3 h after recovery in NTS-free medium. (Result 1B) Membrane-associated NTSR1 in
biotinylation assay after different treatments. * P<0.05 (for 100 nM NTS with miR-21 0 , miR-233 and miR-331-5p), * * P<0.01 (for 100 nM NTS with all other groups) when compared to vehicle control group.
Supplementary Result 2. MiR-133a and AFTPH are not involved in NTS-associated NTSR1 biosynthesis and production. NTSR1 expression levels were examined in NCM460-NTSR1 cells transfected with anti-sense miR-1 33a (Result 2A) or siRNA-against AFTPH (Result 2B) and their respective controls in cell-based ELISA, and no difference between groups was observed.
Supplementary Result 3. Overexpession of miR-133a enhances NTS-induced N F-KB signaling. (Result 3A) IL-8 ELISA on conditioned media from NCM460-NTSR1 cells transfected with control miRNA precursors or miR-1 33a precursors 6 h after vehicle or 100 nM NTS
treatment. (Result 3B) Western blot analysis of ERK1/2 and N F-κΒ phosphorylation in NCM460- NTSR1 cells transfected with control miRNA precursors or miR-1 33a precursors 2 days prior to 100 nM NTS treatment for 5 min and 1 h respectively. * P<0.05 when compared to vehicle control treatment (NT vs. vehicle), #P<0.05 when compared to vehicle control treatment in miR- 133a-overexpressed group.
Supplementary Result 4. Brefeldin A attenuates colony formation in vitro. 38 Anchorage- independent colony formation of untreated or Brefeldin A-treated (2.5 µg/ml, 5 µg/ml, 10 µg/ml) SW480 cells. * P<0.05 when compared to vehicle treatment (NTS- BrA 5 ; NTS+ BrA 0 or 2.5); #P<0.05 when compared to NTS treatment (NTS+ BrA 10 or 5).
Discussion
Since miRs were first discovered in C. elegans 34 , they have been implicated in many physiological functions, including inflammation 3, differentiation 35, apoptosis 3S, and oncogenesis 3 . The results suggest an additional important role for miRs, namely modulation of recycling of a GPCR. We have correlated NTS-induced differential microRNA expression in human colonocytes 3 with the molecular mechanism regulating NTSR1 recycling at the transcriptional level. Furthermore, we show that miR-133a and its previously unrecognized downstream binding target AFTPH are regulated by NTS and associated with NTS/NTSR1-
associated tumorigenesis. MiR-133a has been involved in muscle development 35' 38, cardiac muscle hypertrophy 3 , fibrosis 22, 40 and energy metabolism 40. MyoD and myogenin, two transcription factors associated with muscle differentiation, increase miR-133a expression by binding to its promoter during skeletal muscle differentiation 35' 38. Using a combination of bioinformatic analysis, RT-PCR and ChiP assays, we identified ZEB1 as a NTS-driven negative transcription regulator of miR-1 33a in colonocytes (Fig. 5). The ability of ZEB1 to regulate directly miR-1 33a was not previously recognized. The results reveal that ZEB1 binds directly in miR-1 33a promoter area, suppressing its expression that in turn down-regulates expression of AFTPH, a downstream target of miR-1 33a (Fig. 5). Previous studies have shown that ZEB1 acts as a negative transcriptional regulator of gene expression 41 43. Of note, ZEB1 is associated with epithelial - mesenchymal transition and histone deacetylase down-regulation in colon cancer 44 4 . ZEB1 binding to miR-1 33a promoter may be crucial to the physiological function of NTSR1/miR-133a interactions and related to NF- κΒ activation and IGF-1 receptor transactivation in cancer cells, representing known signaling targets of NTSR1 10, .
NTSR1 recycling is inhibited by miR-1 33a silencing (Fig. 1), but not by antisense treatment of
other NTS-stimulated miRs (Suppl. Result 1). A bioinformatic search identified AFTPH as a potential downstream target of miR-1 33a and cross-species sequence 6 analysis revealed that miR-1 33a/AFTPH interaction may be highly conserved (Fig. 2A). Our observation that AFTPH transcription in vitro is down-regulated after NTS exposure (Figs. 2C and 2D) or miR-1 33a overexpression in the absence of NTS stimulation (Fig. 2F) supports that AFTPH is the
downstream target of miR-1 33a in human colonocytes. NTS-regulated miR-1 33a and AFTPH expression did not induce NTSR1 degradation (Suppl. Result 2) and, more importantly, AFTPH gene silencing promotes NTSR1 recycling (Figs. 3C and 3D). Based on these considerations the data suggest that NTS-induced miR-1 33a expression down-regulates AFTPH expression
that facilitates NTSR1 recycling in human colonocytes. Whether this interaction determines NTSR1 recycling from Rab5a+ early endosomes 23 or the perinuclear TGN (Figs. 3A and 3B) remains to be determined.
Colon cancer development requires multiple steps of histological changes representing different stages of genetic and epigenetic alterations. The results show that NTS promotes tumor growth in colon cancer xenografts that is significantly attenuated by down-regulation of miR-133a (Fig. 6A). Several studies comparing colon tumor tissues and normal tissues suggest that miR-133a expression is reduced in colon cancer tumors 31, 32, 47, 48. On the other hand, in a colon cancer model associated with chronic inflammation, miR-133a is up-regulated in colon tumors when compared to control chronically inflamed epithelium, suggesting that miR-133a expression is induced early in cell transformation 49. Interestingly, we identified for the first time that AFTPH has tumor suppressor properties in colon cancer. Specifically, silencing of AFTPH by siRNA promoted tumor colony formation in vitro (Fig. 6C) and in vivo (Fig. 6E). Although the role of AFTPH in oncogenesis was unknown, AFTPH is known to be a component of the clathrin machinery 2 . A more recent study suggested that AFTPH is involved in Notch signaling 0 . The molecular mechanism by which AFTPH suppresses colon carcinogenesis deserves further investigation.
Receptor trafficking has been associated with the development of drug tolerance in opioid receptor studies 1 , and poor clinical outcomes of neuroblastomas due to altered sensitivity to EGFR inhibitors 2. The data show that NTSR1 recycling, which is regulated by NTS-modulated miR-133a/AFTPH interactions, facilitates NTS- associated cellular responses, such as pro inflammatory signaling (Suppl. Result 3) and colon cancer development (Fig. 6). Thus, interfering with miR-133a/AFTPH-associated NTSR1 recycling may represent a novel approach to inhibit colon cancer development.
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Example 4 : Neurotensin-requlated miR-133a is involved in proinflammatory signaling in human colonic epithelial cells and in experimental colitis This Example describes the role of miR-133a in NT-associated colonic inflammation in vitro and
in vivo. In summary, miR-133a and aftiphilin (AFTPH) levels were measured by quantitative PCR. Antisense (as)-miR-1 33a was administrated intracolonicaly prior to induction of 2 4 , 6- trinitrobenzene sulfonic acid (TNBS)- and dextran sodium sulfate (DSS)-induced colitis. The effect of AFTPH was examined by gene silencing in vitro.
