Microrna-202-3P Inhibits Cell Proliferation by Targeting ADP-Ribosylation Factor-Like 5A in Human Colorectal Carcinoma

Published OnlineFirst December 10, 2013; DOI: 10.1158/1078-0432.CCR-13-1023

Clinical Cancer Human Cancer Biology Research

MicroRNA-202-3p Inhibits Cell Proliferation by Targeting ADP-Ribosylation Factor-like 5A in Human Colorectal Carcinoma

Qifeng Wang1,3,4,5, Zhaohui Huang6, Weijie Guo3,5,7, Shujuan Ni1,3,4,5, Xiuying Xiao8, Lisha Wang1,3,4,5, Dan Huang1,3,4,5, Cong Tan1,3,4,5, Qinghua Xu1,2,3,4,5, Ruopeng Zha3,5,7, Jiwei Zhang6, Weiqi Sheng1,3,4,5, Xianghuo He7, and Xiang Du1,3,4,5

Abstract Purpose: MicroRNAs (miRNA) that are strongly implicated in carcinogenesis have recently reshaped our understanding of the role of non–protein-coding RNAs. Here, we focused on the function and molecular mechanism of miR-202-3p and its potential clinical application in colorectal cancer. Experimental Design: miR-202-3p expression was determined by quantitative reverse transcriptase PCR (qRT-PCR) in 94 colorectal cancer tissues and corresponding noncancerous tissues (NCT). Cell proliferation and colony formation assays in vitro and xenograft experiments in vivo were used to evaluate the effect of miR- 202-3p on colorectal cancer cell proliferation. Luciferase assay and Western blot analysis were performed to validate the potential targets of miR-202-3p after the preliminary screening by online prediction and microarray analysis. The mRNA and protein levels of target genes were detected by qRT-PCR and immunohistochemical staining. The copy number of pre-miR-202 was measured by quantitative PCR. Results: First, miR-202-3p was signiﬁcantly downregulated in 46.7% colorectal cancer samples compared with NCTs. The overexpression of miR-202-3p inhibited colorectal cancer cell growth in vitro and repressed tumorigenesis in nude mice. Then, miR-202-3p downregulated ADP-ribosylation factor-like 5A (ARL5A) protein level by binding to its 30 untranslated region, and knockdown of ARL5A phenocopied the proliferation inhibition effect of miR-202-3p. Furthermore, both of ARL5A mRNA and protein levels were upregulated in colorectal cancer samples compared with NCTs and high ARL5A protein levels predicted a poor prognosis. Conclusions: miR-202-3p might function as a tumor suppressor in colorectal cancer, and ARL5A, the functional target of miR-202-3p in colorectal cancer, is a potential prognostic factor for colorectal cancer. Clin Cancer Res; 20(5); 1146–57. 2013 AACR.

Introduction the posttranscriptional level by binding to their target MicroRNAs (miRNA) are small noncoding RNAs (18–22 mRNAs (1, 2). Since their discovery in 1993, the number nt in length) that regulate the expression of target genes at of verified miRNAs has grown rapidly, and the latest version of miRBase (release 20.0, June 2013, http://www.mirbase. org/) has annotated 2,578 mature miRNA sequences in the Authors' Affiliations: 1Department of Pathology; 2bioMerieux Laboratory, Fudan University Shanghai Cancer Center; 3Department of Oncology, human genome. miRNAs are estimated to regulate up to Shanghai Medical College; 4Institute of Pathology; 5Institutes of Biomedical one third of human genes at the posttranscriptional level 6 Sciences, Fudan University, Shanghai; Wuxi Oncology Institute, the Affili- (3). miRNA deregulation has been demonstrated to play a ated Hospital of Jiangnan University, Wuxi; 7State Key Laboratory of Onco- genes and Related Genes, Shanghai Cancer Institute, Shanghai Jiao Tong role in the pathologic processes of tumorigenesis (4, 5). University School of Medicine; and 8Department of Oncology, Ren Ji Hos- Furthermore, miRNAs serve as potential diagnostic mar- pital, Shanghai Jiao Tong University, Shanghai, China kers, prognostics factors, and therapeutic targets (6–9). Note: Supplementary data for this article are available at Clinical Cancer Although miRNAs have been reported to be involved in Research Online (http://clincancerres.aacrjournals.org/). human cancers, their biologic functions and molecular Q. Wang, Z. Huang, and W. Guo contributed equally to this work. mechanisms remain largely unknown. Corresponding Authors: Xiang Du, Department of Pathology, Fudan Uni- Colorectal cancer is the third most commonly diagnosed versity Shanghai Cancer Center, 270 Dong An Road, Shanghai 200032, China. cancer in men and the second most commonly diagnosed Phone: 86-21-64170067; Fax: 86-21-64170067; E-mail: [email protected]; cancer in women worldwide. Over 1.2 million new cancer and Xianghuo He, State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Ren Ji Hospital, Shanghai Jiao Tong University cases occur annually, resulting in greater than 600,000 School of Medicine, No.25/Ln.2200, Xietu Road, Shanghai 200032, China. deaths each year (10). Despite recent advances in diagnostic Phone: 86-21-64436539; Fax: 86-21-64436539; E-mail: [email protected]. and therapeutic measures, the prognoses of patients with doi: 10.1158/1078-0432.CCR-13-1023 colorectal cancer remain poor. Therefore, it is necessary to 2013 American Association for Cancer Research. clarify the underlying molecular mechanisms and to identify

