ONCOLOGY REPORTS 34: 2797-2805, 2015

MicroRNA-128 suppresses cell growth and metastasis in colorectal carcinoma by targeting IRS1

Lan Wu1, Bo Shi2, Kexin Huang2 and Guoyu Fan3

1Department of Pediatrics, The First Hospital of Jilin University, and 2The Experiment Center, College of Basic Medical Sciences, Jilin University, Changchun, Jilin 130021; 3Department of Oncology, The Center Hospital of Jilin City, Fengman, Jilin 132011, P.R. China

Received June 29, 2015; Accepted August 3, 2015

DOI: 10.3892/or.2015.4251

Abstract. Evidence has shown that microRNAs play impor- cancer-associated mortality (1). Over one million new cases tant roles in tumor development, progression, and metastasis. are detected annually according to the International Agency miR-128 has been reported to be deregulated in different for Research on Cancer (2). is caused by tumor types, whereas the function of miR-128 in colorectal the accumulation of mutations in numerous , including carcinoma (CRC) largely remains to be elucidated. The aim of alterations in oncogenes and tumor‑suppressor genes, which the present study was to investigate the clinical significance, lead to the activation of oncogenes and the inactivation of biological effects and underlying mechanisms of miR-128 in tumor‑suppressor genes (3). Although previous studies have CRC using reverse transcription-quantitative polymerase chain focused on the biological mechanism of colorectal carci- reaction (RT-qPCR) and western blotting. It was found that noma (CRC) and a series of tumor‑suppressor genes and the expression of miR-128 was downregulated in CRC tissues oncogenes have been identified in recent years, the patho- and cell lines as determined by RT-qPCR. Furthermore, the logical process and underlying mechanism of CRC remains expression of miR-128 in tumor tissues was significantly nega- to be elucidated. Therefore, identification of the molecular tively correlated with TNM stage and lymph node metastasis mechanisms by which CRC initiates, progresses, invades and in CRC patients. Functional assay revealed that the overex- recurs is imperative in order to develop novel and effective pression of miR-128 inhibited CRC cell proliferation, colony therapeutic strategies. formation, migration and invasion and promoted apoptosis MicroRNAs (miRNAs) are a large family of endogenous in vitro, and suppressed CRC xenograft tumor growth in vivo. small non-coding RNAs (19-24 nt) that play important In addition, substrate 1 (IRS1), a key mediator regulatory roles in animals and plants by binding to the in oncogenic insulin-like growth factor (IGF) signaling, was 3' untranslated regions (3'UTRs) of target mRNAs, resulting confirmed as a direct target of miR-128 by a luciferase reporter in blocking of translation and/or mRNA degradation (4-6). assay. Western blot analysis indicated that the overexpression Accumulating evidence has shown that miRNAs play diverse of miR-128 significantly downregulated IRS1 expression roles in the regulation of tumor proliferation, invasion, apop- and its downstream Akt signaling in CRC cells. Moreover, tosis and therapy resistance, and act as oncogenes or tumor miR-128 was negatively associated with IRS1 in CRC tissues suppressors depending on the target mRNAs (7-9). Findings compared to adjacent non-tumor tissues. Taken together, these have shown that some miRNAs are involved in CRC develop- data suggested that miR-128RETRACTED serves as a tumor suppressor and ment, progression and metastasis through the regulation of blocks CRC growth and metastasis by targeting IRS1. different cell processes, including apoptosis, migration or invasion (10-12). Thus, miRNAs are presently considered Introduction potential novel targets for CRC therapy. miR-128, an important miRNAs, has been shown to be Colorectal cancer has the third highest incidence of human downregulated in several types of cancer including prostate malignant diseases and is the fourth most common cause of cancer, glioma, head and neck squamous cell carcinoma (HNSCC), and non-small cell , and to inhibit cancer cell growth and invasion when overexpressed (13-15). Concerning CRC, there is currently only one study showing Correspondence to: Dr Lan Wu, Department of Pediatrics, that miR-128 expression was significantly associated with a The First Hospital of Jilin University, 71 Xin Min Street, Changchun, poorer recurrence rate, and that miR-128 directly targeted Jilin 130021, P.R. China NEK2, induced G2-phase cell cycle arrest, and inhibited E-mail: [email protected] cancer cell proliferation in CRC cells (16). However, the Key words: colorectal carcinoma, microRNA-128, insulin-like growth function of miR-128 on CRC cell apoptosis, migration, factor, proli­feration, metastasis invasion and the underlying molecular mechanisms remain relatively unclear. The present study was therefore under- taken to evaluate the potential role of miR-218 in CRC cells. 2798 wu et al: MicroRNA-128 suppresses cell growth and metastasis in CRC by targeting IRS1