NTS increased miR-133a levels in NCM-460 overexpressing NTSR1 (NCM460-NTR1) and HCT-1 16 cells. NTS-induced p38, ERK1/2, c-Jun, and NF-κΒ activation, as well as IL-6, IL-8
and IL- β mRNA expression in NCM-460-NTSR1 cells were reduced in miR-133a-silenced cells, while overexpression of miR-133a reversed these effects. MiR-133a levels were
increased in TNBS (2 d) and DSS (5 d) colitis, while NTSR1 deficient DSS-exposed mice had reduced miR-133a levels, compared to wild-type colitic mice. Intracolonic as-miR-133a attenuated several parameters of colitis as well expression of proinflammatory mediators in the colonic mucosa. In silico search coupled with qPCR identified AFTPH as a downstream target of
miR-133a, while NTS decreased AFTPH expression in NCM-460-NTSR1 colonocytes. Gene silencing of AFTPH enhanced NTS-induced proinflammatory responses and AFTPH levels were downregulated in experimental colitis. Levels of miR-133a were significantly upregulated, while AFTPH levels were downregulated in colonic biopsies of Ulcerative colitis patients compared to controls. Thus, NTS-associated colitis and inflammatory signaling are regulated by miR-133a- AFTPH interactions. The results point to aftiphilin (AFTPH), as a novel downstream target for miR-133a and present evidence for the association of both miR-133a and its downstream target
AFTPH in the pathophysiology of colitis. Targeting of miR-133a or AFTPH provides a novel therapeutic approach in Inflammatory Bowel Disease.
MATERIALS AND METHODS
Cell culture and reagents: NCM460-NTR1 cells were generated from human colonic epithelial NCM460 cells (INCELL, San Antonio, TX, USA) transduced with lentivirus particles as previously described 0 and maintained in M3:D culture media (INCELL) supplemented with 10% fetal bovine serum (FBS, Life Technologies, Grand Island, NY, USA). Human embryonic kidney fibroblasts, HEK293, and colonic cancer HCT-1 16 cells were maintained in Eagle's Minimum Essential Medium (MEM, Life Technologies) and McCoy5a (ATCC, Manassas, VA, USA) respectively and supplemented with 10% FBS. Lipofectamine 2000, lipofectamine RNAimax and OptiMEM were from Life Technologies. Neurotensin was from Bachem Americas (Torrance, CA, USA). TNBS and SR48962 were from Sigma Aldrich (St. Louis, MO, USA) and DSS was from MP Biomedicals (Santa Ana, CA, USA). Goat anti-AFTPH (sc-1 67055), mouse anti-villin and rabbit anti-β tubulin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Transfection experiments: Small interfering RNA (siRNA) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). NCM460-NTSR1 cells were transfected with siRNA against AFTPH (si-AFTPH) using Lipofectamine™ RNAiMAX. All miRs were purchased from Life Technologies. For miR-133a silencing or overexpression, cells were transfected with antisense-miR-133a (as-miR-133a) or miR-133a precursor (miR-133a), respectively using Lipofectamine™ RNAiMAX. Cells transfected with siRNA-A (si-control), antisense-control miR (as-miR-control) or miR-negative control precursor (miR-control) served as controls.
NTS/NTSR1 -mediated phosphoprotein activation and cytokine production: NCM460- NTSR1 cells were transfected with as-miR-133a, miR-133a and si-AFTPH and their respective controls. Cells were incubated in serum-free media overnight and stimulated with NTS (100 nM). Cell lysates were collected for phosphoprotein detection and media were collected 6 h after stimulation for cytokine neasurements. Custom designed Bio-Plex Pro ™ Cell Signaling assay panel (Bio-Rad, Hercules, CA, USA) and Bio-Plex Pro Human Cytokine 27-plex Assay (Bio-Rad) were used for phosphoprotein activation and cytokine production (except IL-8), respectively according to manufacturer's instructions. Animal models and establishment of experimental colitis models: Neurotensin receptor 1 knockout mouse model. Ntsr1 tmD (hereafter called NTSR1KO) were purchased from Jackson Laboratories and bred in our facility. Animals were obtained as fifth generation backcross of 129 onto C57BL6/J. We performed one additional backcross before intercrossing animals as heterozygous NTSR1 KO crosses to generate littermate controls.
TNBS-induced colitis. C57BL6/J wild type mice were purchased from Jackson Laboratories. TNBS was dissolved in 30% ethanol (5 mg/kg) was intracolonicaly administered (50 µ ) to mice
as previously described ' 33. In brief, TNBS was slowly infused via a 1-mL syringe (Becton Dickinson, Laguna Hills, CA, USA) fitted with a polyethylene cannula (Intramedic PE-20 tubing; Becton Dickinson). Colon tissues were collected 48 h after TNBS administration.
DSS-induced colitis. NTSR1 KO and its wild type counterparts or C57BL6/J wild type animals were treated with 5% DSS in drinking water as previously described 13, 34 and colon tissues were collected 5 days after the start of the treatment.
In vivo NTS infusion. NTS was dissolved in PBS supplemented with 1% bovine serum albumin (BSA) 3 and was administered intracolonicaly (300 µg/kg) to C57BL6/J wild type mice twice per day for 4 days. Control mice were infused with 1% BSA in PBS. Colon tissues were collected 5 days after NT administration.
In vivo knockdown of miR-133o. Endogenous expression of miR-133a in colon tissues of C57BL6/J wild type mice was silenced by intracolonic administration of miRCURY ™ LNA Inhibitor probe, in vivo against mmu-miR-133a (20 µg/mouse,Exiqon). In brief, the appropriate amount of oligonucleotides against mmu-miR-133a and its respective control were resuspended in 100 i Opti-MEM with 2 µ lipofectamine 2000 and administered intracolonicaly 24 and 72 h prior to TNBS or DSS treatment.
MicroRNA in situ hybridization: Colon tissues (3 cm from distal end) were obtained from wild type mice 2 d after TNBS treatment. The tissues were immediately fixed with 4% paraformaldehyde, paraffin-embedded and sectioned (5 µηη ) for histological study. 5'-DIG and 3'-DIG labeled detection probes specific for mouse miR-133a (Cat. No. 39270-15) and miRCURY LNA™ microRNA ISH Optimization Kit (FFPE) were purchased from Exiqon and the experiments were performed according to the manufacturer's instructions.
RNA expression studies in patient samples: Total RNAs from colon tissues obtained from patients with UC (n=12), CD (n=8) and normal subjects (n=9) were purchased from Origene (Rockville, MD, USA). Conversion of cDNA of RNA samples was performed as described above and levels of miR-133a and AFTPH were determined by qPCR analysis.