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miR-202-3p Targets ARL5A in Colorectal Cancer

tumor samples indicated that ARL5A is a new independent Translational Relevance prognostic factor in colorectal cancer. MicroRNAs (miRNA) are small, noncoding RNAs that are strongly implicated in carcinogenesis and have Materials and Methods recently reshaped our understanding of the role of Human tissues and cell lines non–protein-coding RNAs. miRNAs have been argued A total of 94 pairs of human primary colorectal cancer to be used as potential diagnostic markers, prognostic tissues and their adjacent NCTs were collected between factors, and therapeutic targets in the pathologic pro- 2005 and 2008 at the Fudan University Shanghai Cancer cesses of tumorigenesis, while the biologic functions and Center. The tissue samples were immediately snap-frozen molecular mechanisms of action of the majority of in liquid nitrogen and were histologically examined in a miR- miRNAs remain largely unknown. In this study, timely manner. All of the human materials were obtained 202-3p , which is frequently downregulated in human with informed consent, and thisprojectwasapprovedby colorectal cancer, was identified as a potent repressor of the Clinical Research Ethics Committee of Fudan Uni- miR-202-3p proliferation. Reexpression of expression versity Shanghai Cancer Center. The clinical information inhibited colorectal cancer proliferation by silencing its of the patients with colorectal cancer is included in ARL5A direct target, ADP-ribosylation factor-like 5A ( ), Supplementary Table S1. HEK-293T cell line and six which was closely related to poor prognosis of patients human colorectal cancer cell lines, including the HCT- with colorectal cancer. Although miRNA research is still 8, HCT-116, LoVo, DLD1, SW480, SW620, were pur- in the early stages, the contribution of miRNAs to disease chased from the American Type Culture Collection processes is becoming clear. Novel and expanded (ATCC). HCT-8 cells were cultured in RPMI-1640 medi- insights into miRNA deregulation will deepen our um, HCT-116 cells were cultured in McCoy’s 5a medium, understanding of the complicated molecular pathogen- and LoVo cells were cultured in F12-K medium. DLD1 esis of colorectal cancer and delineate new options for and HEK-293T cells were cultured in Dulbecco’s Modified colorectal cancer treatment. Eagle Medium (DMEM). SW480 and SW620 cells were maintained in Leibovitz’s L-15 medium. All of these media (Invitrogen) were supplemented with 10% FBS (Gibco). The cells were incubated under the conditions specific biomarkers of colorectal cancer. The timely discovery recommended by ATCC. of the importance of miRNAs is fortunate in this regard. Over the past decade, the contribution of miRNAs (11), DNA and RNA extraction and quantitative real-time such as let-7 (12, 13), miR-17 (14, 15), miR-21 (16–19), RT-PCR miR-31 (20, 21), miR-95 (22), miR-101 (23, 24), miR-135 Tissue genomic DNA was isolated using the DNeasy (25, 26), miR-137 (27), miR-139 (28, 29), miR-143 (30– Blood and Tissue Kit (Qiagen) according to the manu- 32), miR-145 (31, 33), miR-155 (34), miR-183 (35), and facturer’s protocol. Total RNA, including miRNA, was miR-195 (36), to colorectal carcinogenesis has been exam- extracted using the TRIzol reagent (Invitrogen) according ined. In a previous study, we determined that miR-95 is to the manufacturer’s instructions. The concentrations frequently overexpressed in colorectal cancer and acts as an of DNA and RNA were determined using a NanoDrop important inducer of proliferation via the negative regula- ND-1000 (NanoDrop), and aliquots of the samples were tion of SNX1 expression (22). All these data highlight the stored at 80C. Relative DNA copy numbers were importance of miRNAs in tumor development and provide determined by quantitative PCR (qPCR) using SYBR new insights into the molecular mechanisms underlying Premix Ex Taq (TaKaRa). An amount of 50 ng DNA was carcinogenesis. used as template in a final volume of 20 mLwithLINE-1 Our previous microarray data showed that miR-202-3p,a as an internal control (41–43). cDNA was synthesised member of the let-7 family, was downregulated in colorectal with the PrimeScript RT Reagent Kit (TaKaRa) using 500 cancer (22). In addition to colorectal cancer, miR-202-3p ng total RNA as template. qPCR analyses were conducted has also been reportedly deregulated in breast cancer and to quantitate mRNA relative expression using SYBR Pre- cervical squamous cell carcinoma (37, 38) and could inhibit mixExTaq(TaKaRa)withb-ACTIN as an internal control cell proliferation in neuroblastomas (39, 40). These data (44–46). TaqMan microRNA assays (Applied Biosys- suggest that miR-202-3p may play common roles in tumor- tems) were used to determine the expression levels of igenesis. However, the detailed function and molecular miR-202-3p after reverse transcribing by sequence-spe- mechanisms of miR-202-3p in human tumors have not cific primers (Applied Biosystems), and U6 small nuclear been elucidated. In the present work, we confirmed that RNA was used as an internal control (47–50). The results miR-202-3p expression was significantly decreased in of qPCR were defined from the threshold cycle (Ct), and colorectal cancer tissues compared with the noncancerous relative expression levels were calculated by using the DD tissues (NCT) in an expanded colorectal cancer cohort. 2 Ct method (51). PCR was performed using an ABI Moreover, we showed that miR-202-3p could inhibit cell 7900HT instrument (Applied Biosystems). The primers proliferation by directly targeting ADP-ribosylation factor- used for PCR analysis were listed in Supplementary like 5A (ARL5A) in colorectal cancer, and analysis of clinical Table S2.

