microRNA-224 Promotes Cell Proliferation and Tumor Growth ...Human Cancer Biology microRNA-224 Promotes Cell Proliferation and Tumor Growth in Human Colorectal Cancer by Repressing

Human Cancer Biology

microRNA-224 Promotes Cell Proliferation and TumorGrowth in Human Colorectal Cancer by Repressing PHLPP1and PHLPP2

Wen-Ting Liao1,3,4, Ting-Ting Li1,3,4, Zheng-GenWang5, Shu-YangWang1,3,4, Mei-Rong He2, Ya-Ping Ye1,3,4,Lu Qi1,3,4, Yan-Mei Cui1,3,4, Ping Wu1,3,4, Hong-Li Jiao1,3,4, Chi Zhang1,3,4, Yi-Jun Xie1,3,4, Jun-Xian Wang1,3,4,and Yan-Qing Ding1,3,4

AbstractPurpose: To investigate the clinicopathologic significance, role, andmechanism of action of microRNA-

224 (miR-224) in colorectal cancer.

Experimental Design: Real-time PCR was used to quantify miR-224 expression. The association of

miR-224 with the clinicopathologic features and survival was evaluated in 110 colorectal cancer patients.

The role ofmiR-224 in colorectal cancer was investigated using in vitro and in vivo assays. Luciferase reporter

assays were conducted to confirm target gene associations.

Results: miR-224 was overexpressed in colorectal cancer. High-level expression of miR-224 was

significantly associated with an aggressive phenotype and poor prognosis. Overexpression of miR-224

promoted colorectal cancer cell proliferation in vitro and tumor growth in vivo. Specifically, miR-224

accelerated the G1–S phase transition through activation of AKT/FOXO3a signaling, downregulation of

p21Cip1 and p27Kip1, and upregulation of cyclin D1. Moreover, both PH domain leucine-rich-repeats

protein phosphatase 1 (PHLPP1) and PHLPP2, antagonists of PI3K/AKT signaling, were confirmed as bona

fide targets of miR-224. miR-224 directly targeted the 30-untranslated regions of the PHLPP1 and PHLPP2

mRNAs and repressed their expression.

Conclusion: This study reveals functional and mechanistic links between miRNA-224 and the tumor

suppressors PHLPP1 and PHLPP2 in the pathogenesis of colorectal cancer. miR-224 not only plays

important roles in the regulation of cell proliferation and tumor growth in colorectal cancer, but also

has potential as a prognostic marker or therapeutic target for colorectal cancer. Clin Cancer Res; 19(17);

4662–72. �2013 AACR.

IntroductionColorectal cancer is one of the most common causes of

cancer-related deaths worldwide (1). Although progress hasbeen made in diagnostic and therapeutic strategies, theclinical outcome andprognosis of colorectal cancer patientswith advanced stage disease still remains poor (2). Thus,

understanding the molecular mechanisms which regulatethe initiation and progression of colorectal cancer repre-sents a pivotal step in the discovery and exploration of novelmolecular targets, which may help to generate more effec-tive therapies. Similarly to many other solid tumors, theinitiation of colorectal cancer is a multigene and multistepprocess. Activationof oncoproteins or inactivationof tumorsuppressor genes affects multiple aspects of colorectal can-cer tumorigenesis, such as cell proliferation, apoptosis,invasion, andmetastasis (3). However, although numerousmutations are well recognized to be associated with thedevelopment of colorectal cancer, the other genetic andepigenetic alterations responsible for this disease remainlargely unknown.

microRNAs (miRNA) are a class of small-regulatory RNAmolecules that repress protein translation through bindingto the 30-untranslated region (UTR) of their targetmRNAs ina sequence-specific manner. miRNAs are highly conservedacross species and participate in the regulation of a varietyof basic biological processes such as development, cellulardifferentiation, proliferation, apoptosis, and metabolism(4, 5). In recent years, miRNAs have been proven to play

Authors' Affiliations: Departments of 1Pathology and 2Gastroenterology,NanfangHospital, SouthernMedical University; 3Department of Pathology,School of Basic Medical Sciences; 4State Key Laboratory of Oncology inSouthern China, Department of Experimental, Guangzhou, Guangdong;and 5Department of Gastroenterology, The Second Affiliated Hospital,University of South China, HengYang, Hunan, China

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

W.-T. Liao, T.-T. Li, and Z.-G. Wang contributed equally to this work.