Immunohistochemistry: Frozen tissue sections (5 m) obtained from chronic active UC patients (n=3) and their normal control (n=3) were purchased from Origene. The tissues were fixed with 4% paraformaldehyde and blocked with 0.3% Triton X-100 in PBS (PBS-Triton) supplemented with 5% normal bovine serum. Appropriate antibodies were incubated with the tissue sections overnight at 4°C, washed with PBS-Triton and incubated with appropriate secondary antibodies (Santa Cruz Biotechnology). The tissues were imaged using Axio Imager.ZI microscope (Carl Zeiss, Inc).
Supplementary methods for site-directed mutagenesis, luciferase assay, NF-κΒ p65 translocation, measurement of interleukin-8 (IL-8) production, immunoblot analysis, mRNA and miR expression analysis and microRNA profiling in blood samples are described in Law IK, et al. Gut, 2014 Aug 11, PMID:251 12884.
Statistical analysis: All in vitro results derived from at least three sets of experiments, expressed as means ± SD and analyzed with Student's t-Tests. Results from animal experiments and studies in human tissues were analyzed with Student's t-Tests and expressed as means ± SEM. In all statistical comparisons, P<0.05 was used to indicate significant differences.
Site-directed mutagenesis: The seed sequence of miR-133a on AFTPH 3' UTR luciferase reporter (AFTPH 3' UTR) plasmid (Switchgear Genomics, Menlo Park, CA, USA) was deleted by site-directed mutagenesis to generate AFTPH 3'UTR-AmiR-133a. The primers used were as follows: i) AFTPH-del198: (5'-atc agt atg att cag aga agg aca tta tat gaa tgt ctt aca atg g-3'; SEQ ID NO: 8) and ii) AFTPH-del198-antisense (5'-cca ttg taa gac att cat ata atg tec ttc tct gaa tea
tac tga t-3'; SEQ ID NO: 9). Site-directed mutagenesis was done using QuikChange II XL site- directed mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer's instructions.
Luciferase assay: AFTPH 3'UTR, AFTPH 3'UTR-AmiR-133a and the control plasmid, R01_3UTR (Switchgear Genomics, Carlsbad, CA, USA), were transfected to NCM460-NTSR1 cells using lipofectamine 2000 in the presence of as-miR-133a or to HEK293 cells in the presence of miR-133a precursor and their controls. Two days after transfection, NCM460- NTSR1 cells were exposed to NTS (100 nM, 1 h) after overnight incubation in serum-free
media, while luciferase activities in transfected HEK293 cells were measured without NTS exposure. Firefly and Renilla luciferase cell activities were detected using Dual-luciferase reporter assays (Promega, Madison, Wl, USA) according to manufacturer's instructions. The relative AFTPH 3' UTR-associated luciferase activities were calculated by normalizing AFTPH 3' UTR-associated luciferase activities with R01_3' UTR luciferase activity.
NF-κΒ p65 translocation: NCM460-NTSR1 cells were transfected with miR-133a and its control and cell lysates were collected 48 h after transfection. Nuclei were collected using Nuclei EZ Prep (Sigma Aldrich) according to manufacturer's instructions. The nuclei were then lysed with RIPA buffer and total p65 was quantified using NF-KB/p65 ActivELISA ™ (Imgenex, San Diego, CA, USA).
Measurement of interleukin-8 (IL-8) production: NCM460-NTSR1 cells were transfected with miR-133a and its control, and cultured in serum-free media overnight 48 h after transfection. Media were collected and IL-8 production was quantified using Human CXCL8/IL-8 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA).
Immunoblot analyses: NCM460-NTSR1 cells were washed with ice-cold PBS and incubated with radiolabeled immunoprecipitation assay buffer containing the protease inhibitors phenylmethylsulfonyl fluoride and sodium orthovanadate (Santa Cruz) for 5 min. The insoluble debris were removed by centrifugation (12,000 rpm, 15 min, 4°C) and supernatants were analyzed by immunoblot analysis. Equal amount of cell lysates were loaded (~ 35 µg) and transferred to nitrocellulose membrane. The membrane was blocked with Odyssey ® Blocking Buffer (LI-COR, Lincoln, NE, USA). Appropriate antibodies were incubated with the membranes overnight at 4°C, washed with 0.1% Tween 20 in PBS (PBS-T) and incubated with appropriate IRDye ® secondary antibodies (LI-COR). Signals were detected with Odyssey ® Infrared Imaging System (LI-COR) and quantified using Image Studio (LI-COR).
Messenger RNA and miR expression analysis: NCM460-NTSR1 cells were washed once with ice-cold phosphate-buffered saline (PBS) after various treatments. Total RNA were extracted by TRIzol (Life Technologies) and reverse-transcribed into first strand cDNAs using random decamers and reverse transcriptase (Life Technologies) for quantitative PCR (qPCR) analysis. Complementary DNAs for miRNA expression were prepared with Universal cDNA
Synthesis kit II (Exiqon, Woburn, MA, USA) and detected by ExiLENT SYBR ® Green Master Mix (Exiqon) according to the manufacturer's instructions.
MicroRNA profiling in blood samples: Whole blood from UC patients and healthy donors (IRB#12-000420) was subjected to serum and peripheral blood mononuclear cells (PBMCs) isolation by centrifugation (1600g, 15 minutes, 4°C) and Ficoll (Roche) gradient centrifugation (1600g, 10 minutes, 4oC), respectively. RNA was isolated from serum samples using the miRNeasy Serum/Plasma Kit (Qiagen) and from PBMCs with the miRCURY Cell and Plant RNA Isolation Kit (Exiqon) according to the manufacturer's instructions. Eluted RNA from serum samples and PBMCs was further purified and concentrated by using Amicon Ultra YM-3 columns (3000 kDa MWCO, Millipore). RNAs following hybridization reactions were processed using the nCounter Prep Station and subsequently the nCounter Digital Analyzer and analyzed by nSolver software, v 1. 1 (Nanostring Technologies). Normalization was performed using the predominant microRNAs with coefficient of variation less than 70%.
RESULTS
Increased miR-133a levels in human colonic epithelial cells after NTS exposure: NTS exerts its effect on colonic epithelial cells 2' - through its high affinity receptor NTSR1 3. NTSR1 overexpression is observed in colon cancer cell lines 10, 36 , colon tissues from mice with experimental colitis 6' 11, 2 , intestinal tumors 3 and in tissue biopsies from UC patients 6 In a microarray analysis, we previously showed that NTS/NTSR1 coupling in human colonic epithelial NCM460-NTSR1 increased miR-133a expression 0 . In the current study, we first verified this result by examining the levels of miR-133a in human colonic epithelial NCM460- NTSR1 cells and in colon cancer adenocarcinoma HCT-1 16 cells by qPCR. Consistent with previous findings 0 , levels of miR-133a were upregulated after NTS exposure by 2.6±0.43 fold (P<0.01 , Fig. 7A) in NCM460-NTSR1 cells, while in HCT-1 16 cells, levels of miR-1 33a were increased by 1.5±0.07 fold (P<0.01 , Fig. 7B) after addition of NTS. Therefore, NTS increases miR-1 33a expression in two different human colonic epithelial cell lines.