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Vector constructs mal Department of Fudan University. The assay protocols The human pri-miR-202 sequence was amplified from were approved by the Shanghai Medical Experimental Ani- normal human genomic DNA by nested PCR using Primer- mal Care Commission. HCT-116 or LoVo cells stably STAR Premix (TaKaRa). The sequence was then cloned into expressing miR-202 or the vector control were washed from the lentivirus expression vector pGIPZ (Thermofisher) to subconfluent cell culture plates with PBS and then were generate pGIPZ-miR-202. The predicted binding sites in the resuspended with DMEM at a concentration of 1 107 30 untranslated regions (UTR) of the potential target genes cells/mL. A 0.1 mL aliquot of HCT-116 or 0.2 mL aliquot of of miR-202-3p were amplified by nested PCR and cloned LoVo-suspended cells was subcutaneously injected into the into the region directly downstream of a cytomegalovirus right flank of each mouse age 5 weeks (8 mice for each (CMV) promoter–driven firefly luciferase cassette in the group). After transplantation, the growth of the subcutane- pcDNA3.0 vector (pLuc). The mutant-type (MT) 30 UTR of ous tumors was assessed twice a week. Tumor size was ARL5A, which carries the mutated sequence in the comple- monitored by measuring the length and width with calli- mentary site of the seed region of miR-202-3p, was generated pers, and volumes were calculated with the formula: (L from the pLuc-ARL5A 30 UTR–wild-type (WT) plasmid by W2) 0.5, where L is the length and W is the width of each overlap-extension PCR. The sequences of the WT and MT 30 tumor. The mice were sacrificed after a period of 4 to 6 UTRs were confirmed by DNA sequencing. The open read- weeks, and the weight of subcutaneous tumors was ing frame (ORF) of ARL5A was amplified via nested PCR recorded. HCT-116 cells, 24 hours after transfection with and cloned into the pLVX-IRES-Neo vector (Clontech). The miR-202-3p mimics or negative control (NC), were also primers and endonuclease sites used for the vector con- used for xenograft experiments following the procedures structs are shown in Supplementary Table S2. described above (52). The expression levels of miR-202-3p and ARL5A protein of cells used for tumor formation assay Lentivirus production and transduction were examined (Supplementary Figs. S2B and S2C). Virus particles were harvested 48 hours after cotransfect- ing pGIPZ-miR-202 and pLVX-ARL5A with the packaging Cell migration assay plasmid ps-PAX2 and the envelope plasmid pMD2G into For the migration assays, 1 105 HCT-116 or 5 104 HEK-293T cells using Lipofectamine 2000 Reagent (Invi- LoVo cells (stably expressed miR-202 or vector control) in trogen). HCT-116 and LoVo cells were infected with recom- 200 mL serum-free medium were placed onto the top binant lentivirus-transducing units plus 6 mg/mL polybrene chamber of each insert (BD Biosciences). After 36 (HCT- (Sigma). 116) or 8 (LoVo) hours of incubation at 37C, cells adhering to the lower membrane were stained with 0.1% crystal violet Oligonucleotide transfection in 20% methanol, imaged, and counted using an IX71 Small interfering RNA (siRNA) of miR-202-3p target genes inverted microscope (Olympus). (sequence information; Supplementary Table S3), as well as miR-202-3p/5p mimics and inhibitors (anti-miR-202-3p/5p, Microarray analysis chemically modified antisense oligonucleotides designed to Genome-wide expression profiling was performed using specifically target mature miR-202-3p/5p), were synthesised an Agilent whole human genome oligo microarray chip (Ribobio). Oligonucleotide transfection was performed (4 44 K) that contains more than 42,034 genes and using RNAiMAX reagents (Invitrogen) according to the transcripts (Agilent). An amount of 5 105 cells (HCT- manufacturer’s instructions. The final concentration of the 116 or LoVo) were seeded in a 6-cm2 tissue culture dish and miR-202-3p/5p mimics or inhibitors in the transfection were transfected with miR-202-3p or control miRNA as mixture was 50 nmol/L. described above. After propagating for 48 hours, total RNA was extracted from the cells using TRIzol reagent (Invitro- Cell proliferation and colony formation assays gen). The RNA concentration was determined, and the Cell proliferation was quantified using the Cell Counting integrity of RNA was verified using an Agilent 2100 Bioa- Kit-8 (CCK-8; Dojindo Laboratories) according to the man- nalyzer (Agilent). Cy3-labeled cRNA was prepared with an ufacturer’s instructions and the optical density (OD) value Agilent One Color Spike Mix Kit (Agilent) and then hybrid- was obtained for determine the cell numbers. For the colony ized by using a Gene Expression Hybridization Kit (Agilent) formation assays, 1,000 cells per well of HCT-116 and 1,500 at 65C for 17 hours. Signal intensity was calculated from cells per well of LoVo were plated into 6-well plates and digitized images captured by Laser Scanner (Agilent) and incubated in medium containing 10% FBS for 2 weeks. The the chip data were analysed using the GeneSpring GX colonies were fixed with methanol and stained with 0.1% software (Agilent). An mRNA was designated as "down- crystal violet in 20% methanol for 20 minutes. The number regulated" if its expression in miR-202-3p–transfected cells of colonies containing more than 30 cells was counted using was 2-fold lower than that of the corresponding control an inverted microscope. cells.