Corresponding Author: Y.-Q. Ding, Department of Pathology, NanfangHospital and School of Basic Medical Sciences, Southern Medical Uni-versity, Guangzhou 510515, China. Phone: 86-(20)-6164-2148; Fax: 86-(20)-6164-2148; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-13-0244

�2013 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 19(17) September 1, 20134662

on August 15, 2020. © 2013 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst July 11, 2013; DOI: 10.1158/1078-0432.CCR-13-0244

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important roles in the pathogenesis of human cancer.Several miRNA expression profiling studies have been con-ducted in colorectal cancer, and a series of miRNAs (includ-ing miR-20, miR-21, miR-17-5p, miR-15b, miR-181b, andmiR-191) have been showed to be valuable biomarkersassociatedwith tumor progression and the clinical outcomeof colorectal cancer (6–8). In addition, deregulation ofmiRNAs such as miR-21, miR-34a, miR-135, and miR-200 has been shown to modulate the cell viability, prolif-eration, invasion, and metastasis of colorectal cancer cells(9–12). Nevertheless, the role of miRNAs in the regulationof colorectal cancer–associated genes, and thus the role ofmiRNAs in the pathogenesis of colorectal cancer, remainselusive. Therefore, further extensive investigations arerequired to identify miRNAs that are associated with orinvolved in the initiation and progression of colorectalcancer.Herein, we report that upregulation of miR-224 in colo-

rectal cancer is associated with an aggressive phenotype andpoor patient prognosis. Further investigations revealed thatmiR-224 directly targeted the 30-UTRs of PH domain leu-cine-rich-repeats protein phosphatase 1 (PHLPP1) andPHLPP2, which function as tumor suppressors in colorectalcancer, to suppress the expression of these genes, which inturn promoted the proliferation and tumorigenicity ofcolorectal cancer cells.

Materials and MethodsTissue specimens and cell culturesFor the use of clinical materials for research purposes,

prior approval was obtained from the Southern Medical

University Institutional Board (Guangzhou, China). Allsamples were collected and analyzed with the prior writteninformed consent of the patients.

A total of 230 patients with histologically diagnosed withcolorectal columnar adenocarcinoma and undergoing sur-gical treatment were recruited for this study from theDepartment of General Surgery, NanfangHospital (China).Exclusion criteria included smoking, psychiatric disease,cerebrovascular disease, abnormal electrolytes, anemia, orhypertension. A total of 110 cases of archived colorectalcancer tissue samples were collected between March 2005andOctober 2007. 24.5% (27of 110) of patients with rectalcancer received neoadjuvant therapy and 78.2% (86of 110)of patients received postoperative adjuvant therapy. A totalof 43 pairs of colorectal cancer biopsies and their matchedadjacent normal tissues, and 77 cases of fresh colorectalcancer biopsies were collected between January 2011 andMay 2013. All the tissue biopsies were freshly frozen inliquid nitrogen and stored at �80�C until further use. Themedical records of the patients were reviewed to collect thefollowing clinicopathologic information: age, gender, path-ologic stage, Dukes’ stage, T stage, lymph node metastases,and distant metastasis. Survival data were available for thecohort of 110patients. Themedian follow-up timewas 56.7months (range, 2–87 months).

Two colorectal cancer cell lines SW620 andHCT116werepurchased from American Type Culture Collection CellBiology Collection and were maintained in Department ofPathology, Southern Medical University. Cells were cul-tured in RPMI 1640 (Invitrogen) supplemented with10% FBS (HyClone) and 1% penicillinestreptomycin (Invi-trogen) at 37�C with 5% CO-2.

RNA extraction and real-time quantitative PCRFor miRNA quantification, total miRNA was extracted

from the cells and tissues using the mirVana miRNA Isola-tion Kit (Ambion) according to the manufacturer’s instruc-tions, and then cDNA was synthesized from 5 ng of totalRNA using the Taqman miRNA reverse transcription kit(Applied Biosystems). The expression levels of miR-224were quantified using the miRNA-specific TaqMan MiRNAAssay Kit (Applied Biosystems). The relative miR-224expression levels after normalization to U6 small nuclearRNAwere calculated using 2�[(Ct of miR-224) � (Ct of U6)]. Real-time quantitative PCR for target genes was conducted aspreviously described (13). Primer sequences were summa-rized in Supplementary Table S1.