NTS induces proinflammatory signaling in human colonic epithelial cells via miR-1 33a expression: NTS coupling of NTSR1 stimulates inflammatory responses through MAPK 3¾ 3 - and NF-κΒ 40 4 1 - dependent pathways. Here, we examined whether miR-1 33a is involved in NTS-related proinflammatory signaling and cytokine expression in NCM460-NTSR1 cells with an antisense approach using as- miR-1 33a. First, we showed that transfection of as-miR-133a blocked NTS-induced miR-1 33a overexpression in NCM460-NTSR1 cells (Fig. 8A), compared to those transfected with control as-miR, validating this approach. Moreover, NTS stimulation activated p38 and ERK1/2 signaling only in cells transfected with control as-miR, but not as- miR-133a, while NTS-induced c-Jun activation was significantly attenuated in as-miR-133a- transfected cells (Fig. 8B, P<0.05). Moreover, NTS-induced NF-κΒ p65 phosphorylation was blocked in as-miR-133a-transfected cells (Fig. 8C), while NTS-induced IL-6 and IL-8 mRNA (Figs. 8D and 8E, PO.05) and IL-8 secretion were also significantly attenuated upon miR-133a knock-down (Fig. 8F, PO.05). Analysis of the cell culture media showed that NT stimulation increased the production of the proinflammatory IL- β, but reduced the production of the anti inflammatory IL-1 ra (Fig. 8G, PO.05). Transfection of as-miR-133a significantly reduced NT- induced IL-1 β production, while prevented the decrease in IL-1 ra production (Fig. 8G).
Together, these results suggest an important role for miR-133a in the induction of
proinflammatory signaling pathways and cytokine production in response to NTSR1 activation in human colonocytes.
We further verified the proinflammatory role of NTS and miR-133a in NCM460 cells transfected with both NTSR1 and miR-133a. Overexpression of miR-133a further increased NTS-induced ERK1/2, c-Jun (Fig. 9A, PO.05) and NF-κΒ p65 (Fig. 9B, PO.05) activation. In addition, overexpression of miR-133a alone, in the absence of NTS stimulation, promoted nuclear translocation of NF- κΒ p65 (Fig. 9C, PO.05) when compared to the cells expressing control miR. Furthermore, IL-8, IL-1 β and TNF-a mRNA was increased in cells overexpressing miR- 133a when compared to their controls (Fig. 9D, PO.05). For cytokine production, IL-8 secretion was increased upon miR-133a overexpression (Fig. 9E, PO.05). Cells overexpressing miR- 133a had also significantly reduced IL-1 ra production (PO.05), while IL-1 β production was increased slightly, but not significantly (Fig. 9F). Together, these results strongly suggest an
important role for miR-133a in proinflammatory signaling in human colonocytes.
MiR-133a overexpression in vivo is NTS/NTSR1 -dependent: Since, as shown above, NTS
regulates miR-133a in colonocytes in vitro, we next examined whether this effect can be recapitulated in vivo. To do this C57/BL6J wild type (WT) mice received intracolonicaly NTS (300 µg/kg) 35 . After 4 days mice were euthanized and colonic tissues were processed for RNA purification. qPCR analysis showed increased colonic miR-133a levels (by 1.3±0.04 fold, Fig. 10A, PO.05) in mice administered with NTS, compared to intracolonic administration of vehicle. Because NTS is an important mediator of acute colonic inflammation 2· 3 , we also examined
colonic levels of expression of miR-133a in two colitis models. Acute colitis was induced in C57BL6/J WT mice by intracolonic administration of 5 mg/kg TNBS or by addition of 5% DSS in
their drinking water. qPCR analysis showed increased miR-133a levels (by 3.2±1 .01 -fold, Fig. 10B, PO.05) 2 days after TNBS administration, but decreased by 63% at day 7 (Fig. 10B, PO.05). Mice receiving DSS showed a 3.6±0.61-fold increase in miR-133a levels (Fig. 10B, PO.001) at the end of the DSS treatment, compared to controls. To study the mucosal distribution of miR-133a in colon tissue during colitis, in situ hybridization against miR-133a in colon tissue sections obtained from TNBS-treated wild type mice and their control counterparts was performed as described in Methods. The results show that while miR- 133a expression was higher in inflamed colons, the majority of miR-133a expression was
localized in epithelial cells (Fig. 10C, arrows) and less in other cells of the lamia propria (Fig. 10C, arrowheads).
The direct relationship of endogenous NTS/NTSR1 signaling on miR-133a expression in colon tissues during colitis development was further investigated in NTSR1 deficient, NTSR1KO mice. NTSR1KO mice and their WT counterparts were allowed access to 5% DSS in their drinking water and after 5 days mice were euthanized and miR-133a levels were measured. As shown in
Fig. 10D, colonic miR-133a levels were increased in WT mice after DSS treatment (2.4±0.32 fold, P<0.01), but decreased in NTSR1KO mice (P<0.05). Thus, increased miR-133a expression during acute colitis involves NTS/NTSR1 interactions.
Intracolonic silencing of miR-133a attenuates the development of acute colitis: We next determined the role of miR-133a in the development of experimental colitis. C57BL6/J wild type mice were administered intracolonicaly two doses of as-miR-133a or its control 24 h and 72 h prior to intracolonic administration of TNBS (5 mg/kg) or vehicle. Colonic tissues were harvested 48 h post TNBS-treatment for gene expression and histological analysis. Histological examination on colon tissues collected from mice without TNBS-treatment did not show any differences (Fig. 11A). As expected, TNBS administration induced acute colonic inflammation in mice administered with as-miR-control, while administration of as-miR-133a prior to TNBS treatment attenuated colitis development (Fig. 11A). In addition, quantitative PCR analysis showed that intracolonic administration of as-miR-133a significantly reduced endogenous colonic expression of miR-133a (P<0.05, Fig. B). Mice treated with as-miR-133a and TNBS showed a significant reduction in colon weight after normalization against colon length (Fig. 11C, P<0.05), neutrophil infiltration, and an improvement in mucosal integrity which resulted in a significantly improved histological score (Fig. 11D, P<0.05). Proinflammatory cytokine production was generally increased in colon tissues from treated with TNBS. Pre-treatment with as-miR-133a prior to TNBS administration blocked increased expression of the neutrophil product lipocalin 2 (Icn2), and TNF-a, a major proinflammatory cytokine in colitis. The production of cxcM, an important neutrophil chemoattractant (Fig. 11E , P<0.05) was significantly
reduced in mice administered with as- miR-133a, when compared to control as-miR- administered counterparts (Fig. 11E). We also investigated the effect of as-miR-133a treatment in the DSS-induced colitis model. Administration of as-miR-133a and its control was performed at 24 h and 72 h before DSS treatment. Mice administered with as-miR-133a and DSS showed reduced weight loss 5 days after DSS treatment (Fig. 13A, P<0.05) while levels of TNF-a and
IL- β were increased 5 d after DSS-induced colitis and significantly reduced in mice administered with as-miR-133a (Figs. 13B and 13C,_P<0.05). In conclusion, these results show that miR-133a is involved in the pathophysiology of colitis.