In the present study, we identified that miR-128 was Transfection. The miR-128 mimics (miR-128) and corre- downregulated in CRC tissue and cell lines, suppressed cell sponding negative control (miR-NC) were purchased from proliferation, migration and invasion, induced apoptosis GenePharma (Shanghai, China). Transfection was performed in vitro, and inhibited tumor growth in vivo. We also showed using Lipofectamine 2000 (Invitrogen) according to the the potential tumor suppressor role of miR-128 involved in manufacturer's instructions. CRC by identifying the new targeting IRS1. Furthermore, we showed that miR-128 directly targets the 3'UTR of IRS1 Cell growth and colony formation assays. Transfected to regulate its expression and downstream signaling . (2x103 cells/well) were plated in 96-well plates and were Our data suggest a new perspective on how miR-128 is cultured at the indicated time points (24, 48 and 72 h) when involved in CRC. cell growth was estimated by the Cell Counting Kit-8 (CCK-8) (Dojindo, Japan) according to the manufacturer's instructions. Materials and methods For colony formation assay, 1x103 transfected cells/well were seeded into a 6-well plate and cultured for 14 days. The cells Human tissue specimens. Forty-five paired CRC and adjacent were fixed with 4% paraformaldehyde for 20 min and counted normal tissues were collected from the First Hospital of Jilin after staining with 1% crystal violet. The percentage colony University (Changchun, China). CRC tissues were obtained formation was calculated by the adjusting control (miR-NC or from patients undergoing resection, and adjacent colon tissues si-NC) to 100%. were obtained from the distal normal colon tissue of CRC cancers. None of the patients received chemotherapy or radio- Cell apoptotic analysis. Cell apoptotic analysis was performed therapy prior to surgery. Tissues were immediately frozen in using the phycoerythrin (PE)-Annexin V apoptosis detection liquid nitrogen, and stored at -80˚C for subsequent experiments. kit (BD Pharmingen, San Jose, CA, USA). Briefly, the cells The patient clinicopathological characteristics were collected were seeded in 6-well plates at 4x105 cells/well. Twenty-four and are listed in Table I. Informed consent was obtained from hours after transfection, the cells were suspended and those all the patients prior to the study. The study was approved by that adhered, were collected and labeled with Annexin V for the Ethics Committee of Jilin University (Changchun, China). 15 min in dark place. Propidium iodide (PI) (50 µg/ml) was added to each sample prior to the cell apoptosis. Distribution Cell lines and culture. The human LoVo, CaCoS2, SW1116, was analyzed using the FACSCalibur flow cytometer SW480, and HCT-116 CRC cell lines and a normal colonic (BD Biosciences, Mansfield, MA, USA). cell line (NCM460) were purchased from the Cell Bank of In addition, the activity of caspase-3 and -8 was detected the Chinese Academy of Sciences (Shanghai, China). The as an additional indicator of apoptosis using caspases colori- cells were incubated in Dulbecco's modified Eagle's medium