miR-204-5pInhibits Proliferation andInvasion andEnhances ... · for colorectal cancer. Clin Cancer Res; 20(23); 6187–99. 2014 AACR. Introduction Colorectal cancer is the third most

Biology of Human Tumors

miR-204-5p Inhibits Proliferation and Invasion and EnhancesChemotherapeutic Sensitivity of Colorectal Cancer Cells byDownregulating RAB22A

Yuan Yin1, Binbin Zhang1,2, Weili Wang3, Bojian Fei3, Chao Quan1, Jiwei Zhang1, Mingxu Song1, Zehua Bian1,Qifeng Wang4, Shujuan Ni4, Yaling Hu1, Yong Mao1, Leyuan Zhou1, Yugang Wang5, Jian Yu6, Xiang Du4,Dong Hua1, and Zhaohui Huang1

AbstractPurpose: miR-204-5p was found to be downregulated in colorectal cancer tissues in our preliminary

microarray analyses. However, the function of miR-204-5p in colorectal cancer remains unknown. We

therefore investigated the role, mechanism, and clinical significance of miR-204-5p in colorectal cancer

development and progression.

Experimental Design:Wemeasured the expression of miR-204-5p and determined its correlation with

patient prognoses. Ectopic expression in colorectal cancer cells, xenografts, and pulmonary metastasis

models was used to evaluate the effects of miR-204-5p on proliferation, migration, and chemotherapy

sensitivity. Luciferase assay and Western blotting were performed to validate the potential targets of miR-

204-5p after the preliminary screening by a microarray analysis and computer-aided algorithms.

Results: miR-204-5p is frequently downregulated in colorectal cancer tissues, and survival analysis

showed that the downregulation of miR-204-5p in colorectal cancer was associated with poor prognoses.

Ectopic miR-204-5p expression repressed colorectal cancer cell growth both in vitro and in vivo. Moreover,

restoring miR-204-5p expression inhibited colorectal cancer migration and invasion and promoted tumor

sensitivity to chemotherapy. Mechanistic investigations revealed that RAB22A, a member of the RAS

oncogene family, is a direct functional target of miR-204-5p in colorectal cancer. Furthermore, RAB22A

protein levels in colorectal cancer tissues were frequently increased and negatively associated withmiR-204-

5p levels and survival time.

Conclusions: Our results demonstrate for the first time that miR-204-5p acts as a tumor suppressor in

colorectal cancer through inhibitingRAB22A and revealRAB22A to be anewoncogene andprognostic factor

for colorectal cancer. Clin Cancer Res; 20(23); 6187–99. �2014 AACR.

IntroductionColorectal cancer is the third most common cancer and

the third leading cause of cancer death in women and

men worldwide (1). The development of colorectal can-cer involves multiple factors, including cancer inhibitors(tumor suppressors), and cancer inducers (oncogenes).During the past decade, a type of non–protein-codingRNA molecules known as microRNAs (miRNA) has beenlinked to cancer development by acting as either tumorsuppressors or oncogenes (1, 2). These miRNAs are gen-erally endogenous, noncoding RNAs of 19 to 24 nucleo-tides in length (3). Deregulation of miRNAs has beenimplicated in the development and progression of nearlyall tumor types, including colorectal cancer (2). Gener-ally, miRNAs play critical roles in the negative regulationof gene expression by base pairing to complementary sitesin the 30-untranslated regions (UTR) of their targetmRNAs (4).

In colorectal cancer,manymiRNAs showaberrant expres-sion patterns (5–7), and some have been shown to beinvolved in tumorigenesis by targeting tumor-associatedgenes (7–12). In addition, miRNAs appear to be promisingtumor biomarkers (13, 14). Our previous expression pro-filing data revealed that miR-204-5p (previously known

1Wuxi Oncology Institute, the Affiliated Hospital of Jiangnan University,Wuxi, Jiangsu, China. 2Oncology Institute, the Fourth Affiliated Hospital ofSoochowUniversity,Wuxi, Jiangsu,China. 3Department of SurgicalOncol-ogy, the Affiliated Hospital of Jiangnan University, Wuxi, Jiangsu, China.4Department of Pathology, Fudan University Shanghai Cancer Center,Shanghai, China. 5Department of Urology, University of Michigan Com-prehensive Cancer Center, Ann Arbor, Michigan. 6State Key Laboratory ofOncogenes and Related Genes, Shanghai Cancer Institute, Shanghai JiaoTong University School of Medicine, Shanghai, China.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Y. Yin and B. Zhang contributed equally to this article.

Corresponding Author: Zhaohui Huang, Wuxi Oncology Institute, theAffiliated Hospital of Jiangnan University, 200 Huihe Road, Wuxi, Jiangsu214062, China. Phone: 86-51088682087; Fax: 86-51088682087; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-14-1030

�2014 American Association for Cancer Research.

ClinicalCancer

Research

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as miR-204 before mirBase release 19.0) was one of themiRNAs that wasmost significantly downregulated in colo-rectal cancer tissues compared with adjacent noncanceroustissues (NCT; ref. 7). Several studies have also shown thatmiR-204 is frequently downregulated in other cancers,suggesting a common role of miR-204 in human tumori-genesis (15–23). However, the role of miR-204-5p in colo-rectal cancer remains undefined.