Plasmids and transfectionTo generate a miR-224 expression vector, a 281 bp geno-

mic fragment covering the region coding for pri-miR-224and its upstream and downstream regions was PCR ampli-fied and cloned into the pLvthm vector (Addgene). The full-length PHLPP1 30-UTR is 1,012 bp long and the PHLPP2 30-UTR is 3,883 bp long. The miR-224 binding site in thePHLPP1 30-UTR is located at 5,917 to 5,924 bp, and7,540 to7,546 bp in the PHLPP2 30-UTR. The region of the humanPHLPP1 30 UTR from5,857 to 6,318bp and PHLPP2 30-UTR

Translational RelevanceDysregulation of microRNA-224 (miR-224) has been

implicated in the pathogenesis of hepatocellular carci-noma and breast cancer. However, the biological func-tions of miR-224 in the development and progression ofcolorectal cancer remains unknown. This study revealedthat miR-224 is overexpressed in colorectal cancer. Highlevel of miR-224 expression is associated with aggressivephenotype of colorectal cancer and is an independentprognostic factor for poor survival of patients withcolorectal cancer. Overexpression of miR-224 enhancesthe proliferation and tumorigenicity of colorectal cancercells both in vitro and in vivo. In addition, overexpressionof miR-224 leads to downregulation of p21Cip1 andp27Kip1, and upregulation of cyclin D1 via activation ofAKT/FOXO3a signaling. Moreover, both PH domainleucine-rich-repeats protein phosphatase 1 (PHLPP1)and PHLPP2, antagonists of PI3K/AKT signaling, wereconfirmed as bona fide targets of miR-224. Our datahighlight an important role for miR-224 in the patho-genesis of colorectal cancer, and miR-224 could beconsidered as a potential prognostic marker or thera-peutic target for colorectal cancer.

miR-224 Target PHLPP1 and PHLPP2 in Colorectal Cancer

www.aacrjournals.org Clin Cancer Res; 19(17) September 1, 2013 4663




from 7,525 to 7,835 bp were generated by PCR amplifica-tion and subcloned into the SacI/XmaI sites of the pGL3-basic luciferase reporter plasmid (Promega). The primersused to generate these constructs are listed in Supplemen-tary Table S2. The miR-224 mimics, negative control, andanti-miR-224 inhibitors were purchased fromGenecopoeia(Genecopoeia Co. Ltd.) and transfected into colorectalcancer cells using Lipofectamine 2000 reagent (Invitrogen)according to the manufacturer’s instructions.

Western blottingProtein lysates were prepared, subjected to SDS-PAGE,

transferred onto PVDF membranes, and blotted accordingto standard methods using anti-PHLPP1 (ab71972), anti-PHLPP2 (ab71973; Abcam), anti-p21 (#2947), anti-p27(#3688), anti-cyclinD1 (#2978), anti-p-AKT (#4056), anti-AKT (#9272) antibodies (Cell Signaling Technology), anti-p-FOXO3a (BS5019), or anti-FOXO3a (BS5520) antibodies(Bioworld Technology). Anti-a-tubulin monoclonal anti-body (T6199; Sigma) was used as a loading control.

MTT assay, colony formation assay, soft-agar colonyformation assay, flow cytometry, andimmunohistochemistry

The MTT assay, colony formation assay, soft-agar col-ony formation assay, flow cytometry, and immunohis-

tochemistry (IHC) were conducted according to previ-ously described methods (13). Further details are provid-ed in the Supplementary Materials and Methods section.

Luciferase assaysCells were seeded in triplicate in 24-well plates (1 � 105/

well) and cultured for 24 hours. The pGL3-luciferase report-er gene plasmids pGL3.0-PHLPP1-30-UTR, pGL3-PHLPP2-30-UTR, or the control-luciferase plasmidwere cotransfectedinto the cells with the control pRL-TK Renilla plasmid(Promega) using Lipofectamine 2000 Reagent (Invitrogen).Luciferase and Renilla activities were assayed 48 hours aftertransfection using the Dual Luciferase Reporter Assay Kit(Promega) following the manufacturer’s instructions. Allexperimentswere conducted at least 3 times and the data arepresented as mean � SD.