Tumor formation assay in a nude mouse model Luciferase assay Athymic male BALB/c nude mice were maintained under Approximately 8,000 HEK-293T cells per well and 12,000 speciﬁc pathogen-free conditions in the Experimental Ani- HCT-116 cells per well were plated into 96-well plates and

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were cotransfected with 50 nmol/L of miR-202-3p mimic (or colorectal cancer. Consistent with the microarray data, miR- NC), 50 ng of the luciferase reporter, and 5 ng of the pRL- 202-3p expression was remarkably downregulated in 44 of CMV Renilla luciferase reporter using 0.5 mL per well Lipo- the 94 (46.7%) colorectal cancer tissues when compared fectamine 2000 (Invitrogen). LoVo cells, plated into 96-well with the corresponding NCTs (P < 0.001; Fig. 1A). However, plates with 12,000 cells per well, were cotransfected with there was no significant association between miR-202-3p 100 nmol/L of miR-202-3p mimic (or NC), 200 ng of the expression in colorectal cancer tissues and gender, age, luciferase reporter, and 20 ng of the pRL-CMV Renilla tumor location, tumor size, histologic classification, nodal luciferase reporter using 1.25 mL per well X-tremeGENE status, or survival (P > 0.05; data not shown). In addition, Reagent (Roche). After a 48-hour incubation, the Firefly and miR-202-3p T:N expression ratios were weakly associated Renilla luciferase activities were quantified using a dual- with OS of patients with colorectal cancer (Supplementary- luciferase reporter assay (Promega). Fig. S1A).