metric protease assay kits (Millipore Corporation, Billerica, (DMEM) supplemented with 10% fetal bovine serum (FBS) MA, USA) according to the manufacturer's instructions. (both from Gibco-BRL, Gaithersburg, MD, USA), 100 U/ml penicillin and 100 mg/ml streptomycin at 37˚C in an atmo- Wound‑healing assay. A wound‑healing assay was also sphere of 5% CO2. performed for analysis of cell migration in vitro. Briefly, SW480 cells were transfected with miR-128 mimics or Reverse transcription-quantitative polymerase chain reac- miR-NC, cultured in 6-well plates (5x105 cells/well) and tion. Total RNA of tissues and cells were isolated using incubated overnight. An artificial homogenous wound was TRIzol reagent according to the manufacturer's instructions scratched into the monolayer using a sterile plastic micropi- (Invitrogen, Carlsbad, CA, USA). The concentration of all pette tip. After wounding, the debris was removed by washing RNAs was measured using a spectrophotometer (Thermo the cells with phosphate-buffered saline (PBS), and complete Fisher Scientific, Inc., Waltham, MA, USA), and 1 µg RNA RPMI-1640 medium with 10% FBS was added. Cell migration was used for complementaryRETRACTED DNA (cDNA) synthesis using towards the wounded area was observed and photographed the M-MLV reverse transcriptase kits (Promega, Madison, after 24 h. Wound closure (%) was calculated as the area of WI, USA) according to the manufacturer's instructions. qPCR migrated cells divided by the wounded area at 0 h. Individual was performed using SYBR Premix Ex Taq (Takara, Dalian, cells were quantified as an average of at least five fields for China). The miR-128 and U6 primers were purchased from each experiment. Qiagen (Valencia, CA, USA). Primers for IRS1 were: 5'-CAA CTGGACATCACAGCAGAA-3' (sense), and 5'-ACTGAA Cell invasion assay. Cell invasion was performed by Transwell ATGGATGCATCGTACC-3' (antisense). Primers for β-actin assay (BD Biosciences) according to the manufacturer's were: 5'-AGCAGCATCGCCCCAAAGTT-3' (sense), and instructions. Transfected cells (2x105) in serum-free medium 5'-GGGCACGAAGGCTCATCATT-3' (antisense). Ampli­ were added to each upper insert pre-coated with Matrigel fication was performed using the ABI PRISM 7000 Sequence matrix. Medium (600 µl) with 10% FBS was added to the Detection System (Applied Biosystems, Foster City, CA, lower chamber to serve as a chemoattractant. Forty-eight hours USA) and the amplification procedure consisted of 40 cycles after incubation, the non-invasive cells were removed from the (95˚C for 10 sec, 58˚C for 10 sec, 72˚C for 30 sec) following an upper surface of the Transwell membrane with a cotton swab, initial denaturation at 95˚C for 30 sec. The fold-change in and the invasive cells on the lower membrane surface were target mRNA or miRNA expression was calculated using the fixed in methanol, and stained with 0.2% crystal violet for 2-ΔΔCt method following normalization to β-actin or U6 10 min. Images of five randomly selected fields of the fixed expression, respectively. cells were captured and the cells were counted. ONCOLOGY REPORTS 34: 2797-2805, 2015 2799