In this study, we confirmed that the miR-204-5pexpression is significantly decreased in colorectal cancerusing an expanded cohort. This decrease was attributedto the hypermethylation of its promoter. The downregu-lation of miR-204-5p in colorectal cancer was associatedwith poor patient prognosis. Functional analyses showedthat the reintroduction of miR-204-5p into colorectalcancer cells markedly suppresses cell proliferation andinvasion both in vitro and in vivo and increases thesensitivity of colorectal cancer cells to oxaliplatin (OXL),5-fluorouracil (5-FU), and cisplatin (DDP). Furthermechanistic investigations revealed that miR-204-5pinhibits colorectal cancer development and progressionby directly targeting RAB22A (a member of the RASoncogene family), which appears to be a new prognosticfactor in colorectal cancer. Collectively, these data high-light the tumor-suppressive role of miR-204-5p and theconsequences of its inactivation on the oncogenic activityof RAB22A. These factors might contribute, at least inpart, to the poor response of colorectal cancer to con-ventional chemotherapy.

Materials and MethodsCell lines and clinical samples

Human colorectal cancer cell lines, including Caco2,DLD1, HCT8, HCT116, HT29, LoVo, SW480, and SW620,

were purchased from ATCC. HCT116 and LoVo cellsusing for functional and mechanism studies in this studywere tested and authenticated using short tandem repeat(STR) assay by Genewiz. All of the media (Hyclone) weresupplemented with 10% FBS (Gibco). The cells wereincubated under the conditions recommended by ATCCas we described previously (7).

A total of 272 paired colorectal cancer and NCT tissueswere obtained from Fudan University Shanghai CancerCenter (Shanghai, China) and the Affiliated Hospital ofJiangnan University (Jiangsu, China; Supplementary TableS1). All of the samples were gathered according to theInstitutional Review Board–approved protocol and thewritten informed consent from each patient.

DNA and RNA isolationGenomicDNAwas isolated using theGeneral AllgGenKit

(Cwbio) according to the manufacturer’s protocol. TotalRNA was extracted using TRIzol reagent (Invitrogen). Theconcentrations of DNA and RNA were determined using aNanoDrop 2000 (Thermo).

Real-time qRT-PCRcDNA was synthesized using the PrimeScript RT Reagent

Kit (TaKaRa). QRT-PCR analyses were conducted to quan-titate the relative mRNA expression using SYBR Premix ExTaq (TaKaRa), with b-actin as an internal control. Stem-loop qRT-PCR assays using TaqMan miRNA probes(Applied Biosystems) were performed to quantify the levelsof the mature miRNAs. The reactions were incubated in 96-or 384-well optical plates at 95�C for 10 minutes, followedby 40 cycles of 95�C for 15 seconds and 60�C for 1 minute.After the reactions, the cycle threshold (Ct) data weredetermined using default threshold settings, and the meanCt was determined from the duplicate PCRs. A comparativeDCt method was used to compare each condition with thecontrols, and the values are expressed as 2�DCt. The relativelevels of miRNAs were normalized to the levels of U6, aubiquitously expressed small nuclear RNA. The relativeDNA copy numbers were determined as described previ-ously (24). All of the primers used are listed in Supplemen-tary Table S2.

Promoter methylation analysisThe gDNAwas bisulfite-modified as previously described

(25). Bisulfite-treated DNA was amplified with bisulfitesequencing PCR (BSP) primers. The purified BSP productswere directly sequenced and the methylation status of eachCpG site was determined as described previously (26). Toperformmethylation-specific PCR (MSP) analysis, bisulfite-treated DNA was subjected to PCR using methylation-spe-cific primers, and the PCR products were analyzed by 2%agarose gel electrophoresis. BSP and MSP primers weredesigned using MethPrimer (Supplementary Table S2 andSupplementary Fig. S1A; ref. 27). HCT116 and LoVo Cellswere treated with 10 mmol/L 5-aza-dC (Sigma), a demethy-lating agent, for 72 hours, and then subjected to the quan-titation of miR-204-5p.

Translational RelevanceMicroRNAs (miRNA) expression aberration has been

observed in almost all human cancers, thus offering agroup of potential diagnostic markers, prognosticfactors, and therapeutic targets in tumorigenesis. Wenow present data showing that miR-204-5p, which isfrequently downregulated in colorectal carcinoma tis-sues, functions as a tumor suppressor in colorectalcarcinoma. Ectopic miR-204-5p expression repressedcolorectal carcinoma cell growth, migration, and inva-sion and promoted tumor sensitivity to chemotherapy.RAB22A, a member of the RAS oncogene family, wasidentified as a direct functional target of miR-204-5p incolorectal carcinoma. Low miR-204-5p and highRAB22A expression are associated with poor prognosesin patients with colorectal carcinoma, suggesting thatthey could serve as valuable prognostic factors for sur-vival. In addition, therapeutic miR-204-5p overexpres-sion could be useful for the treatment of colorectalcarcinoma by targeting RAB22A.