Western blot analysis Downregulation of miR-202-3p in colorectal cancer Harvested proteins were first separated by 10% SDS–PAGE tissues caused by DNA deletion and then transferred to nitrocellulose membranes (Bio-Rad To confirm the hypothesis that downregulation of miR- Laboratories). The membranes were blocked with 5% nonfat 202-3p in colorectal cancer tissues was partly caused by copy milk and incubated with a mouse anti-ARL5A polyclonal number variation of its coding sequence, we determined the antibody at a dilution of 1:500 (Abgent) or a mouse anti– pre-miR-202 copy number by qPCR in 37 pairs of the b-ACTIN monoclonal antibody at a dilution of 1:1,000 colorectal cancer tissues and NCTs. Interestingly, we found (Sigma). The membranes were subsequently incubated with that the copy number of pre-miR-202 was lower in 64.9% a goat anti-mouse horseradish peroxidase secondary anti- (24 of 37) and higher in 13.5% (5 of 37) of the colorectal body (Sigma). The protein complex was detected using cancer tissues compared with their NCT counterparts (P < enhanced chemiluminescence reagents (Pierce). Endoge- 0.05; Fig. 1B). Subsequent analysis showed that the pre-miR- nous b-ACTIN was used as the internal control. 202 copy numbers were related to the expression levels of mature miR-202-3p in both the colorectal cancer and NCT Immunohistochemical staining samples (P < 0.05; Fig. 1C and D). Tissue arrays were constructed using the 94 paired colorectal cancer tissues and NCTs. Immunohistochemical miR-202-3p represses colorectal cancer cell staining was performed on 4 mm sections of paraffin- proliferation in vitro and in vivo embedded tissues to determine the expression level of The consistently low expression of miR-202-3p in colo- ARL5A protein. In brief, the slides were incubated in ARL5A rectal cancer suggests it contributes to tumorigenesis. The antibody (Abgent) diluted 1:200 at 4 C overnight. The expression of miR-202-3p was examined in 6 different subsequent steps were performed using the EnVision FLEX colorectal cancer cell lines (SupplementaryFig. S1B). A cell High pH visualisation system according to the manufac- proliferation assay revealed that overexpression of miR-202- turer’s instructions (Dako). The ARL5A staining intensity 3p significantly reduced the growth rate of HCT-116 and measurements are presented in Supplementary Fig. S2. LoVo cells (P < 0.01; Fig. 2A), and a colony formation assay confirmed that miR-202 overexpression represses the pro- Statistical analyses liferation of colorectal cancer cells (P < 0.01; Fig. 2B). In The results are presented as the mean values SEM. contrast, silencing of miR-202-3p expression with siRNA Overall survival (OS) rates were calculated actuarially significantly promoted the growth of HCT-116 and LoVo according to the Kaplan–Meier method with log-rank test cells (P < 0.01; Fig. 2A). Besides that, miR-202-5p did not and were measured from the day of surgery. Differences show significant effect on the colorectal cancer cell growth x2 between groups were estimated using the test, the Student (Supplementary Fig. S1C). To assess the function of miR- t U test, the Mann–Whitney test, and the repeated-measures 202 in vivo, a tumor formation assay in a nude mouse model ANOVA test. Relationships were explored by the Spearman was performed using HCT-116 and LoVo cells stably expres- P < correlation. A value of 0.05 was considered statistically sing miR-202 after lentiviral infection. Compared with the significant. SPSS 16.0 package (IBM) and Graphpad prism empty vector control, overexpression of miR-202 remark- 5.0 software (GraphPad Software) were used for statistical ably repressed tumorigenesis in nude mice (P < 0.01; Fig. 2C analyses and scientific graphing, respectively. and D). Furthermore, overexpressed miR-202-3p by mimics transfecting in HCT-116 cells also repressed tumor growth Results in vivo (Supplementary Fig. S2A). In addition, transwell Expression of miR-202-3p is decreased in human assay in HCT-116 or LoVo cells indicated that miR-202-3p colorectal cancer tissues may not significantly affect colorectal cancer migration miRNA expression profiling previously demonstrated (Supplementary Fig. S1D). that miR-202-3p is downregulated in colorectal cancers compared with NCTs (22). To further confirm this result, Screening of candidate target genes of miR-202-3p the expression level of mature miR-202-3p was examined To explore the potential mechanism by which miR-202- using qRT-PCR in an expanded cohort of 94 patients with 3p represses the proliferation of colorectal cancer cells, we

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Figure 1. miR-202-3p expression was frequently reduced in colorectal cancer (CRC) as a result of DNA CNV. A, comparison of miR-202-3p expression levels in 94 matched pairs of colorectal cancer tissues and NCTs. The qRT-PCR results showed that miR-202-3p expression was signiﬁcantly downregulated in tumor tissues (U6 small nuclear RNA used as an internal control). miR-202-3p expression was upregulated in 16.0%, downregulated in 46.7%, and unchanged in 37.2% of the colorectal cancer samples. B, pre-miR-202 copy numbers were determined for 37 matched colorectal cancer and NCT samples using qPCR. The copy number of pre-miR-202 was decreased in the tumor tissues compared with the NCTs (LINE-1 used as the internal control). The copy numbers of pre-miR-202 DNA were increased in 13.5%, decreased in 69.4%, and unchanged in 21.6% of the colorectal cancer samples compared with NCTs. C and D, miR-202-3p expression was positively correlated with the pre-miR-202 DNA copy number in both the colorectal cancer and the NCT samples.

performed a microarray analysis to search for candidate 202-3p (Fig. 3B). Then the recombinant plasmids with the 30 genes regulated by miR-202-3p. Compared with the vector UTR sequences of these 10 genes containing the predicted controls, a total of 823 downregulated genes (>2-fold binding site sequence of miR-202-3p were constructed for change) were identiﬁed in the miR-202-3p–transfected luciferase assays. A panel of 4 candidate genes (ARL5A, LoVo cells (Supplementary Table S4). By comparing all of LRIG3, HECTD2, and CPEB4) was selected because of their the downregulated genes with the candidate genes predicted relatively decreased luciferase activity in miR-202-3p–trans- by the TargetScan and Miranda programs, a total of 18 fected cells (Fig. 3C). Subsequently, a preliminary cell downregulated genes were selected out (Fig. 3A). Then the proliferation assay was performed on HCT-116 and LoVo expression of these 18 genes was validated using qRT-PCR in cells transfected with the pertinent siRNAs (to eliminate the both miR-202-3p-transfected HCT-116 and LoVo cells and off-target effects of siRNAs by qRT-PCR; Supplementary Fig. 10 of these 18 genes were indeed downregulated by miR- S3B). ARL5A, LRIG3, and CPEB4 were found to be

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miR-202-3p Targets ARL5A in Colorectal Cancer