MiR-133a directly regulates AFTPH expression through binding its 3' UTR in colonic epithelial cells: MiRs act as gene-silencers by inhibiting the expression of mRNA transcripts
through binding to the 3' UTRs of target genes 4 . To identify candidate genes involved in the
NTSR1-miR-133a response, we searched for genes with possible miR-133a binding sites in their 3' UTRs. In silico search using 3 online databases [TargetScanHuman (www.targetscan.org); miRBase (www.mirbase.org) and PicTar (http://pictar.mdc-berlin.de)], identified aftiphilin (AFTPH) with a miR-133a binding site at its 3' UTR highly conserved across species (Fig. 12A).
The NTSR1-miR-133a-AFTPH interaction was first validated in NCM460-NTSR1 cells transfected with as-miR-133a and exposed to NTS. NTS exposure downregulated AFTPH mRNA levels, as shown in quantitative PCR analysis (Fig. 12B, P<0.05) and AFTPH 3'UTR- regulated luciferase activity assay (Fig. 12C, P<0.05) when compared with control cells. To confirm the direct interaction between miR-133a and AFTPH, we deleted the miR-133a binding sequence on the AFTPH 3' UTR and showed that NT-induced downregulation of AFTPH 3' UTR
luciferase activity was not observed in the absence of the miR-133a binding site (Fig. 12D).
Overexpression of miR-133a in HEK293 cells significantly reduced AFTPH-3' UTR-associated luciferase activity in the absence of NTS stimulation (Fig. 12E, P<0.05). Moreover, treatment with the NTSR1 specific antagonist of SR48962 prevented the reduction in AFTPH protein expression induced by NTS (Fig. 12F). The above results strongly indicate that AFTPH is a downstream target of miR-133a in colonocytes after NTS exposure, suggesting that NTS/NTSR1 signaling suppresses AFTPH expression through miR-133a overexpression.
To confirm our in vitro findings in the in vivo setting, we compared expression of AFTPH mRNA
in colon tissues from control, NTS- and TNBS-exposed (2d) mouse colon. The results show significantly decreased colonic AFTPH mRNA expression in NTS-administered (Fig. 12G, P<0.05) and TNBS-injected mice (Fig. 12H, P<0.05) compared to vehicle-exposed control
colon, consistent with the increased miR-133a levels observed in both models (Figs. 10A and
11F). In NTSR1KO mice exposed to DSS, only a small (by 1.28± 0.17fold, n=5 per group) but not significant increase in AFTPH levels were observed. Along these lines, silencing of miR-
133a by intracolonic administration of as-miR-133a increased AFTPH levels both in normal and inflamed colon tissues, when compared to their control counterparts (Fig. 12H, P<0.05).
AFTPH gene silencing promotes colonic proinflammatory responses in human colonic epithelial cells: To examine the functionality of AFTPH in NTS-mediated proinflammatory signaling, we silenced expression of AFTPH in NCM460-NTSR1 cells with si-RNA transfection prior to NTS stimulation. The results show that AFTPH gene silencing enhanced NTS-induced c-Jun and NF-κΒ activation (Figs. 13A and 13B, P<0.05), consistent with our findings for a role of miR-133a in NTS-mediated signaling activation. In addition, gene silencing of AFTPH increased basal IL-1 β production (Fig. 13C, P<0.05), while ameliorated NTS-mediated IL-1 ra
reduction in human colonic epithelial cells (Fig. 13C), in line with our results from miR-133a overexpression (Fig. 9F).
Levels of miR-133a and AFTPH are dysregulated in colon of ulcerative colitis patients: To
confirm our mouse findings we compared levels of miR-133a and AFTPH in colonic tissue samples from UC and normal colon tissues. Levels of miR-133a were significantly increased by 2.6±1 .82 fold (P=0.048, Fig. 14A) in UC patients with active disease, while AFTPH mRNA levels were decreased by 0.72±0.17 fold (P=0.0073, Fig. 14A) in the same patients. In contrast, levels of miR-133a obtained from CD patients did not show significant difference compared with normal controls (n=8 per group). In addition, immunohistochemistry of colon tissues from UC patients showed reduced AFTPH expression in the colonic mucosa when compared to normal colon tissues (Fig. 14B), with AFTPH expressed primarily in colonic epithelial cells, and co- localized with villin, an epithelial cell marker (Fig. 14B). The above results suggested that as in mouse colitis, miR-133a and AFTPH expression are inversely dysregulated in UC patients.
DISCUSSION
Coupling of NTS to its high affinity receptor NTSR1 triggers MAPK 38' 3 and NF-κΒ 40' 4 signaling and promotes intestinal inflammation 2·5· 3 . We have previously shown that NTS
alters expression of several microRNAs, including miR-133a in human colonic epithelial cells, and presented evidence that microRNAs represent an important functional component of the
effects of NTS in the intestine 10 . In this current Example, we examined the role of miR-133a in the NTS-associated intestinal inflammatory circuit in vitro and in vivo. The results demonstrate that expression of miR-133a is increased following NTS stimulation of colonocytes in vitro and
normal mouse colon in vivo. We also present strong evidence that miR-133a is involved in the inflammatory cascade activated by NTS/NTSR1 interactions in human colonocytes and that
silencing of endogenous miR-133a in the colon diminishes acute experimental colitis in two different mouse models. Lastly, the results point to AFTPH as a novel downstream target of miR-133a that mediates intestinal proinflammatory signaling and cytokine transcription following
NT-miR-133a interactions. Thus, the NT-regulated miR-133a and its target AFTPH may represent new targets for colonic inflammatory responses in vivo.