Luciferase assay. The human IRS1 3'UTR oligonucleotides Table I. Correlation between clinicopathological characteristics containing the wild-type (Wt) or mutant (Mut) miR-128 and miR-128 expression in 45 patients with CRC. binding site were cloned into the pGL3 vector (Ambion, Austin, TX, USA) at the NheI and XhoI sites. For the luciferase miR-128 expression ------assay, SW480 cells were inoculated into 24-well plates and No. of Low High transfected with 100 ng of luciferase reporter vectors (WT/ Characteristics cases (n, %) (n, %) P-value Mut) and 50 nM of miR-128 or miR-NC. Forty-eight hours after transfection, firefly and Renilla luciferase activities Age (years) >0.05 were measured using the Dual-Luciferase Reporter Assay <55 24 15 (62.5) 9 (37.5) (Promega). ≥55 21 12 (57.1) 9 (42.9) Western blotting. was extracted from cells or tissue Gender >0.05 using Cell Lysis Buffer (Cell Signaling Technology, Danvers, Male 23 13 (56.5) 10 (43.5) MA, USA). After centrifugation for 15 min at 4˚C at 14,000 x g, Female 22 14 (63.6) 8 (36.4) the upper supernatant was collected and the concentrations of TNM stage <0.001 protein were determined with a bicinchoninic acid protein I-II 30 13 (43.3) 17 (56.7) assay kit (Pierce, Rockford, IL, USA). Proteins (30 µg) were III-IV 15 14 (93.3) 1 (6.7) electrophoresed in SDS-polyacrylamide gels (Invitrogen) and transferred to polyvinylidene fluoride membranes (Millipore, Tumor size (cm) >0.05 Bedford, MA, USA). After blocking with 5% non-fat milk, <5 26 17 (65.4) 9 (34.6) the membranes were incubated with specific primary anti- ≥5 19 10 (52.6) 9 (47.4) bodies overnight at 4˚C, inclucing anti-IRS1, anti‑Bcl-2, Lymph node <0.001 anti-MMP-2, anti-cyclinD1, anti-Akt, anti-pAkt and anti- metastasis β‑actin (all antibodies from Santa Cruz Biotechnology, Inc., No 29 12 (41.3) 17 (58.7) Santa Cruz, CA, USA; all diluted at 1:1,000). The membranes Yes 16 15 (93.8) 1 (6.2) were washed three times with TBST buffer, incubated with the corresponding horseradish peroxidase (HRP)-conjugated TNM, tumor node metastasis; CRC, colorectal carcinoma. secondary antibody (Santa Cruz Biotechnology, Inc.) for 2 h at room temperature and then exposed to X-ray film (Denville Scientific) using chemiluminescent reagents. β-actin was used as the internal control. sample. P<0.05 was considered to indicate a statistically significant result. Tumor xenograft treatment model. Animal experimental procedures were approved by the Institutional Animal Care Results and Use Committee of Jilin University. Twenty 6-week-old male BALB/C nude mice were obtained from the Experimental Expression of miR-128 is downregulated in CRC tissue and Animal Center of Changchun Institute for Biological Sciences cell lines. In order to confirm the involvement of miR-128 in (Changchun, China). The animals were kept and the experi- CRC, we examined the relative expression level of miR-128 in ments were performed in accordance with the European 45 CRC tissues and corresponding adjacent non-tumor tissues Community guidelines for the use of experimental animals using RT-qPCR. The results indicated that miR-128 was greatly (86/609/EEC). decreased in CRC tissues when compared with adjacent non- An equal number of SW480 cells (2x106) stably expressed tumor tissues (42/45, 93.3%, P<0.01) (Fig. 1A). To investigate in miR-128 mimic or miR-NCRETRACTED were suspended in 100 µl the clinical significance of miR-128 in CRC, 45 patients were serum‑free RPMI-1640 medium and injected subcutaneously divided into two groups according to the median value (3.36) into the right rear flank of each mouse (n=10) to establish a of the miR-128 expression level in CRC tissues: high‑miR-128 CRC xenograft model. Tumor volumes were measured every group (n=18) and low-miR-149 group (n=27). By statistical 7 days using calipers along the two major axes after treatment. assay we showed that the level of miR-128 expression in tissues Tumor volumes were calculated as: V = 0.5 x L (length) x W2 was significantly correlated with lymph node metastasis and (width). The mice were sacrificed 35 days after injection. The TNM stage, which are all indicators of poor prognosis (all tumor tissues were dissected and weighed. An aliquot of tumor P<0.05), but not with other clinicopathological characteristics, tissues was collected for analysis of the expression of IRS1 and such as age, gender and tumor size (Table I). miR-128 using previously described methods (16). In addition, a panel of human CRC cell lines was first analyzed to quantify the expression level of miR-128. The Statistical analysis. Data are expressed as means ± standard results showed that the expression level of miR-128 was down- deviation (SD) from three independent experiments. Statistical regulated in CRC cell lines when compared with that of the analyses were performed with software GraphPad Prism 5.0 normal colonic cell line (NCM460) (Fig. 1B). Additionally, the software (GraphPad Software, San Diego, CA, USA). The expression level of miR-128 in the SW480 cell line was lowest, differences between groups were analyzed using the Student's thus, we selected this cell line for subsequent experiments. t-test. The reverse relationship between IRS1 and miR-128 These observations suggested that miR-128 plays a key role in expression was assessed by Spearman's correlation in CRC CRC development. 2800 wu et al: MicroRNA-128 suppresses cell growth and metastasis in CRC by targeting IRS1