Figure 2. miR-202-3p represses colorectal cancer cell growth in vitro and in vivo. A, miR-202-3p overexpression represses the proliferation of HCT-116 and LoVo cells. miR-202-3p knockdown enhanced the growth rate of HCT-116 and LoVo cells. The OD values were examined and the number of cells was determined using the Cell Counting Kit-8 Assay (, P < 0.05; , P < 0.01; , P < 0.001). B, the effect of miR-202 on colony formation was evaluated by crystal violet staining of miR-202 lentiviral vector-infected HCT-116 and LoVo cells. miR-202 overexpression represses the colony formation rates (P < 0.01). C and D, the effect of miR-202 on tumor formation in a nude mouse xenograft model. Lentiviral vector- and miR-202–infected HCT-116 cells (1 106) or LoVo cells (2 106) were injected s.c. into the right ﬂank of each nude mouse. The tumor formation rate was 8 of 8 (Lenti-vector group) and 6 of 8 (Lenti-miR-202 group) for HCT-116 cells, and was 7 of 8 (NT group) and 5 of 8 (Lenti-miR-202 group) for LoVo cells. The tumor volume and weight of the miR- 202-3p group was signiﬁcantly decreased compared with the control group (, P < 0.05; , P < 0.01; , P < 0.001). associated with cell growth, and depleting ARL5A expres- Upregulation of ARL5A is closely correlated with miR- sion showed the most obvious growth repression effect (Fig. 202-3p expression and poor prognosis in colorectal 3D). In addition, ARL5A mRNA expression was upregulated cancer in colorectal cancer tissues compared with NCTs in small To study the relationship between miR-202-3p and samples, whereas LRIG3 mRNA was not (n ¼ 30; Supple- ARL5A in human colorectal cancer, we measured the mRNA mentary Fig. S3C). and protein expression levels of ARL5A in the 94 paired colorectal cancer and NCT samples using qRT-PCR and ARL5A is a direct target of miR-202-3p in colorectal immunohistochemistry methods, respectively. Compared cancer with the matched NCTs, ARL5A mRNA expression was To determine whether ARL5A is a direct target of miR-202- upregulated in colorectal cancer tissues (P < 0.05; Fig. 3p in colorectal cancer, we constructed a recombinant 5A) and inversely correlated with miR-202-3p levels (Sup- plasmid containing the ARL5A 30 UTR sequence with a plementary Fig. S4B). ARL5A protein was overexpressed in mutated binding site sequence (Fig. 4B). Luciferase activity 56.4% (53 of 94) of the colorectal cancer tissues compared was no longer reduced in cells (HEK-293T, HCT-116, LoVo) with the matched NCTs (P < 0.05; Fig. 5B) and clearly transfected with the mutant 30 UTR plasmid versus the WT 30 negatively correlated with miR-202-3p expression levels UTR plasmid (Fig. 4A). In concordance with these results, (P < 0.05; Fig. 5C). More importantly, enhanced immuno- endogenous ARL5A protein levels were downregulated in reactivity of ARL5A in colorectal cancer tissues was inversely miR-202-3p–overexpressing cell lines and were restored correlated with OS and suggested a poor prognosis for the when the expression of miR-202-3p was repressed (Fig. 4C). patients with colorectal cancer (P < 0.01; Fig. 5D). In

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Figure 3. Screening of candidate target genes of miR-202-3p in colorectal cancer. A, initial screening of miR-202-3p target genes in HCT-116 and LoVo using a microarray assay and bioinformatics predictions. A total of 18 downregulated genes were selected out. B, validation of the microarray results in both HCT-116 and LoVo cells using qRT-PCR. A panel of 10 genes were indeed downregulated by miR-202-3p. C, four candidate genes (ARL5A, LRIG3, HECTD2, and CPEB4) were selected due to their relatively decreased luciferase activity in miR-202-3p–transfected cells (-1, -2, -3 following the gene names represent the 30 UTR plasmids cloned with different predicted binding sites). D and E, proliferation assays performed on HCT-116 and LoVo cells transfected with siRNA directed against 4 candidate genes. Depleted ARL5A expression showed the most obvious growth repression effect.

addition, higher ARL5A protein levels in colorectal cancers proliferation, which could not be restored by either exog- compared with NCTs also indicated poor prognosis (Sup- enous or endogenous overexpression of miR-202-3p (Fig. plementary Fig. S4C). No association was observed between 6C and D). the intensity of ARL5A staining and tumor size, grade, stage, or lymph node metastasis (P > 0.05). Discussion miR-202-3p represses proliferation by directly It is well known that miRNAs are key components of targeting ARL5A in colorectal cancer tumorigenesis, as they participate in many cellular processes To further clarify whether targeting of ARL5A might including cell proliferation, differentiation, and death. The mediate the inhibition of cell proliferation in miR-202- effects of miRNAs are based on their regulation of the 3p–overexpressing colorectal cancer cells, we performed a expression of many cancer-related genes through posttran- series of functional restoration assays using HCT116 and scriptional repression. As we previously reported, a panel of LoVo cells. First, we inhibited ARL5A expression with siRNA miRNAs was altered in colorectal cancer tissues, suggesting and found that ARL5A-depleted cells exhibited decreased that variations in the expression of miRNAs are common proliferation, which strongly indicated that silencing events in colorectal tumorigenesis (1, 5, 11). We previously ARL5A phenocopied the proliferation-repressing effect of reported that miR-95, which seems to be an oncogenic miR-202-3p (Fig. 6A). Next, cotransfection experiments miRNA in colorectal cancer, promoted cell proliferation by using ARL5A and miR-202-3p siRNAs also showed that directly targeting SNX1 (22). In addition to upregulated miR-202-3p knockdown did not enhance the growth rate miRNAs, downregulated miRNAs also contribute notably of colorectal cancer cells when ARL5A expression had been to tumorigenesis. miR-139-5p, miR-145, miR-125a, and silenced by siRNA (Fig. 6B). Moreover, ARL5A overexpres- miR-133a have been reported to be tumor suppressors sion (overexpressed ORF without 30 UTR) promoted cell whose precise underlying mechanisms have been identiﬁed