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Clin Cancer Res; 20(23) December 1, 2014 Clinical Cancer Research6188




Plasmid and siRNAThe human pri-miR-204 sequence was amplified from

normal human gDNA and cloned into the lentivirusexpression vector pWPXL to generate pWPXL-miR-204.The 30UTRs of potential miR-204-5p target geneswere amplified from gDNA using PrimerSTAR Premix(TaKaRa). The amplified 30UTRs were then cloned intothe region directly downstream of a CMV promoter–driven firefly luciferase cassette in a pcDNA3.0 vector(p-Luc). The mutant 30UTR of RAB22A, which carriedthe mutated sequence in the complementary site for theseed region of miR-204-5p, was constructed on the basisof the p-Luc-RAB22A 30UTR-WT plasmid by overlap-extension PCR. The open reading frame (ORF) of RAB22Awas amplified and cloned into pWPXL. Duplex siRNAswere purchased from GenePharma.

Lentivirus production and transductionThe pWPXL, pWPXL-miR-204, and pWPXL-RAB22A

plasmids were cotransfected into HEK-293T cells alongwith the packaging plasmid ps-PAX2 and the envelopeplasmid pMD2G using Lipofectamine 2000 (Invitrogen).Virus particles were harvested 48 hours after cotransfec-tion. Then, the particles were individually used to infectHCT116 and LoVo cells. The cells were then harvested at 3days after infection for Western blotting and qRT-PCRvalidation.

Cell proliferation assay and colony formation assayFor the cell proliferation assay, 1,000 cells were plated

in 96-well plates, incubated and detected with the CellCounting Kit-8 (CCK-8, Dojindo) according to the man-ufacturer’s instructions. For the colony formation assay,1,000 cells were placed in each well of a 6-well plate andmaintained in media containing 10% FBS for 2 weeks.The colonies were fixed with methanol and stained with0.1% crystal violet in 20% methanol for 20 minutes. Thenumber of colonies was counted using an invertedmicroscope.

Cell migration and invasion assayThe migration ability of colorectal cancer cells was tested

in a Transwell Boyden Chamber (8-mm pore size, BDBiosciences) as previously described (12). For the cell inva-sion assay, the polycarbonate membranes of the uppercompartment of the chambers were precoatedwith amatrixgel.

Assessment of chemotherapy sensitivity and apoptosisLoVo or HCT116 cells stably expressing miR-204 or

the control vector were treated with oxaliplatin (range,0–24 mg/mL), and cell inhibition was then assessed byCCK-8 assay. The half-maximal inhibitory concentration(IC50) was calculated. For the apoptosis analysis, colorectalcancer cells were treated with 5 mg/mL oxaliplatin for 48hours. The cells were then harvested and subjected toapoptosis analysis using an Annexin V-FITC and propidiumiodide labeling kit (Invitrogen).

Tumor formation in nude mouseLoVo or HCT116 cells stably expressing miR-204 or the

blank vector were subcutaneously injected into either flankof the same athymicmale BALB/c nudemouse at 5 weeks ofage. The mice were sacrificed after a period of 4 to 6 weeksand examined for the growth of subcutaneous tumors. Forthe in vivo metastasis assays, 2 � 106 LoVo cells stablyexpressing miR-204 or the control vector were suspendedin 100 mL DMEM and were injected into the caudal vein ofeach nude mouse. The nude mice were maintained underspecific pathogen-free conditions in the Experimental Ani-mal Department of Fudan University.

Microarray analysisExpression profiling was performed using an Agilent

human whole genome oligo microarray chip (4 � 44 K;Agilent). A total of 5 � 105 HCT116 cells were seeded in 6-cm2 tissue culture plates and transfected with the miR-204-5p or the negative control (NC) as described above. Afterpropagation for 48 hours, total RNA was extracted for theexpression profiling analyses. The microarray profiling wasperformed as described in our previous work (12), and theresults had been submitted to GEO (GSE59897).

Luciferase reporter assayHEK-293T or HCT116 cells were cultured in 96-well

plates and cotransfected with 50 nmol/L of miR-204-5pmimic (or NC), 50 ng of luciferase reporter, and 10 ng ofpRL-CMV Renilla luciferase reporter using Lipofectamine2000. Forty-eight hours after transfection, the luciferaseactivities were assayed using a luciferase assay kit(Promega).

Western blottingThe levels of RAB22A were analyzed by Western blotting

using a rabbitmonoclonal anti-humanRAB22A antibody ata dilution of 1:1,000 (Abcam). Normalization was per-formed by blotting the same samples with an antibodyagainst b-actin (Abcam).

ImmunohistochemistryTissue microarrays were constructed using paired colo-

rectal cancer and NCT tissues from 2 different colorectalcancer cohorts. Immunohistochemical (IHC) staining wasperformed on 4-mm sections of paraffin-embedded tissuesamples to detect the expression levels of RAB22A protein.In brief, the slides were incubated in RAB22A antibodydiluted to1:200 at 4�Covernight. The subsequent stepswereperformed using the EnVision FLEX High pH 9.0 Visuali-zation System (DAKO).

Statistical analysesThe results are expressed as the mean � SEM. The data

were subjected to Student t tests, the Mann–WhitneyU test,or the Kruskal–Wallis test unless otherwise specified (c2 testor Spearman correlation). The survival curves were plottedaccording to the Kaplan–Meier method, with the log-ranktest applied for comparisons. Cox proportional hazards

miR-204-5p Targets RAB22A in Colorectal Cancer

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regression analysis was used to estimate the HRs and the95% confidence intervals (CI). A value of P < 0.05 wasconsidered to be statistically significant. The SPSS 16.0package (IBM) was used for the statistical analyses.