Colorectal cancer xenograft model in nude miceAnimal experiments were conducted as previously

described (13). Details are provided in the SupplementaryMaterials and Methods.

Statistical analysisAll statistical analyses were conducted using SPSS13.0

for Windows. The two-tailed paired Student t test was usedfor analyzing 2 groups. The Mann–Whitney U test and

Figure1. miR-224 is overexpressed in colorectal cancer. A, real-timePCRanalysis ofmiR-224 expression in 43paired humancolorectal cancer tissues and thematched adjacent normal tissues; miR-224 expression was normalized to U6 and expressed relative to the matched adjacent normal tissues. Each barrepresents the mean of 3 independent experiments. B, mean expression of miR-224 in the 43 paired human colorectal cancer tissues and the matchedadjacent normal tissues. C,mean expression ofmiR-224 by T classification. ��P < 0.01. D, Kaplan–Meier overall survival analysis for colorectal cancer patientsstratified by high miR-224 expression (>median; n ¼ 51) or low miR-224 expression (<median; n ¼ 59).

Liao et al.

Clin Cancer Res; 19(17) September 1, 2013 Clinical Cancer Research4664




Spearman’s correlation analyses were used to analyze therelationship between miR-224 expression and the clinico-pathologic features of colorectal cancer. Survival curveswere plotted by the Kaplan–Meier method and comparedwith the log-rank test. The significance of various survival-related variables was assessed using a multivariate Coxregression model. P < 0.05 was considered statisticallysignificant.

ResultsmiR-224 is overexpressed in colorectal cancerThe expression of miR-224 was detected in 43 pairs of

colorectal cancer biopsies and their matched adjacentnormal tissues, and 77 cases of fresh colorectal cancerbiopsies by real-time PCR revealed that miR-224 wassignificantly overexpressed in 76.7% (33/43) of the colo-rectal cancer tissue samples examined (T/N > 2-fold)compared to the matched adjacent normal tissues fromthe same patient, with up to 68-fold increases observed inthe colorectal cancer tissue samples (Fig. 1A). Student ttest revealed that the miR-224 expression levels weresignificantly lower in the adjacent normal tissues thanthe colorectal cancer tissues samples (P ¼ 0.004, Fig. 1B).In addition, miR-224 was expressed at relatively lowlevels in tumors with an early T classification (T1 andT2), and markedly increased in T3 tumors and furtherelevated in T4 tumors (Fig. 1C).

High levels of miR-224 expression are associated withprogression and poor prognosis in colorectal cancerTo further investigate the clinicopathologic and prog-

nostic significance of miR-224 expression in colorectalcancer patients, the levels of miR-224 were quantified in acohort of 107 colorectal cancer tissue samples using real-time PCR. The median relative expression level of miR-224 in all 110 colorectal cancer samples was chosen as thecut-off point for separating tumors with low expression ofmiR-224 and high expression of miR-224. Mann–Whit-ney U tests and Spearman correlation analysis showedthat low expression of miR-224 was significantly associ-ated with pathologic stage, Dukes’ stage, T classification,and distant metastasis in colorectal cancer (P < 0.05; Table1 and Supplementary Table S3). However, there were nosignificant correlations between the miR-224 expressionlevel and age, gender, histology, or N classification incolorectal cancer. Kaplan–Meier survival analysis revealedthat patients with low miR-224 expression levels had abetter clinical outcome (Fig. 1D). Multivariate survivalanalysis indicated that the miR-224 expression level, Tstage, and pathologic stage were independent prognosticfactors for outcome in patients with colorectal cancer(Supplementary Table S4).

Overexpression of miR-224 enhances the proliferationand tumorigenicity of colorectal cancer cells in vitroand in vivoWe transfected the colorectal cancer cell lines SW620 and

HCT116 with hsa-miR-224 mimic oligonucleotides and

examined the effects on cellular proliferation. MTT andcolony formation assays revealed that overexpression ofmiR-224 significantly increased the growth rate of bothcolorectal cancer cell lines, compared to negative con-trol–transfected cells (Supplementary Fig. S1A and S1B).Moreover, the expression of Ki-67, a well-known markerof proliferation, was dramatically increased in miR-224–overexpressing SW620 and HCT116 cells compared withnegative control–transfected cells (Supplementary Fig.S1C). Furthermore, the BrdUrd incorporation assay showedthat a higher percentage ofmiR-224–overexpressing SW620and HCT116 cells contained newly synthesized DNA, com-paredwith negative control cells (Supplementary Fig. S1D).