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miR-202-3p Targets ARL5A in Colorectal Cancer

Figure 4. miR-202-3p suppresses ARL5A expression by directly targeting its 30 UTR. A, relative luciferase activity assays of luciferase reporters with ARL5A WT or MT 30 UTR were performed in cells (HEK-293T, HCT-116, and LoVo) after cotransfection with pGIPZ-miR-202 plasmids. Luciferase activity was determined 48 hours after transfection and normalized to the Renilla luciferase activity. The luciferase activity generated by pLUC in each experiment was set as 1. B, schematic of the WT or MT 30 UTRs of the ARL5A vector constructs. The complementary site of the seed region of miR-202-3p was selected for mutation. C, endogenous ARL5A expression was suppressed by miR-202-3p. The protein levels of ARL5A were determined by Western blot assays in HCT-116 and LoVo cells infected with miR-202-3p or control lentiviruses, the miR-202-3p inhibitor (anti-miR-202-3p), or the negative control (anti-NC). b-ACTIN served as an internal control. as they relate to the development of colorectal cancer Mature miRNAs are generated from primary precursors (31, 53–55). encoded by genes involving two subsequent RNA cleavage With the aim of finding new colorectal cancer-associated steps (56). When we overexpressed miR-202-3p by lentiviral miRNAs, we focused on miR-202-3p (previously named overexpression of the pre-miR-202 sequence, the intrinsic miR-202 before miRBase release 16.0), a target that was mature miR-202-5p was simultaneously overexpressed with found to be significantly downregulated in colorectal cancer a complementary sequence of miR-202-3p. For that reason, samples in our previous profiling data (22). miR-202-3p is we also detected the effect of miR-202-5p on cell growth and highly conserved across animal species and is now consid- found that overexpressing of miR-202-5p did not affect ered a member of the let-7 family, which consists of tumor colorectal cancer cell proliferation (Supplementary Fig. suppressors that target k-RAS and MYCN (12, 39, 40). Con- S1C). Thus, we excluded the side effect of miR-202-5p sistent with the role of let-7 family members, in this study, overexpression on functional assays using cells that stably we found that miR-202-3p repressed cell proliferation and expressed miR-202. colony formation in colorectal cancer via its direct target, Copy number variants (CNV) have been described as ARL5A. In addition to affecting proliferation, the role of notable structural variations in the human genome that miR-202-3p in metastasis is another important aspect that affect gene expression and contribute to phenotypic differ- must be considered in future tumor-related research. How- ences (57, 58). However, only a few studies have closely ever, we did not conduct in-depth study on the influence of examined the CNVs of miRNAs and recognised their poten- miR-202-3p on tumor metastasis and only found that it did tial consequences. The coding sequence of miR-202-3p is not significantly affect the migration of HCT-116 or LoVo located on chromosome 10q26.3, a region considered a cells (Supplementary Fig. S1D). chromosome fragile site that is frequently deleted in

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Wang et al.

Figure 5. ARL5A was overexpressed in colorectal cancer and its level closely correlated with the level of miR-202-3p. A, ARL5A mRNA expression level was significantly increased in the 94 colorectal cancer tissues compared with matched NCT samples determined by qRT-PCR. b-ACTIN mRNA served as the internal control. B, immunohistochemical staining of ARL5A in the tumor tissues and the corresponding normal colon epithelia. Brown cytoplasmic ARL5A staining was observed in colorectal cancer tissues but was nearly absent in the normal epithelia (original magnification, 100 or 400). ARL5A expression is frequently increased in the tumor tissues (n ¼ 94) compared with the matched NCTs, with overexpression in 56.4%, reduced expression in 25.5%, and unchanged expression in 18.1% of the colorectal cancer tissues. Statistical analyses of the cases grouped according to the ARL5A 2 scoring. Statistical significance was assessed using the x test. C, the level of expression of ARL5A correlated negatively with the miR-202-3p expression levels in the tumor tissues. D, OS analysis based on the expression level of ARL5A. The groups were ranked according to the ARL5A staining intensity. Patients who scored 0, 1, or 2 were included in the low-expression group, whereas those who scored 3 were included in the high-expression group. The percentage of OS in the ARL5A low-expression group was significantly higher than that of patients in the ARL5A high-expression group.