ResultsDownregulation of miR-204-5p predicts poor survivalin colorectal cancer

miR-204-5p emerged as one of the most prominentlydownregulatedmiRNAs in colorectal cancer in our previousmicroarray data (7). To validate the result, we examined themiR-204-5p levels in an expanded colorectal cancer cohortusing qRT-PCR. Consistent with the microarray data, miR-

204-5p expression was frequently downregulated in colo-rectal cancer tissues compared with the correspondingNCTs (P < 0.0001, Fig. 1A and B). These cases were thendivided into 3 groups (high: the 25% highest, low: the 25%lowest, or not significantly changed) for survival analysisbased on their miR-204-5p levels. We found that miR-204-5p expression was significantly associated with overallsurvival, and patients with low miR-204-5p expression hadshorter overall survival than patients with highmiR-204-5pexpression (P ¼ 0.017, Fig. 1C). After adjusting for age,gender, tumor size, stage, and grading,multivariate analysesconfirmed thatmiR-204-5p expressionwas an independentprognostic factor for colorectal cancer survival (HR, 0.303;

Figure 1. miR-204-5p expressionwas frequently downregulated in colorectal cancer (CRC). A andB,miR-204-5p expressionwas examined by qRT-PCR in 80paired CRC and adjacent NCTs. miR-204-5p expression was markedly downregulated in tumor tissues compared with the corresponding NCTs. C, survivalanalysis based on the expression level of miR-204-5p. miR-204-5p expression was examined in 140 CRCs, and these cases were then divided into3 groups (high, low, or not significantly changed groups) based on their miR-204-5p levels in tumors. D, survival analysis based on the data from the TCGAdatabase. miR-204-5p expression was positively correlated with the overall survival.

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95% CI, 0.147–0.622; P ¼ 0.001). No significant associa-tion was found between miR-204-5p expression in colorec-tal cancer and tumor size, location, stage, and grading.To further evaluate the impact of miR-204-5p on prog-

nosis, we analyzed the miR-204-5p expression data fromThe Cancer Genome Atlas (TCGA) database. A total of 159colorectal cancer s were selected for further analysis. Asignificant positive correlation was found between miR-204-5p levels and survival time (P ¼ 0.007, Fig. 1D). Afteradjusting for age, gender, T stage, N stage, TNM stage, andvascular/lymphovascular invasion, Cox multivariate anal-yses showed that miR-204-5p expression was an indepen-dent prognostic factor for colorectal cancer survival. Tumorswith high miR-204-5p expression levels (>median) exhib-ited a decreased risk of death (HR, 0.182; 95% CI, 0.067–0.499, P ¼ 0.001).

Downregulation of miR-204-5p caused by promoterhypermethylation in colorectal cancer cellsThe coding sequence of miR-204 is located within an

intronic region of the TRPM3 gene. TRPM3 and miR-204share the same transcriptional regulatory motif and arederived from a single transcription unit (28, 29). Previousstudy reported that the promoter CpG islands of TRPM3were hypermethylated in glioma (21). We next sought toinvestigate whether the promoter hypermethylation alsooccurred in colorectal cancer andwhether it was responsiblefor the downregulation of miR-204-5p. The BSP resultsshowed that most of the 8 colorectal cancer cell lines werehypermethylated in the TPRM3 promoter (SupplementaryFig. S1B). The expression of miR-204-5p was linearly cor-related with that of TRPM3 in these colorectal cancer celllines (Supplementary Fig. S1C–S1E). We then examinedpromoter methylation of TRPM3 in colorectal cancer tis-sues. The results showed that the methylation ratio ofTRPM3 in colorectal cancer tissues (23 of 28, 82.1%) wassignificantly higher than that inNCTs (14 of 28, 50.0%; P¼0.011), and TRPM3 was unmethylated in 5 normal colonicepithelia samples. To further examine the effect of DNAmethylation on miR-204 expression, HCT116 and LoVocells were treated with the DNAmethyltransferase inhibitor5-aza-dC, and the expression of miR-204-5p in the treatedcells was significantly restored (Supplementary Fig. S1F).Together, these data demonstrate that promoter hyper-methylation is an important mechanism for silencingmiR-204 expression in colorectal cancer.Copy number variants (CNV) also cause aberrant expres-

sion of genes, including miRNAs (12). We analyzed theCNV of miR-204 in 48 paired colorectal cancer s and NCTsand observed no significant changes between them (datanot shown).