We next examined the effect of miR-224 on the tumor-igenicity of colorectal cancer cells using an anchorage-inde-pendent growth assay. Overexpression of miR-224 signifi-cantly enhanced the anchorage-independent growth abilityof SW620 and HCT116 cells, as indicated by increasedcolony number and size (Fig. 2A). To test whether miR-224 could promote the growth of colorectal cancertumors in vivo, we engineered SW620 and HCT116 cellsto stably overexpress miR-224. These stably miR-224–overexpressing and control cells were subcutaneously

Table 1. Correlation between clinicopathologicfeatures and miR-224 expression in 110colorectal cancer tissues

miR-224expression

Characteristics Low High P value

Age�mean (52) 29 17 0.095>mean (52) 30 34

GenderMale 38 30 0.550Female 21 21

Pathologic stage1 9 1 0.0142 42 373 8 13

Dukes stageA 8 4 0.023B 21 16C 22 10D 8 21

T classification1–2 17 7 <0.0013 37 244 5 20

N classification0 29 21 0.4041 30 30

Distant metastasisNo 51 30 0.001Yes 8 21






inoculated into nude mice. As shown in Fig. 2B, thetumors in the SW620/miR-224 group grew more rapidlythan the tumors in the SW620/vector group (P ¼ 0.004).Similar results were obtained using HCT116 cells (P <0.001; Fig. 2D). IHC staining confirmed that the tumorsof the control group displayed much lower Ki-67 indexesthan the tumors from the miR-224–overexpressing group(Fig. 2C and E).

Upregulation of miR-224 accelerates colorectal cancercell-cycle progression and regulated cell-cycle factors:AKT/FOXO3a activation in colorectal cancer cells wasinvolved

We determined the cell-cycle distribution of miR-224–overexpressing SW620andHCT116 cells byflowcytometry,to explore the possible mechanism by which miR-224regulates colorectal cancer cell proliferation. A significantdecrease in the percentage of cells in the G1–G0 phase and

increase in the percentage of cells in the S-phase wereobserved in miR-224–overexpressing cells (Fig. 3A). Theexpression levels of a number of critical cell-cycle regulatorswere also detected. As expected, p21Cip1 and p27Kip1werestrikingly downregulated, whereas cyclin D1 was signifi-cantly upregulated at both the protein and mRNA levels inmiR-224–overexpressing cells (Fig. 3B and C). It has beenwell documented that the expression of p27 Kip1 and cyclinD1 can be transcriptionally regulated by FOXO3a and, inturn, the transcriptional activity of FOXO3a is modulatedby AKT phosphorylation (14). Thus, we hypothesized thatupregulation of miR-224 may activate AKT/FOXO3a sig-naling. As shown in Fig. 3B and C, the phosphorylationlevels of both FOXO3a and AKT increased in miR-224–overexpressing colorectal cancer cells. In addition, FOXO3aactivity was strongly repressed by overexpression ofmiR-224, as showed by a FOXO-luciferase reporter gene(Fig. 3C).

Figure 2. Upregulation of miR-224promotes the proliferation andtumorigenicity of colorectal cancercells in vitro and in vivo. A,anchorage-independent colonyformation assays. Representativemicrographs are shown (left); thenumbers of colonies containingmore than 50 cells (right) werescored. Each bar represents themean of 3 independentexperiments. �P < 0.05. B, D, tumorxenograft model. Cells wereinjected into the hindlimbs of nudemice (n ¼ 6). Data points arepresented as themean�SD tumorvolume. C, E, histopathology ofxenograft tumors. The tumorsections were under H&E stainingand IHC staining using antibodyagainst Ki-67.