endometrial and brain tumors (59, 60). We observed that for the first time, that ARL5A is a potential oncogene in the copy numbers of pre-miR-202 were decreased in most colorectal cancer. However, the detailed mechanism by colorectal cancer or NCT samples and were positively cor- which ARL5A influences colorectal cancer cell growth has related with miR-202-3p expression, most likely leading to not yet been elucidated. Further study about other cell the downregulation of miR-202-3p. The analysis of more growth–related genes in colorectal cancer (LRIG3 and clinical samples is necessary to confirm these findings. CPEB4) may be carried through in the future work. ARL5A, located on human chromosome 2q23.3, and Taken together, miR-202-3p, which is frequently down- ARL5A protein in eukaryotes belongs to the ARF family, regulated in human colorectal cancer, was identified as a which are members of the Ras gene superfamily of GTP- potent repressor of proliferation. Reexpression of miR-202- binding proteins that are involved in a variety of processes, 3p inhibited colorectal cancer proliferation by silencing its such as cellular communication, endoplasmic reticulum direct target, ARL5A, which was closely related to prognosis binding, vesicle transport, and protein synthesis (61, 62). of patients with colorectal cancer. Although miRNA ARL5A is upregulated in liver carcinoma according to a research is still at the early stages, the contribution of previous report (62). In our study, ARL5A was identified as a miRNAs to disease processes is becoming clear. Novel and direct target of miR-202-3p using a series of assays. We expanded insights into miRNA deregulation will deepen our demonstrated that ARL5A is upregulated in colorectal can- understanding of the complicated molecular pathogenesis cer tissues, promotes proliferation, and is a prognostic of colorectal cancer and delineate new options for colorectal factor in patients with colorectal cancer. These data suggest, cancer treatment.

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miR-202-3p Targets ARL5A in Colorectal Cancer

Figure 6. miR-202-3p represses colorectal cancer cell growth by downregulating ARL5A expression. The cell proliferation rate was determined 96 hours after transfection, and the level of ARL5A protein expression was detected 48 hours after transfection. A, ARL5A knockdown using siRNA signiﬁcantly repressed colorectal cancer cell proliferation; overexpression of miR-202-3p did not suppress cell proliferation in ARL5A-depleted HCT-116 or LoVo cells. B, miR-202-3p knockdown restored the endogenous expression of ARL5A protein and promoted colorectal cancer cell growth, but did not promote cell proliferation in ARL5A-depleted HCT-116 or LoVo cells. C and D, overexpression of ARL5A (ORF without 30 UTR) signiﬁcantly promoted cell growth and abrogated the miR-202-3p–induced cell growth inhibition in HCT-116 and LoVo cells. The miR-202-3p was overexpressed by mimics transfection (C) or lentivirus (lenti) transduction. To get ARL5A ORF and/or miR-202 overexpressed stable cells, HCT-116 or LoVo cells were infected with Lenti-ARL5A ORF and/or Lenti-miR-202 virus particles (D).

Disclosure of Potential Conﬂicts of Interest Acknowledgments No potential conﬂicts of interest were disclosed. The authors thank Didier Trono (School of Life Sciences, Ecole Poly- technique Federale de Lausanne, 1015 Lausanne, Switzerland) for the Authors' Contributions generous gift of the psPAX2 and pMD2.G plasmids. Conception and design: Z. Huang, X. He, X. Du Development of methodology: Z. Huang, W. Guo, S. Ni, L. Wang, Grant Support D. Huang This study was partially supported by grants from the National Natural Acquisition of data (provided animals, acquired and managed Science Foundation of China (81071791, 81000867, and 81272299), the patients, provided facilities, etc.): Q.Wang,Z.Huang,X.Xiao, Science and Technology Commission of Shanghai Municipality (10DJ1 L. Wang, J. Zhang 400501), and the Medical Key Professionals Program of Jiangsu Province Analysis and interpretation of data (e.g., statistical analysis, biosta- (RC2011031). tistics, computational analysis): Q. Wang, Z. Huang, W. Guo, Q. Xu The costs of publication of this article were defrayed in part by the payment Writing, review, and/or revision of the manuscript: Q. Wang, Z. Huang, of page charges. This article must therefore be hereby marked advertisement in S. Ni, X. He, X. Du accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Administrative, technical, or material support (i.e., reporting or orga- nizing data, constructing databases): Q. Wang, X. Xiao, C. Tan, R. Zha, W. Sheng Received April 16, 2013; revised October 22, 2013; accepted October 23, Study supervision: X. He 2013; published OnlineFirst December 10, 2013.

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MicroRNA-202-3p Inhibits Cell Proliferation by Targeting ADP-Ribosylation Factor-like 5A in Human Colorectal Carcinoma

Qifeng Wang, Zhaohui Huang, Weijie Guo, et al.

Clin Cancer Res 2014;20:1146-1157. Published OnlineFirst December 10, 2013.

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