miR-204-5p inhibits colorectal cancer cell proliferationin vitro and in vivoCell proliferation assays revealed that miR-204-5p over-

expression significantly reduced the growth rates ofHCT116 and LoVo cells (P < 0.01, Fig. 2A), whereas silenc-ing miR-204-5p expression significantly promoted the

growth of colorectal cancer cells (P < 0.01, Fig. 2B). Colonyformation assays confirmed the proliferation-repressingfunction of miR-204 in colorectal cancer cells (P < 0.01,Fig. 2C). A tumor formation assay in a nude mouse modelwas performed to evaluate the in vivo function of miR-204and revealed that miR-204 overexpression significantlyrepressed the tumorigenesis of colorectal cancer cells com-pared with the vector control (P < 0.01, Fig. 2D). Becausepri-miR-204 couldproduce bothmiR-204-3p andmiR-204-5p, we checked the expression and function of miR-204-3pin colorectal cancer cells. The expression of miR-204-3p incolorectal cancer cells and tissues was much lower thanthat of miR-204-5p, and overexpression of miR-204-3pshowedno significant effect on colorectal cancer cell growth(Supplementary Fig. S2A–S2D) and migration (data notshown). Furthermore, we inhibited miR-204-3p in LoVoand HCT116 cells stably expressing miR-204 using anti-miR-204-3p and did not observe significant effect of miR-204-3p on the miR-204-5p–mediated cell growth (Supple-mentary Fig. S2E and S2F). Collectively, these data indicatethatmiR-204-5p has a growth-suppressive function in colo-rectal cancer.

miR-204-5p inhibits colorectal cancer cell migrationand invasion in vitro and in vivo

To determine whether miR-204-5p could modulate themetastasis ability of colorectal cancer, we first examined theeffect ofmiR-204-5pon colorectal cancer cellmigration andinvasion using a Transwell assay. As shown in Fig. 3A, miR-204-5p–transfected cells exhibited considerably slowermigration and invasion compared with the control. Wethen assessed the impact of miR-204-5p on in vivo metas-tasis using a mouse model of pulmonary metastasis. Over-expression ofmiR-204-5p significantly reduced the numberof lung metastasis sites (Fig. 3B), further validating that theinvasive behavior of colorectal cancer could be suppressedby miR-204-5p.

miR-204-5p increases sensitivity of colorectal cancercells to chemotherapy

Because low miR-204-5p expression was significantlyassociated with poor patient survival and increased cellgrowth, we assessed the potential effect of miR-204-5p onthe sensitivity of colorectal cancer cells to oxaliplatin, one ofthe most commonly used drugs in colorectal cancer treat-ment. The results revealed that the IC50 of miR-204-5p–overexpressed colorectal cancer cells was significantly lowerthan that of the control (LoVo: 2.961 vs. 1.501 mg/mL;HCT116: 2.749 vs. 1.012 mg/mL; P < 0.01, Fig. 3C), suggest-ing that miR-204-5p strongly increases the sensitivity ofcolorectal cancer cells to oxaliplatin. To further confirm thatmiR-204-5p enhances oxaliplatin-induced cytotoxicity, weexamined the apoptosis of oxaliplatin-treated colorectalcancer cells. Following treatment with 5 mg/mL of oxalipla-tin for 48 hours, significantly increased apoptosis ratioswere observed in miR-204-5p–overexpressed HCT116 andLoVo cells, relative to the controls (P < 0.01, Fig. 3D).






To investigate whethermiR-204-5p overexpression couldaffect apoptosis in colorectal cancer cells exposed to che-motherapy agents other than oxaliplatin, we exposed colo-rectal cancer cells to 5-FU and DDP, which are also com-monly used in colorectal cancer treatment. Following treat-ment with 5-FU or DDP, significantly increased apoptosiswas observed in miR-204-overexpressing colorectal cancercells (Supplementary Fig. S3). Taken together, these obser-vations suggest that miR-204-5p, through enhanced apo-ptosis, significantly increases the sensitivity of colorectalcancer cells to chemotherapy.

Identification of RAB22A as a target of miR-204-5pGenomic-wide expression profiling was first performed

in miR-204-5p- or NC-transfected HCT116 cells using a

microarray (GSE59897). A total of 1,367 downregulatedtranscripts (>2-fold change) were identified in miR-204-5p–transfected cells compared with the control (Supple-mentary Table S3). Using 2 computer-aided algorithms,TargetScan and PicTar, we identified 123 genes that arepotentially regulated by miR-204-5p. By comparing all ofthedownregulated geneswith the candidate genespredictedby the programs, 15 downregulated genes were selected(Fig. 4A). Because it is generally accepted that miRNAs exerttheir function by inhibiting the expression of their targetgenes, miR-204-5pmay execute its tumor-suppressive func-tion by downregulating targets that normally have tumor-promoting functions. On the basis of this rationale, 4candidate genes (BCL2, CCPG1, KLF12, and RAB22A) wereselected from the 15 genes. Luciferase reporter assays were

Figure 2. miR-204-5p inhibits colorectal cancer cell proliferation in vitro and in vivo. A and B, miR-204-5p overexpression repressed the proliferation of LoVoand HCT116 cells (A), whereas knockdown of miR-204-5p enhanced the cell growth rate of LoVo and HCT116 cells (B). The CCK-8 assay was used todetermine the cell growth rate (�, P < 0.05; ��, P < 0.01). C, miR-204-overexpressed LoVo and HCT116 cells exhibited decreased colony formation ratescompared with the control cells. D, the effect of miR-204 on tumor formation in a nude mouse xenograft model. LoVo (2 � 106) and HCT116 (1 � 106) cellsstably expressing miR-204 or the control were injected subcutaneously into the right flank of each nudemouse. The growth and tumor weight of themiR-204group were significantly decreased compared with the control. NC, negative control.