The present study focused on understanding the role of miR-133a in NTS-related colonic inflammation. NTS/NTSR1 signaling promotes acute colitis 2' , while NTSR1 deficiency reduces
the severity of experimental colitis 5, 3 . In addition, NTS and NTSR1 are overexpressed in colon
tissues from UC patients 6, and as shown in the current study, NTSR1KO mice have reduced miR-133a colonic levels compared to wild-type mice. On the other hand, miR-133a and its downstream target expression have been associated with different disease states, such as
colorectal cancer 21 28, myocyte development, and hypertrophy 42 44 , and fibrosis in the cardiac muscle and liver 45' 46. However, its role in colonic inflammation has never been studied. Several studies have shown that miR-133a modulates ERK 2 , 30 and PI3K/Akt 21, 3 signaling pathways or directly targets molecules involved in these pathways 22 . Consistent with the above findings, we have shown that miR-133a modulates ERK1/2 and p38 activation upon NTS stimulation. We also demonstrate for the first time that miR-133a directly regulates NF- κΒ activation and NTS exposure, together with miR-133a overexpression, in cells increased the production of
proinflammatory IL- β and suppressed that of the anti-inflammatory IL-1 ra. IL-1 ra antagonizes IL-1 β in colitis in vivo 47, 48. Interestingly, a reduced IL-1 ra/IL-1 β ratio is considered as an important factor for the severity of inflammation in IBD patients 4 . Taken together, the results suggest that NTS/NTSR1 coupling triggers proinflammatory signaling pathways by modulating miR-133a levels in human colonic epithelial cells.
The results showed that miR-133a levels were significantly increased in the colonic mucosa of mice with acute TNBS- and DSS-induced colitis as well as colon tissues of UC patients with active disease, compared to controls. MiR-133a was primarily expressed in colonic epithelial cells as shown by in situ hybridization in mouse colon with increased expression of this miR during TNBS-induced colitis. However, miR-133a was not detectable in sera from UC patients and their normal counterparts. To study the role of endogenous miR-133a in colitis, we introduced as-miR-133a intracolonicaly in the two experimental colitis models to avoid perturbation of normal cellular functions of non-target tissues. The results showed that mice receiving intracolonic as-miR-133a had reduced endogenous miR-133a levels, improved mucosa histology and reduced proinflammatory cytokine expression in colon tissues of mice with colitis. Intracolonic delivery of antisense oligonucleotides is largely confined to colonic epithelial cells in vivo 5 , the results suggest that miR-133a levels in colonic epithelial cells can influence the pathophysiology of colitis. However, since expression of miR-133a has also been identified in circulating monocytes 5 1 , and in sub-epithelial cells of the colonic mucosa,
participation of miR-1 33a in inflammatory signaling in colonic lamina propria cell types cannot be excluded.
We identified a novel miR-133a target, AFTPH, in which its expression level is regulated by NTS/NTSR1 interactions. Several pieces of evidence support the identification of AFTPH as a
miR-1 33a downstream target. Incubation of human colonic epithelial cells with NTS decreases AFTPH mRNA levels as well as AFTPH 3'UTR-regulated luciferase activity. Most importantly,
expression of AFTPH with a deleted miR-1 33a binding sequence on its 3' UTR is no longer
downregulated in response to NTS exposure. The presence of AFTPH has not been previously recognized in the intestine and its cellular function is not well-characterized. AFTPH is essential to intracellular trafficking-with binding sites for clathrin and adaptor proteins (AP-1 , AP-2) 52. AFTPH is localized in trans-golgi network (TGN) 3 , and its gene silencing leads to unregulated exocytosis of Weibel-Palade bodies in endothelial cells 4 without altering trans-golgi network morphology 5. However, the role of AFTPH or TGN in proinflammatory signaling cascades has not been investigated. Here, we demonstrate that AFTPH levels are significantly downregulated in colon tissues from UC patients, as well as in the colon of mice with TNBS- and DSS-induced colitis. At the cellular level, AFTPH gene silencing activates the NF- κΒ pathway and promotes proinflammatory cytokine IL- β production in vitro. NF-κΒ activation is involved in IL- β expression 6 and administration of NF- κΒ inhibitor reduce IL-1 β production and ameliorates colonic inflammation in mice . Thus, dysregulated AFTPH expression may modulate proinflammatory responses,_representing a novel regulator in proinflammatory signaling pathways and colitis development. Moreover, measurements of its expression levels, together with measurements of the levels of miR-1 33a, may serve as biomarkers in UC.
Overall, the results represent a previously unrecognized paradigm whereby activation of a neuropeptide receptor (NTSR1) in the colonic mucosa stimulates the expression of a miR (miR- 133a) that modulates the development of colitis. The therapeutic potential of miR-1 33a silencing and stabilization of colonic AFTPH levels against colitis deserves further investigation.
References cited in Example 4 :
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9 . da Silva L , et al. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 201 1;1813:1863-1871 . 10. Bakirtzi K , et al. Gastroenterology 201 1;141: 1749-1 761.e1. 11. Margolis KG, Gershon MD. Curr Opin Gastroenterol 2009;25:503-1 1. 12. Zhao D, Pothoulakis C. Peptides 2006;27:2434-44. 13. Bugni JM, et al. Int J Cancer 2012;130:1798-805. 14. McKenna LB, et al. Gastroenterology 2010;139:1654-64, 1664 el 15. Bartel DP. Cell 2009;136:215-33. 16. Wu F, et al. Gastroenterology 2008;135:1624-1635 e24. 17. Bian Z , et al. J Pathol 201 1;225:544-53. 18. Fasseu M , et al. PLoS ONE 201 0;5. 19. Takagi T, et al. Journal of Gastroenterology and Hepatology 2010;25:S129-S133. 20. Wu F, et al. Inflamm Bowel Dis 2010;16:1729-38. 21. Josse C, et al. Am J Physiol Gastrointest Liver Physiol 2013. 22. Wang H , et al. Eur J Cancer 2013;49:3924-35. 23. Arndt GM, et al. BMC Cancer 2009;9:374. 24. Sarver AL, et al. BMC Cancer 2009;9:401 . 25. Dong Y , et al. Mol Cancer Res 2013;1 1:1051-60. 26. Ma Y , et al. Int J Cancer 2012;130:2077-87. 27. Necela BM, et al. PLoS ONE 201 1;6:e18501 . 28. Li Z , et al. Oncol Lett 2012;3:346-350. 29. Li Q , et al. Am J Physiol Heart Circ Physiol 201 0;298:H 1340-7. 30. Feng Y, et al. Cell Death Dis 2013;4:e934.