Figure 1. Expression of miR-128 is downregulated in CRC tissue and cell lines. (A) Expression of miR-128 was downregulated in 45 CRC tumor tissue (T) compared with adjacent non-tumor tissues (N). (B) The expression level of miR-128 was downregulated in the five CRC cell lines compared with the normal colonic cell line (NCM460). *P<0.05, **P<0.01. CRC, colorectal carcinoma.

RETRACTED Figure 2. Overexpression of miR-128 inhibits CRC cells proliferation and colony formation. (A) miR-128 mimic restored miR-128 expression in SW480 cells. (B) Cell proliferation was determined by CCK8 assay in SW480 cells following transfection with miR-128 mimic or miR-NC. (C) Cell colony formation was determined in SW480 cells following transfection with miR-128 mimics or miR-NC. *P<0.05, **P<0.01 vs. miR-NC. CRC, colorectal carcinoma.

miR-128 inhibits the proliferation and colony formation performed to assess the role of miR-128 in cancer cell growth. of CRC cells in vitro. The decreased expression of miR-128 Compared with the miR-NC group, the number of SW480 in CRC tissues suggested miR-128 was a tumor suppressor. colonies was significantly reduced by restoration of miR-128 To examine the role of miR-128 in CRC growth, miR-128 (P<0.05, Fig. 2C). Taken together, the results indicated that mimic or miR-NC was transfected into SW480 cells. The miR-128 inhibited the proliferation and colony formation of RT-qPCR assay results showed that transfection of miR-128 CRC cells in vitro. mimics significantly increased miR-128 expression in SW480 cells (Fig. 2A). Then, we investigated the effects of miR-128 induces apoptosis of CRC cells in vitro. The ability miR-128 restoration on cell proliferation by CCK-8 assay. of miR-128 to induce apoptosis in CRC cell lines was evalu- As shown in Fig. 2B, the proliferation of CRC cells was ated by co-staining with Annexin V and PI. The staining suppressed following transfection with miR-128 compared demonstrated that miR-128 significantly induced apoptosis in to cells transfection with miR-NC. Colony forming was then SW480 cells compared with the miR-NC groups (Fig. 3A). ONCOLOGY REPORTS 34: 2797-2805, 2015 2801

Figure 3. Overexpression of miR-128 promotes CRC cell apoptosis and caspase-3 and -8 activities. (A) Cell apoptosis was analyzed by FACS analysis in SW480 cells following transfection with miR-128 mimic or miR-NC. (B) Caspase-3 and (C) -8 activities were measured in SW480 cells following transfection with miR‑128 mimics or miR-NC. *P<0.05, **P<0.01 vs. miR-NC. CRC, colorectal carcinoma.

To determine the potential mechanism of cell apoptosis measured. It was found that exogenous miR-128 expression in vitro, the activity of caspase-3 and -8 was detected in obviously suppressed the luciferase activity of wild-type SW480 cells following transfection with 128 mimic or miR-NC. IRS1 site. However, the activity of the mutant IRS1 site was It was found that caspase-3 and -8 activity was significantly not affected (Fig. 5B), which suggested that IRS1 is directly increased in the miR-128 treatment groups compared to the targeted by miR-128. The RT-qPCR and western blot analysis miR-NC groups (P<0.05, Fig. 3B and C). was performed to measure substrate 1 (IRS-1) on the mRNA and protein level in SW480 cells transfected miR-128 inhibits migration and colony invasion of CRC cells with miR-128 mimic. We found that the expression of IRS1 in vitro. The above results showed that the level of miR-128 was downregulated in the mRNA (Fig. 5C) and protein levels expression in tissues was significantly correlated with lymph (Fig. 5D) under miR-128 mimic treatment, which indicates that node metastasis. Therefore, we hypothesized that the over- miR-128 directly binds to IRS1 and inhibits the expression of expression of miR-128 hadRETRACTED an inhibitory effect on CRC cell IRS1. Given that IRS1 was the target of miR-128, we examined migration and invasion. To confirm this hypothesis, migration the expression of IRS1 in the 45 CRC samples and adjacent and invasion assays were performed in SW480 cells transfected non-tumor tissues. The results of RT-qPCR showed that the with miR-128 mimic by wound‑healing and Transwell assays, expression level of IRS-1 mRNA was markedly increased respectively. The results showed that the overexpression of in CRC tissue comparing to the adjacent non-tumor tissues miR-128 significantly decreased the migration and invasion (41/45, 91.1%, P<0.001) (Fig. 5E) and was inversely associated abilities of SW480 cells (P<0.05; Fig. 4A and B). with the expression of miR-128 (Fig. 5F).

IRS1 is a direct target of miR-128. Potential targets of miR-128 miR-128 reduces downstream AKT signaling. It is known were predicted using bioinformatic databases (TargetScan that Akt signaling is a major molecular pathway under IRS1, and PicTar). IRS1 with a critically conserved binding site and that Akt phosphorylates and affects multiple downstream was selected for further expression and function confirma- effectors, such as cyclin D1, Bcl-2 and MMP2, which involved tion (Fig. 5A). To verify whether IRS1 is a direct target of in cell cycle progression, apoptosis and invasion. In the present miR-128 in CRC, a human IRS 3'UTR fragment containing study, we found phosphorylated Akt expression was markedly the binding sites of miR-128 or the mutant sites (Fig. 5A) was reduced after transient miR-128 mimic in the SW480 CRC cloned into the pGL3 vector. The vector along with miR-128 cell line (Fig. 6), suggesting that miR-128 suppressed Akt mimic or miR-NC were co-transfected into SW480 cells, signaling via IRS1 reduction. We also detected cyclin D1, cultured for 48 h, and luciferase activities in those cells were Bcl-2 and MMP2 protein expression in SW480 cells trans- 2802 wu et al: MicroRNA-128 suppresses cell growth and metastasis in CRC by targeting IRS1