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then conducted to determine the influence of miR-204-5pon the expression of these 4 genes. The results revealed thatmiR-204-5p could inhibit the expression of the reportergene in recombinant plasmids containing the 30UTRs ofRAB22A and BCL2, particularly RAB22A (Fig. 4A). To testwhether RAB22A is a direct target of miR-204-5p, the wild-type and mutant RAB22A 30UTRs were independentlycloned into p-Luc (Fig. 4B). As shown in Fig. 4C, themutantRAB22A 30UTR was completely refractory to the miR-204-5p–mediated luciferase reporter repression in both HEK-293T and HCT116 cells. In concordance with these results,RAB22A protein expression was significantly decreased inmiR-204-5p–overexpressed colorectal cancer cells andenhanced in miR-204-5p–depleted cells (Fig. 4D). In addi-tion, although about 20% decreased luciferase activity wasobserved in the recombinant plasmid containing the BCL230UTR (Fig. 4A), we did not observed significant down-regulation of BCL2 protein in miR-204-5p–overexpressedcolorectal cancer cells (data not shown).

RAB22A expression is increased in colorectal cancerand inversely correlated with miR-204-5p expressionand survivalTo further study the relationship between miR-204-5p

and RAB22A in human colorectal cancer, we assessed theRAB22A protein expression in 140 paired colorectal cancerand NCT samples using IHC (Supplementary Fig. S4A). Asindicated in Fig. 5A and B, 93 of 140 (66.4%) tumorsshowed increased RAB22A expression compared withpaired NCTs. The RAB22A protein levels in the colorectalcancer tissues were inversely correlated with the miR-204-5p levels (P < 0.001, Fig. 5C), suggesting that miR-204-5pacts as a regulator of RAB22A expression in clinical colo-rectal cancer tumors.Survival analyses revealed that increased RAB22A protein

levels (score 2 or 3) were associated with shorter survivaltime (P ¼ 0.019, Fig. 5D). After adjusting for age, gender,nodal status, tumor differentiation, tumor size, and tumorstage, multivariate analyses showed that RAB22A expres-sion was an independent risk factor for survival. Tumorswith higher RAB22A expression presented a higher risk ofdeath (HR, 4.229; 95% CI, 2.018–8.861, P < 0.0001). Tofurther validate these results, the impact of RAB22A onprognosis was evaluated in an independent test cohort.RAB22A detection was available in 132 tumors and 106NCTs, and more than half of tumors (58.5%) showedincreased RAB22A expression compared with paired NCTs(Supplementary Fig. S4B). Thenegative correlationbetweenRAB22A expression and survival time was confirmed usingboth univariate (P ¼ 0.002, Fig. 5D) and multivariateanalyses (HR, 2.695; 95% CI, 1.258–5.777, P ¼ 0.011).Collectively, RAB22A appears to be a new prognostic factorfor colorectal cancer.

Role of miR-204-5p in tumorigenesis via directlytargeting RAB22ATo determine the functional significance of RAB22A in

themiR-204-5p–induced phenotype, we performed a series

of restoration assays usingHCT116 and LoVo cells.We usedsiRNA to knockdown RAB22A expression, and a constructcontaining theRAB22AORF to increase RAB22A expression(Supplementary Fig. S5A–S5C). As for the cell proliferationassay, siRNA-mediated RAB22A silencing could phenocopythe proliferation-repressing effect of miR-204-5p, whereasanti-miR-204-5p could not restore cell proliferation inRAB22A-depleted colorectal cancer cells (Fig. 6A). Further-more, we revealed that RAB22A ORF overexpression couldsignificantly abrogate the inhibitory effect of miR-204-5pon cell proliferation (Fig. 6B). Likewise, RAB22A overex-pression could restore miR-204-5p–induced apoptosis andmigration-repression in colorectal cancer cells (Fig. 6C andD). Taken together, these results imply that miR-204-5pexerts tumor-suppressive function in colorectal cancer viadirectly targeting RAB22A.

DiscussionAs important gene regulators, miRNAs are predicted to

regulate more than 60% of human protein-coding genes(30). According to the latest version of miRBase (Release20: June 2013), 2,578 human mature miRNAs have beenannotated; however, the functions and molecularmechanisms of most of these miRNAs remain unknown.

We and other groups have shown that miR-204 isdownregulated in multiple human cancers (7, 15–23).CpG methylation represents an important mechanismresponsible for the inactivation of genes, including miR-NAs. miR-204 is an intronic miRNA located in the TRPM3gene. We observed that the promoter region of TRPM3/miR-204 is frequently hypermethylated in colorectal can-cer cell lines and colorectal cancer tissues. Aberrant pro-moter methylation of TRPM3/miR-204 was also observedin NCT tissues but not in normal colonic epithelia,suggesting that TRPM3/miR-204 methylation is likely tobe an early event in colorectal cancer tumorigenesis.Expression of miR-204-5p is in proportion to that ofTRPM3 in colorectal cancer cells, suggesting that miR-204 and TRPM3 share common regulatory mechanisms,consistent with previous reports (21, 28). In addition,CNV also results in aberrant expression of genes, includ-ing miRNAs (12). Imam and colleagues reported that the9q21.12 chromosomal region containing miR-204 isfrequently lost in ovarian cancers, breast cancers, andpediatric renal tumors (31). We observed no significantchanges in the copy numbers of the miR-204 genebetween the colorectal cancer and NCT samples, suggest-ing that CNV may not be a key mechanism leading to thedownregulation of miR-204. These findings must beconfirmed in large colorectal cancer cohorts.