3 1. Cui W, et al. FEBS J 2013;280:3962-74. 32. Zhao D, et al. J Biol Chem 201 1;286:6092-9. 33. Koon HW, et al. Am J Pathol 2010;177:2300-9. 34. Koon HW, et al. Am J Pathol 2008;173:400-10. 35. Evers BM, et al. Dig Dis Sci 1992;37:426-31 . 36. Maoret JJ, et al. Biochem. & Biophys. Research Communications 1994;203:465-471 . 37. Gui X , et al. Peptides 2008;29:1609-15. 38. Zhao D, et al. J Biol Chem 2004;279:43547-54. 39. Zhao D, et al. Int J Cancer 2007;120:1652-6. 40. Zhao D, et al. Am J Physiol Cell Physiol 2003;284:C1 397-404. 4 1. Zhao D, et al. Mol Pharmacol 2005;67:2025-31 . 42. Rao PK, et al. Proc Natl Acad Sci U S A 2006;103:8721-6. 43. Chen X , et al. J Biol Chem 2009;284:5362-9. 44. Hua Y, et al. J Cell Mol Med 2012;16:83-95. 45. Roderburg C, et al. Journal of Hepatology 201 3;58:736-742. 46. Castoldi G , et al. Journal of Cellular Physiology 2012;227:850-856. 47. Cominelli F, et al. J Clin Invest 1990;86:972-980. 48. Ferretti M , et al. J Clin Invest 1994;94:449-453. 49. Casini-Raggi V, et al. The Journal of Immunology 1995;154:2434-40. 50. Huang Z , et al. Gut 2013.
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Throughout this application various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to describe more fully the state of the art to which this invention pertains.
From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims. What is claimed is:
1. A method for detection of inflammatory bowel disease in a subject, the method comprising:
(a) contacting a specimen obtained from the subject with reagents for assaying for miR133a;
(b) measuring the amount of miR-1 33a present in the specimen as compared to a control sample; and
(c) determining the presence of inflammatory bowel disease when an elevated amount of miR133a is present in the specimen compared to the control sample.
2 . The method of claim 1, wherein the specimen is intestinal biopsy tissue or intestinal fluid.
3 . The method of claim 2 , wherein the intestinal biopsy tissue comprises colon.
4 . The method of claim 1, wherein the inflammatory bowel disease comprises ulcerative colitis or Clostridium difficile colitis.
5 . The method of claim 1, wherein the measuring comprises polymerase chain reaction assay.
6 . The method of claim 1, wherein the reagents comprise a probe that specifically binds to SEQ ID NO: 1.
7 . A method for monitoring the efficacy of treatment of inflammatory bowel disease in a subject, the method comprising:
(a) contacting a specimen obtained from the subject at a first time point with reagents for assaying for miR-1 33a;
(b) contacting a specimen obtained from the subject at a second time point with reagents for assaying for miR-1 33a, wherein the subject has been treated for inflammatory bowel disease prior to the second time point;
(c) measuring the amount of miR-1 33a present in the specimens obtained at the first and second time points; and
(d) determining whether a decreased amount of miR-1 33a is present in the specimen obtained at the second time point compared to the specimen obtained at the first time point, which decreased amount of miR-1 33a is indicative of effective amelioration of the inflammatory bowel disease.
8 . The method of claim 7 , wherein the specimen is intestinal biopsy tissue or intestinal fluid.
9 . The method of claim 8 , wherein the intestinal biopsy tissue comprises colon.
10. The method of claim 7 , wherein the inflammatory bowel disease comprises ulcerative colitis or Clostridium difficile colitis.
11. The method of claim 7 , wherein the measuring comprises polymerase chain reaction assay.
12. The method of claim 7 , wherein the reagents comprise a probe that specifically binds to SEQ ID NO: 1.
13. The method of claim 1, further comprising measuring the amount of AFTPH and/or neurotensin receptor 1 (NTSR1) in the specimen as compared to a control sample, wherein an elevated amount of NTSR1 and/or a decreased amount of AFTPH in the specimen is indicative of IBD.
14. A method for detection of inflammatory bowel disease in a subject, the method comprising:
(a) contacting a specimen obtained from the subject with reagents for assaying for aftiphilin (AFTPH);
(b) measuring the amount of AFTPH present in the specimen as compared to a control sample; and
(c) determining the presence of inflammatory bowel disease when a reduced amount of AFTPH is present in the specimen compared to the control sample.
15. The method of claim 14, wherein the specimen is intestinal biopsy tissue or intestinal fluid.
16. The method of claim 15, wherein the intestinal biopsy tissue comprises colon.
17. The method of claim 14, wherein the inflammatory bowel disease comprises ulcerative colitis or Clostridium difficile colitis.
18. The method of claim 14, wherein the measuring comprises polymerase chain reaction assay. 19. The method of claim 14, wherein the reagents comprise a probe that specifically binds to SEQ ID NO: 2 .
20. The method of claim 14, wherein measuring comprises immunoassay.
2 1. A method for monitoring the efficacy of treatment of inflammatory bowel disease in a subject, the method comprising:
(a) contacting a specimen obtained from the subject at a first time point with reagents for assaying for AFTPH;
(b) contacting a specimen obtained from the subject at a second time point with reagents for assaying for AFTPH, wherein the subject has been treated for inflammatory bowel disease prior to the second time point;
(c) measuring the amount of AFTPH present in the specimens obtained at the first and second time points; and
(d) determining whether a decreased amount of AFTPH is present in the specimen obtained at the second time point compared to the specimen obtained at the first time point, which decreased amount of AFTPH is indicative of effective amelioration of the inflammatory bowel disease.
22. The method of claim 2 1, wherein the specimen is intestinal biopsy tissue or intestinal fluid.
23. The method of claim 22, wherein the intestinal biopsy tissue comprises colon.
24. The method of claim 2 1, wherein the inflammatory bowel disease comprises ulcerative colitis or Clostridium difficile colitis.
25. The method of claim 2 1, wherein the measuring comprises polymerase chain reaction assay.
26. The method of claim 2 1, wherein the reagents comprise a probe that specifically binds to SEQ ID NO: 1.
27. A method of treating inflammatory bowel disease in a subject, the method comprising administering to the subject a therapeutically effective amount of an inhibitor of miR-1 33a.
28. The method of claim 27, wherein the administering is intracolonic or intravenous.
29. The method of claim 27, wherein the inhibitor of miR1 33a is an anti-sense miR-1 33a oligonucleotide. 30. The method of claim 28, wherein the antisense oligonucleotide is a locked-nucleic acid- based antisense miR-133a oligonucleotide.
3 1. The method of claim 29, wherein the antisense miR-1 33a oligonucleotide is administered via a lentiviral vector.
32. The method of claim 27, wherein the inflammatory bowel disease comprises ulcerative colitis, Crohn's disease or Clostridium difficile colitis.
33. A method of determining whether a patient suffering from inflammatory bowel disease has ulcerative colitis or Crohn's disease, the method comprising performing the method of claim 1 or 14, wherein an elevated amount of miR-1 33a and/or a decreased amount of AFTPH relative to a control sample is indicative of ulcerative colitis.