Figure 4. Overexpression of miR-128 inhibits CRC cell migration and invasion. (A) Cell migration was analyzed by wound healing in SW480 cells following transfection with miR-128 mimic or miR-NC. (B) Cell invasion was analyzed by Transwell chamber in SW480 cells following transfection with miR-128 mimic or miR-NC. *P<0.05, **P<0.01 vs. miR-NC. CRC, colorectal carcinoma.

RETRACTED

Figure 5. miR-128 targets the 3'UTR of IRS-1 and downregulates its expression. (A) Predicted binding sites for miR-128 in the 3'UTR of IRS-1 and the muta- tions in the binding sites are shown. (B) Luciferase assay in SW480 cells co-transfected with miR-128 or miR-NC and a luciferase reporter plasmid (Wt/Mut 3'UTR IRS1). (C) Measurement of IRS1 mRNA expression levels in SW480 cells by RT-qPCR following transfection with the miR-128 mimics or miR‑NC. The β-actin was used as an internal control. (D) Measurement of IRS1 expression levels in SW480 cells by western blot analysis following transfection with the miR-128 mimics or miR-NC. β-actin was used as an internal control. (E) RT-qPCR analysis of IRS1 expression in human CRC tissue samples (N) and their matched normal tissues (T). β-actin was used as an internal control. (F) The reverse relationship between IRS1 and miR-128 expression was assessed using the Spearman's correlation in the CRC sample. *P<0.05, **P<0.01 vs. miR-NC. 3'UTR, 3' untranslated region; IRS-1, insulin receptor substrate 1; CRC, colorectal carcinoma. ONCOLOGY REPORTS 34: 2797-2805, 2015 2803

smaller in the miR-128 group compared with those in the miR-NC group (Fig. 7A-C), indicating that miR-128 over- expression suppressed CRC tumor growth in vivo. We also detected miR-128 and IRS1 expression in xenograft tumors. We found that miR-128 expression level was upregulated in xeno- graft tumors (Fig. 7D), while the mRNA and protein level of IRS1 was downregulated in xenograft tumors (Fig. 7E and F). These results suggested that miR-128 suppressed tumor growth in nude mice by inhibiting IRS1.

Discussion

Figure 6. miR-128 regulates AKT signaling pathway and its downstream Emerging evidence has suggested that miRNAs play gene. Western blotting determined AKT, p-AKT, cyclin D1, Bcl-2 and critical roles in the initiation, promotion and progression of MMP-2 protein expression in SW480 cells transfected with miR-128 mimic human cancers by regulating target (7,8). or miR-NC. β-actin was used as an internal control. Therefore, miRNAs are promising diagnostic and prognostic markers and therapeutic targets for various types of cancer, including colorectal cancer (CRC) (10,12). For example, fected with miR-128 mimic by western blotting. We found that Wang et al showed that the overexpression of miR-378-5p the expression of cyclin D1, Bcl-2 and MMP2 decreased as a in CRC cells significantly decreased the proliferation and consequence of downregulated Akt phosphorylation (Fig. 6). induced apoptosis by regulating the RAS/RAF/MEK/ERK Taken together, these data suggested that miR-128-induced pathway by targeting BRAF, suggesting that miR-378-5p is a IRS1 underexpression potentially reduced downstream AKT potentially promising therapeutic agent in CRC (18). Qin et al signaling. reported that miR-145 and paxillin are significant biomarkers for proliferation and metastasis and served as targets for the miR-128 suppresses tumor growth in nude mice by inhib- development of antiproliferative and antimetastastic strategy iting IRS1. To examine the possibility of miR-128 as a in the therapeutic interventions of CRC (19). Fang et al therapeutic agent to control CRC, an SW480 xenograft tumor suggested that miR-301a promotes CRC progression by model was established in BALB/C nude mice. The stably directly downregulating SOCS6 expression, and miR-301a is a transfected human CRC SW480 cells (SW480/miR-128 or novel biomarker for the prevention and treatment of CRC (20). SW480/miR-NC) were implanted subcutaneously into nude Zhao et al reported that miR-194 acted as a tumor suppressor mice to allow tumor formation. At five weeks post-injection, in CRC by targeting the PDK1/AKT2/XIAP pathway, and the xenograft tumor volumes and weight were significantly may be a significant diagnostic and prognostic biomarker for