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Inhibition of miR-224 reduces the growth of colorectalcancer cellsTo confirm the effects of miR-224 on the proliferation of

colorectal cancer cells, endogenous miR-224 was inhibitedby ectopically expressing a miR-224 inhibitor in SW620and HCT116 cells. As shown in Supplementary Fig. S2,inhibition of miR-224 evidently increased the mRNAexpression levels of both p21Cip1 and p27Kip1, anddecreased the level of cyclin D1 mRNA in both cell lines.In addition, inhibition of miR-224 dramatically increasedthe percentage of cells in theG0–G1 phase anddecreased thepercentage of cells in the S-phase, indicating that silencing

of miR-224 induced G1–S arrest in colorectal cancer cells(Fig. 4A). Moreover, MTT assays (Fig. 4B) and the colonyformation assay (Fig. 4C) confirmed that inhibition ofmiR-224 reduced the growth rate of SW620 and HCT116colorectal cancer cells, compared to negative control–trans-fected cells.

miR-224 directly targets the tumor suppressorsPHLPP1 and PHLPP2 in colorectal cancer cells

Recently, the PHLPP phosphatase family membersPHLPP1 and PHLPP2 have been identified to directlydephosphorylate and serve as negative regulators of AKT

Figure 3. Overexpression of miR-224 promotes the G1–S transition and cell-cycle progression in colorectal cancer cells. A, flow cytometric analysis ofthe percentage of cells in various phases of the cell cycle. B, Western blotting analysis of indicated proteins. The protein expression levels were quantified bycomparing the gray level of each band using Quantity One Software. C, relative FOXO3a luciferase reporter gene activity and real-time PCR analysisof the expression ofp21, p27, and cyclin D1mRNA;GAPDHwas used as a loading control. Error bars represent themean�SDof 3 independent experiments;�P < 0.05.






(15). This prompted us to investigate whether the modu-lation of AKT/FOXO3a signaling activity by miR-224 weredue to regulation of PHLPP1 and PHLPP2. The 3 bioinfor-matic algorithms (miRBase, Pictar, and TargetScan) wereused to predict miR-224 target sites. Gene ontology enrich-ment analysis showed that PHLPP1 and PHLPP2 weretheoretical target genes of miR-224 (Fig. 5A). Westernblotting analyses showed that the protein levels of bothPHLPP1 and PHLPP2 were dramatically downregulated inmiR-224–overexpressing cells, whereas PHLPP1 andPHLPP2 were upregulated after inhibition of miR-224 inboth cell lines (Fig. 5B). RNA-immunoprecipitation (RIP)analysis revealed that overexpression of miR-224 increasedthe recruitment of PHLPP1 mRNA and PHLPP2 mRNA tomiRNP complexes (Fig. 5C). To further analyze the rela-tionship between miR-224 and PHLPP1 and PHLPP2, thePHLPP1 or PHLPP2 30-UTR fragments containing the wild-type or mutant miR-224 binding sites were subcloned intothe pGL3.0-Baisic luciferase reporter vector. As shownin Fig. 5D, a dose-dependent reduction in both wild-typePHLPP1 and PHLPP2 reporter gene luciferase activity wasobserved upon overexpression of miR-224 in both colorec-tal cancer cell lines, whereas inhibition of miR-224increasedwild-type PHLPP1 and PHLPP2 luciferase activity,compared to negative control–transfected cells. However,when miR-224-binding sites in the 30-UTRs were mutated,we observed a dramatic relief of silencing of PHLPP1 and

PHLPP2 genes, suggesting that these are probably truemiR-224 target sites (Fig. 5D).

Repression of PHLPP1 and PHLPP2 play essential rolesin miR-224–induced proliferation of colorectal cancercells

Further analyses revealed that overexpression of PHLPP1or PHLPP2 dramatically abrogated the miR-224–mediatedregulation of AKT and FOXO3a phosphorylation (Fig. 6AandB) andmodulation of the cell-cycle regulators p21, p27,and cyclin D1 (Supplementary Fig. S3). In addition, flowcytometry analysis showed that overexpression of PHLPP1or PHLPP2 in miR-224–overexpressing colorectal cancercancer cells decreased the percentage of cells in S-phase (Fig.6C). MTT assays indicated that overexpression of PHLPP1or PHLPP2 significantly reduced the growth rate of miR-224–overexpressing SW620 and HCT116 cells (Fig. 6D).