miR-204 has been reported to function as a tumor sup-pressor in a variety of human cancers through differentmechanisms (15–23), suggesting its extensive function intumorigenesis. For example, miR-204 inhibited tumorgrowth in renal clear cell carcinoma (17) and pancreaticcancer (32) and suppressed invasion in endometrial cancer(23), glioma (21), gastric cancer (16), intrahepatic






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cholangiocarcinoma (16), and head and neck tumor (18).In addition, miR-204 could affect chemoresistance in neu-roblastoma and gastric cancer cells by targeting BCL2(22, 33). Our data showed that miR-204-5p inhibited cellproliferation and metastasis of colorectal cancer both invitro and in vivo. In addition, miR-204-5p enhanced apo-ptosis in colorectal cancer and sensitized colorectal cancercells to multiple drugs. These data indicate a key role formiR-204-5p in colorectal cancer tumorigenesis and itspotential application for colorectal cancer treatment. Pre-vious reports have shown the involvement of miR-204-5pin epithelial-to-mesenchymal transition (EMT) in gastriccancer and intrahepatic cholangiocarcinoma cells (16).Interestingly, we observed that the overexpression of

miR-204-5p inhibited EMT in LoVo cells but not inHCT116cells (data not shown), suggesting that the involvement ofmiR-204-5p in EMT of colorectal cancer may not be ageneral phenomenon.

In the subsequent mechanistic study, we demonstratedthat miR-204-5p directly targets RAB22A to inhibit prolif-eration and invasion and enhance apoptosis in colorectalcancer. In miR-204-5p–transfected cells, ectopic RAB22Aexpression can rescue the proliferation ability attenuated bymiR-204-5p, whereas RNAi-mediated knockdown ofRAB22A phenocopied the proliferation-promoting effectof miR-204-5p. Li and colleagues performed preliminaryluciferase reporter assay to validate the targeting of miR-204-5p to the 30UTRs of RAB22A under stress conditions in

Figure 4. Screening for candidate target genes of miR-204-5p in colorectal cancer. A, initial screening of miR-204-5p target genes using a microarray assay,bioinformatics predictions, and the luciferase reporter assay. Four downregulated genes (BCL2, CCPG1, KLF12, and RAB22A) were selected from the 15genes in the initial screening based on the functional analysis of these genes, and their 30UTRswere assessed using the luciferase reporter assay. Luciferaseactivity was determined 48 hours after transfection and normalized toRenilla luciferase activity. B, the putative miR-204-5p binding sequence in theRAB22A30UTR. Amutation was generated in the site complementary to the miR-204-5p seed region of theRAB22A 30UTR, as indicated. C, analyses of the luciferaseactivity of the luciferase reporter plasmids containing eitherwild-type (WT) ormutant-type (MT)RAB22A 30UTRs inHEK-293TandHCT116 cells. D, the proteinlevels of RAB22A were determined by Western blotting in LoVo and HCT116 cells transfected with miR-204-5p mimic, miR-204-5p inhibitor, or thecorresponding negative control (NC). b-Actin served as an internal control.

Figure 3. Ectopic miR-204-5p expression inhibits cell migration and invasion and increases the sensitivity of colorectal cancer cells to oxaliplatin. A, miR-204-5poverexpression repressed themigration and invasionof LoVoandHCT116cells (P<0.01). B, the effect ofmiR-204on tumormetastasis in a lungmetastasismodel of nude mouse. LoVo cells stably expressing miR-204 or the control (2 � 106) were injected into the caudal vein of each nude mouse, and lungmetastasis was detected by hematoxylin and eosin staining. White arrows show the sites with metastasis. Overexpression of miR-204 significantly reducedthe number of lungmetastases (P < 0.0001). C, miR-204-5p overexpression increases the sensitivity of LoVo andHCT116 cells to oxaliplatin. The IC50 ofmiR-204-5p–overexpressed colorectal cancer cells was significantly lower than that of the control (LoVo: 2.961 vs. 1.501 mg/mL; HCT116: 2.749 vs. 1.012 mg/mL,P < 0.01). D,miR-204-5p overexpression enhanced oxaliplatin-induced apoptosis. LoVo andHCT116 cells were treatedwith 5 mg/mL oxaliplatin for 48 hours.






Figure 5. RAB22A protein levels were overexpressed in colorectal cancer and inversely correlated with miR-204-5p levels. A, IHC staining of RAB22A in 140tumor tissues and adjacent NCTs. Brown cytoplasmic RAB22A staining was observed in colorectal cancer cells but was nearly absent in normal colonicepithelia. B, RAB22A protein expression was frequently increased in the tumor tissues compared with the matched NCTs (66.4%). C, the expressionlevels of RAB22A were negatively correlated with the miR-204-5p expression levels in the colorectal cancer tissues (P¼ 0.0005). �, the median value of miR-204-5p levels in 140 colorectal cancer tissues was used to divide these cases into 2 groups with low or high miR-204-5p levels, respectively. D, survivalanalysis based on the expression levels of RAB22A in 2 different CRC cohorts. The groups were ranked according to the RAB22A staining intensity.The percentage of overall survival in lowRAB22Aexpression (scored 0or 1)was significantly higher than that of patientswith highRAB22Aexpression (scored2 or 3; P ¼ 0.019 for the training set, P ¼ 0.002 for the testing set).