RETRACTED

Figure 7. miR-128 inhibits tumor growth tumorigenicity in vivo by inhibiting IRSI. (A) Images of tumors derived for different groups. (B) Tumor growth curves from different groups. (C) Weight of tumors in nude mice from different groups. (D) miR-128 expression was determined in tumor tissue by RT-qPCR. (E and F) IRS1 mRNA and protein expression in tumor tissues was determined by RT-qPCR and western blotting, respectively. β-actin was used as an internal control. *P<0.05, **P<0.01 vs. miR-NC. IRS-1, insulin receptor substrate 1. 2804 wu et al: MicroRNA-128 suppresses cell growth and metastasis in CRC by targeting IRS1

CRC (21). In the present study, we studied the role of miR-128 3'UTR of IRS1 mRNA in CRC cells, and that overexpression in CRC. The results showed that miR-128 was downregulated of miR-128 was able to efficiently reduce the expression of in CRC tissues and cell lines, and its expression was IRS1 in CRC cells. These results suggest that IRS1 is directly significantly correlated with lymph node metastasis and TNM targeted by miR-128. stage, which agreed with previous findings (17). In addition, IRS1 transmits signals from insulin or IGF receptor and the overexpression of miR-128 in CRC cells significantly activates the PI3K/Akt and MAPK pathways, both of which decreased cell proliferation, migration and invasion in vitro, were critical in tumor initiation and progression (33). In the and suppressed tumor growth in vivo. These data suggested present study, we focused on the effect of downregulated that miR-128 acts as a novel biomarker or therapeutic agent IRS1 on Akt signaling, since the constitutive activation of for the prevention and treatment of CRC. Akt signaling play important roles in cell proliferation, cell miR-128, a type of brain-enriched miRNA, has been cycle progression, apoptosis and invasion in CRC (34,35). The shown to play important roles in the development of the data from the present study show that the overexpression of nervous system and the maintenance of its normal physiolog- miR-128 in CRC cells inhibited pAkt expression, reduced ical functions (22). Accumulating evidence has demonstrated the expression of several Akt-regulated proteins including that the aberrant expression of miR-128 was involved in the cyclin D1, Bcl-2 and MMP2. These results suggested that prolife­ration, differentiation, apoptosis, invasion and metas- miR-128 inhibited CRC growth and metastasis by targeting tasis of various tumor cells (13-16). For instance, miR-128 the IRS1‑regulating AKT signaling pathway. has been shown to inhibit tumor progression, angiogenesis In conclusion, the present study provides evidence that and lymph angiogenesis in non-small‑cell lung cancer by miR-128 expression is downregulated in CRC tissues and blocking ERK, AKT and p38 signaling pathways (16). cell lines, and that its expression was significantly correlated Recent findings have shown that the upregulation of miR-128 with lymph node metastasis and TNM stage. In addition, the inhibited HNSCC growth by directly mediating its targets overexpression of miR-128 in CRC cells inhibited prolifera- Paip2, BAG-2, H3F3B, BMI-1 and BAX in proliferation and tion, migration and invasion, induced cell apoptosis in vitro, apoptotic pathways (15). Additionally, miR-128 expression is and suppressed tumor growth in vivo by targeting IRS1 and downregulated in glioma tissue and cell lines, and restoration its downstream AKT signaling. These results suggest that of miR-128 repressed growth and mediated differentiation miR-128 is a novel candidate for CRC therapeutics. of glioma-initiating neural stem cells by targeting oncogenic receptor kinases epithelial growth factor receptor Acknowledgements and platelet-derived growth factor receptor-α and repressed gliomagenesis (14,23,24). Although it was recently shown The present study was supported by the Health Department of that miR-128 induced G2-phase cell cycle arrest and inhibited Jilin Province (2010SO20). cancer cell proliferation in CRC cells by targeting NEK2 (17), its roles in human CRC remain largely unclear, particularly References for migration and invasion. To investigate the functions 1. 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