We further investigated the expression levels of miR-224,PHLPP1, and PHLPP2 in clinical colorectal cancer samples.Analyses of the 43 paired colorectal cancer samples andmatched adjacent normal tissues in which miR-224 wasupregulated revealed that miR-224 expression correlatednegatively with PHLPP1 and PHLPP2 mRNA expression(Supplementary Fig. S4). We also measured the expressionof PHLPP1 and PHLPP2 in the xenograft tumors formed bySW620/vector cells, SW620/miR-244 cells, HCT116/vectorcells, and HCT116/miR-244 cells. Real-time PCR and IHC

Figure 4. Inhibition of miR-224 inhibits cell-cycle progression and suppresses the proliferation of colon cancer cells. A, flow cytometric analysis of cell-cycledistribution. B, C, inhibition ofmiR-224 inhibits cell growth as determined byMTT assays (B) and colony formation assays (C). Error bars representmean�SDfrom 3 independent experiments; �P < 0.01.

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showed that PHLPP1 and PHLPP2mRNA and protein wereexpressed at lower levels in the tumors generated frommiR-224–overexpressing cells, compared to the vector controlcells (Supplementary Fig. S5).

DiscussionmiRNAs are a large family of gene regulators that nega-

tively regulate their target mRNAs in a sequence-specificmanner (16). miRNAsmay also function as tumor suppres-sors or oncogenes (17). Recent evidence has shown thatmiRNAs play essential roles inmultiple biological processesrelated to cancer, including cell differentiation, prolifera-tion, tumorignesis, angiogenesis, invasion, and metastasis(18–20). Upregulation of miR-224 has only been observedin a few tumor types, such as hepatocellular carcinoma,pancreatic ductal carcinoma, breast and renal cancer (21–24). Importantly, in hepatocellular carcinoma, p65/NF-kBhas been shown to act as a direct transcriptional regulator ofmiR-224 expression, which links upregulation of miR-224with cellmigration and invasion (21). In addition,miR-224can promote cell invasion and the expression of metastasis-related genes by targeting the Raf kinase inhibitor protein(RKIP) in human breast cancer cells (22). However, it wasuncertain whether dysregulation ofmiR-224was associatedwith the progression of colorectal cancer. In this study, wefound that miR-224 was upregulated in colorectal cancertissues, and high miR-224 expression levels were found tobe significantly associated with aggressive characteristicsandpoor patient prognosis in colorectal cancer. In addition,ectopic overexpression of miR-224 promoted cell prolifer-

ation, proliferation, and tumorigenesis in colorectal cancercells. Thus, our data are consistent with previously pub-lished studies which associated changes in miR-224 expres-sion with tumor formation and progression (21, 22),implicating miR-224 as an oncomir.

More specifically, we showed that the molecular mech-anism by which miR-224 promotes colorectal cancer cellproliferation was due, at least in part, to acceleration of theG1–S phase transition, downregulation of p21Cip1 andp27Kip1, and upregulation of cyclin D1 in cells overexpres-sing miR-224. Previous studies revealed that the PI3K/AKTsignal transduction cascade was required for cell-cycle pro-gression through the G1-phase (25). In addition, activationof PI3K/AKT decreases the cellular levels of p21Cip1 andp27Kip1, and induces cyclin D1 expression, thereby pro-moting cell proliferation (14, 26). The modulation of thesecell-cycle regulators by PI3K/AKT is accomplished eitherdirectly or indirectly through inhibiting the phosphoryla-tion and activation of FOXO3a (14, 27, 28). Our datashowed that miR-224 activates PI3K/Akt signaling andpromotes cell survival through modulation of FOXO3a,p21Cip1, p27Kip1, and cyclin D1 expression. This pathwayrepresents a new mechanism which may possibly underliethe development of colorectal cancer.

We further explored the mechanism by which miR-224could activate the PI3K/Akt signaling cascade. Bioinformat-ic algorithms predicted that both isoforms of PHLPP,PHLPP1 and PHLPP2, were bona fide target genes of miR-224. PHLPP1 and PHLPP2 belong to a novel family ofSer/Thr protein phosphatases and play central roles in

Figure 5. miR-224 directly targetsthe 30-UTRs of PHLPP1 andPHLPP2. A, predicted miR-224target sequences in the 30-UTRsof PHLPP1 and PHLPP2. Thenucleotides mutated in the 30-UTRs of PHLPP1 and PHLPP2are highlighted in red. B, Westernblotting analysis of PHLPP1and PHLPP2 expression in theindicated cells. The proteinexpression levels were quantifiedby comparing the gray level ofeach band using Quantity OneSoftware. C, RIP analysis, asassessedby immunoprecipitationof Ago1 in HCT116 cells. IgGimmunoprecipitation was used asa negative control. D, luciferaseactivity assays of the indicatedcells. Error bars represent themean � SD of 3 independentexperiments; �, P < 0.05.