Yin et al.





human trabecular meshwork cells without further study(34).RAB proteins, includingmore than 60members inmam-

mals, constitute a Ras superfamily of GTPases (35). They areusually activated by binding GTP in the transport vesiclesand then hydrolysed to generate GDP-bound RABs aftermembrane fusion (36). Recent data suggest an emergingrole forRABGTPases inhuman cancer (37).RAB22A is a lessstudied member of the RAB family that plays a role in theendocytic pathway (38). Although RAB22A has recentlybeen reported to be upregulated in hepatocellular carcino-ma, cholangiohepatoma (39), and melanoma (40), little isknown about its role in human tumorigenesis. Here, werevealed that increased RAB22A expression in colorectal

cancer predict poor prognosis and showed that RAB22Aoverexpression could significantly increase cell growth,migration, and resistance to chemotherapy-induced apo-ptosis. These data suggest, for the first time, thatRAB22A is apotential new oncogene and prognostic factor for colorectalcancer. The detailed role andmechanism by which RAB22Apromotes colorectal cancer development and progressionshould be investigated in future work.

Resistance to conventional chemotherapy remains a sig-nificant obstacle in the successful treatment of colorectalcancer. Most chemotherapeutic agents, including oxalipla-tin, 5-FU, DDP, and adriamycin, ultimately kill tumor cellsby inducing apoptosis, regardless of distinct antitumormechanisms (41). miR-204-5p could promote the

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Figure 6. miR-204-5p represses colorectal cancer cell growth and migration by targeting RAB22A in colorectal cancer. A, knockdown of RAB22A by siRNAsignificantly repressed colorectal cancer cell proliferation, and upregulation of miR-204-5p did not suppress cell proliferation in RAB22A-depletedLoVo or HCT116 cells. In contrast, miR-204-5p silencing promoted cell growth but did not promote cell proliferation in RAB22A-depleted LoVo or HCT116cells. B, upregulation of RAB22A promoted cell growth and abrogated miR-204-5p–induced growth inhibition in LoVo and HCT116 cells. C, upregulation ofRAB22A reduced oxaliplatin-induced apoptosis and abrogatedmiR-204-5p–enhanced apoptosis in LoVo and HCT116 cells. D, upregulation of the RAB22Apromoted cell migration and abrogatedmiR-204-5p–inducedmigration inhibition in LoVo and HCT116 cells. Significant differences are indicated (�, P < 0.05;��, P < 0.01; ��, P < 0.001).






sensitivity of colorectal cancer cells to the most commonlyused drugs for colorectal cancer by targeting RAB22A, sug-gesting a central role for miR-204-5p/RAB22A signaling inapoptosis regulation. This pathway could be a promisingnew target for preventive and therapeutic strategies of colo-rectal cancer.

In conclusion, we have identified an important tumor-suppressive miRNA, miR-204-5p, that is frequently down-regulated inhuman colorectal cancer.miR-204-5pplays keyroles in colorectal cancer development and progression, byrepressing colorectal cancer cell growth, migration, andinvasion as well as by promoting colorectal cancer cellsensitivity to chemotherapeutic drugs through directly tar-getingRAB22A.We alsodemonstrated thatmiR-204-5p andRAB22A are potential prognostic factors for colorectal can-cer. Thus, our study demonstrates the importance of miR-204-5p/RAB22A signaling in colorectal cancer tumorigen-esis and suggests that targeting the signalingmay represent anew therapeutic approach for human colorectal cancer.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: Y. Yin, Y. Wang, D. Hua, Z. HuangDevelopment of methodology: Y. Yin, J. Zhang, Q. Wang, Y. WangAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): Y. Yin, B. Zhang,W.Wang, B. Fei, J. Zhang, Y. Hu,Y. Mao, L. Zhou, D. HuaAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): Y. Yin, B. Fei, C. Quan, Z. Bian, Y. Mao,Y. Wang, X. Du, Z. HuangWriting, review, and/or revision of the manuscript: Y. Yin, W. Wang,Z. HuangAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): Y. Yin, B. Zhang, J. Zhang, M. Song,Y. Wang, J. YuStudy supervision: Y. Yin, S. Ni, Y. Wang, X. Du, D. Hua, Z. Huang

Grant SupportThis study was partially supported by grants from the National Natural

Science Foundation of China (nos. 81000867, 81272299, 81372368, and81301784) and the Medical Key Professionals Program of Jiangsu Province(RC2011031).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received May 4, 2014; revised August 16, 2014; accepted September 2,2014; published OnlineFirst October 7, 2014.

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2014;20:6187-6199. Published OnlineFirst October 7, 2014.Clin Cancer Res Yuan Yin, Binbin Zhang, Weili Wang, et al. Downregulating RAB22AChemotherapeutic Sensitivity of Colorectal Cancer Cells by miR-204-5p Inhibits Proliferation and Invasion and Enhances

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miR-204-5pInhibits Proliferation andInvasion andEnhances ... · for colorectal cancer. Clin Cancer Res; 20(23); 6187–99. 2014 AACR. Introduction Colorectal cancer is the third most