34. A method of treating colon cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of an inhibitor of miR-1 33a.
35. The method of claim 34, wherein the administering is intracolonic, intratumoral, or intravenous.
36. The method of claim 34, wherein the inhibitor of miR-1 33a is an anti-sense miR-1 33a oligonucleotide.
INTERNATIONAL SEARCH REPORT International application No. PCT/US2014/055493 A. CLASSIFICATION OF SUBJECT MATTER C12Q l/68(2006.01)i, C12N 15/113(2010.01)i, A61K 31/7105(2006.01)i, A61P 35/00(2006.01)i
According to International Patent Classification (IPC) or to both national classification and IPC B. FIELDS SEARCHED Minimum documentation searched (classification system followed by classification symbols) C12Q 1/68; C40B 30/04; C12N 15/1 13; A61K 3 1/7105; A61P 35/00
Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched Korean utility models and applications for utility models Japanese utility models and applications for utility models
Electromc data base consulted during the international search (name of data base and, where practicable, search terms used) eKOMPASS(KIPO internal) & Keywords: inflammatory bowel disease, miR-133a, aftiplulin (AFTPH)
DOCUMENTS CONSIDERED TO BE RELEVANT
Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.
A US 2011-0117111 Al ( WO e t a l . ) 19 May 2011 7-12, 21-26 See abst ract ; c l aims 1-15; and t able 13 .
A US 2013-0225440 Al (FRIEDMAN e t a l . ) 29 August 2013 7-12, 21-26 See abst ract ; and c l aims 1-11 .
A US 2013-0143764 Al (OGIER-DENIS et a l . ) 06 June 2013 7-12, 21-26 See abst ract ; and c l aims 5-12.
A ARNDT e t a l . , 'Charact e i zat i on of global mi croRNA express i on reveals 7-12, 21-26 oncogeni c potent i a l of miR-145 in metast at i c co l orectal cancer ' BMC (Bi oMed Cent ral ) Cancer , Vo l .9 , Art i c l e No .374 ( int ernal pages 1-17) (2009) See the who l e document .
A COSKUN e t a . , 'Mi croRNAs in inf l ammat ory bowe l d i sease-pathogenes i s , 7-12, 21-26 d i agnost i cs and therapeut i cs' Wor l d Journal of Gastroent erol ogy (WJG) , Vo l . 18 , Issue No. 34 , pp .4629-4634 (2012) See the who l e document .
Further documents are listed in the continuation of Box C. See patent family annex.
* Special categories of cited documents: "T" later document published after the international filing date or priority "A" document defining the general state of the art which is not considered date and not in conflict with the application but cited to understand to b e of particular relevance the principle or theory underlying the invention "E" earlier application or patent but published on or after the international "X" document of particular relevance; the claimed invention cannot b e filing date considered novel or cannot b e considered to involve an inventive "L" document which may throw doubts on priority claim(s) or which is step when t e document is taken alone cited to establish the publication date of another citation or other "Y" document of particular relevance; the claimed invention cannot b e special reason (as specified) considered to involve an inventive step when the document is "O" document referring to an oral disclosure, use, exhibition or other combined with one or more other such documents, such combination means being obvious to a person skilled in the art "P" document published prior to the international filing date but later "&" document member of the same patent family than the priority date claimed Date of the actual completion of the international search Date of mailing of the international search report 14 January 2015 (14.01.2015) 14 January 2015 (14.01.2015)
Name and mailing address of the ISA/KR Authorized officer International Application Division Korean Intellectual Property Office KIM, Seung Beom 189 Clieongsa-ro, Seo-gu, Daejeon Metropolitan City, 302-701, Republic of Korea - Facsimile No. ++82 42 472 3473 Telephone No. +82-42
FormPCT/ISA/210 (second sheet) ( y 2009) INTERNATIONAL SEARCH REPORT International application No. PCT/US2014/055493
Box No. II Observations where certain claims were found unsearchable (Continuation of item 2 of first sheet)
This international search report has not been established in respect of certain claims under Article 17(2)(a) for the following reasons:
Claims Nos.: 1-6, 13-20, 27-36 because they relate to subject matter not required to be searched by this Authority, namely: Claims 1-6, 13-20 and 27-36 pertain to methods for treatment of the human body by therapy, as well as diagnostic methods, and thus relate to a subject matter which this International Searching Authority is not required, under PCT Article 17(2)(a)(i) and PCT Rule 39.1(iv), to search.
I I Claims Nos.: because they relate to parts of the international application that do not comply with the prescribed requirements to such an extent that no meaningful international search can be carried out, specifically :
3 . Claims Nos.: because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a).
Box No. Ill Observations where unity of invention is lacking (Continuation of item 3 of first sheet)
This International Searching Authority found multiple inventions in this international application, as follows:
I IAs all required addtional search fees were timely paid by the applicant, this international search report covers all searchable claims.
I IAs all searchable claims could be searched without effort justifying an additional fees, this Authority did not invite payment of any additional fees.
I IAs only some of the required additional search fees were timely paid by the applicant, this international search report covers only those claims for which fees were paid, specifically claims Nos. :
4. I INo required additional search fees were timely paid by the applicant. Consequently, this international search report restricted to the invention first mentioned in the claims; it is covered by claims Nos.:
Remark on Protest | | The additional search fees were accompanied by the applicant's protest and, where applicable, the payment of a protest fee. I IThe additional search fees were accompanied by the applicant's protest but the applicable protest fee was not paid within the time limit specified in the invitation. I INo protest accompanied the payment of additional search fees.
Form PCT/ISA/210 (continuation of first sheet (2)) (My 2009) INTERNATIONAL SEARCH REPORT International application No. PCT/US2014/055493
C (Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT
Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.
PX LAW et a l . , 'Neurot ens in-regul t ed mi - 133 a i s invo lved in 7-12, 21-26 proinf lammatory s ignal l ing in human co loni c epi the l i a l ce l ls and in exper iment al col i t is ' Gut , DOI : 10. 1136/gut jnl-2014-307329 ( internal pages 1-10) (e-pub . 11 August 2014) See the whole document .
Form PCT/ISA/210 (continuation of second sheet) (My 2009) INTERNATIONAL SEARCH REPORT International application No. Information on patent family members PCT/US2014/055493
Patent document Publication Patent family Publication cited in search report date member(s) date
US 2011--0117111 Al 19/05/2011 WO 2009-120877 A2 01/10/2009 wo 2009-120877 A9 25/02/2010
us 2013--0225440 Al 29/08/2013 AU 2011-312504 Al 23/05/2013 CA 2812940 Al 12/04/2012 EP 2622105 A2 07/08/2013 EP 2622105 A4 05/03/2014 IL 225519 DO 27/06/2013 JP 2014-500707 A 16/01/2014 WO 2012-047631 A2 12/04/2012 wo 2012-047631 A3 31/05/2012
us 2013--0143764 Al 06/06/2013 EP 2603605 Al 19/06/2013 JP 2013-535971 A 19/09/2013 WO 2012-020126 Al 16/02/2012
Form PCT/ISA/210 (patent family annex) ( y 2009)