maintaining cell survival suppression through negativelyregulating the signaling pathways activated by AKT, PKC,MAPK, andMst1 (29–32). Evidence has recently emerged tosuggest that PHLPP1 and PHLPP2 act as tumor suppressorgenes, and their expression is frequently depleted in avariety of human cancers, including breast cancer, prostatecancer, and colorectal cancer (26, 33). As Ser/Thr proteinphosphatases, PHLPP1 and PHLPP2 can directly dephos-phorylate AKT to inhibit AKT signaling activity, whichpromotes apoptosis (15, 30, 33). Overexpression ofPHLPP1 and PHLPP2 in cancer cell lines decreases cellproliferation and tumorigenesis both in vitro and in xeno-graft models (26, 30, 33–37). In colorectal cancer cells,stable overexpression of PHLPP1 or PHLPP2 blocked theG2–M transition or induced G1 cell-cycle arrest, thus

decreasing the rate of cell proliferation (26). Collectively,these studies suggest that restoration of the expression orfunction of the PHLPPs may represent a potential noveltherapeutic intervention strategy for colorectal cancer.However, precise details of the mechanisms which regulatePHLPP1 and PHLPP2 remain elusive.

Our results also confirmed that both PHLPP1 andPHLPP2were direct targets of miR-224. In addition, ectopicoverexpression of PHLPP1or PHLPP2 (without the 30-UTR)significantly abrogated the miR-224–induced proliferationof colorectal cancer cells in vitro. Finally, expression analysesof miR-224, PHLPP1, and PHLPP2 in clinical colorectalcancer tissues revealed significant negative correlationsbetween miR-224 and the expression of PHLPP1 andPHLPP2. Taken together, these results suggest that the

Figure 6. PHLPP1 and PHLPP2 cooperatively play a crucial role in miR-224-induced colon cancer cell growth. A, Western blotting analysis of PHLPP1 andPHLPP2 expression in the indicated cells. B, Western blotting analysis of indicated proteins in miR-224-overexpressing cells transfected with PHLPP1,PHLPP2 (without the 30-UTRs), or empty vector. The protein expression levels were quantified by comparing the gray level of each band usingQuantity One Software. C, flow cytometric analysis of cell-cycle distribution in the indicated cells. D, MTT analyses of indicated cells. Each bar represents themean � SD of 3 independent experiments; �P < 0.05.

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effects of miR-224 on the proliferation of colorectal cancercells may be mediated via downregulation of PHLPP1 andPHLPP2 via miR-224 directly targeting the 30-UTRs of thesegenes. Thus, our current study uncovers what we believe tobe a novel mechanism leading to downregulation ofPHLPP1 and PHLPP2 in cancer cells.In summary, this study provides, for the first time, an

essential link between miR-224–mediated tumor growthand downregulation of PHLPP1 and PHLPP2 in colorectalcancer. Our findings suggest an important role for miR-224in theproliferationof colorectal cancer cells.Understandingthe precise roles played by miR-224 in the initiation andprogression of colorectal cancer will not only increase ourunderstanding of the biology of this tumor type, but mayalso allow the development of a novel therapeutic strategybased on inhibition of miR-224.

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

Authors' ContributionsConception and design: W.-T. Liao, Z.-G. Wang, C. Zhang, Y. DingDevelopment of methodology: T.-T. Li, Z.-G. Wang, S.-Y. Wang, Y.-P. Ye,P. Wu, H.-L. Jiao, C. Zhang, Y. Ding

Acquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): T.-T. Li, M.-R. He, Y.-M. Cui, P.Wu, C. Zhang, Y.-J.Xie, J.-X. Wang, Y. DingAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): L. Qi, Y.-M. Cui, C. Zhang, Y. DingWriting, review, and/or revisionof themanuscript:W.-T. Liao, C. Zhang,Y. DingAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): T.-T. Li, P. Wu, C. Zhang, Y. DingStudy supervision: C. Zhang, Y. Ding

Grant SupportThis work was supported by the National Natural Science Foundation of

China (Nos. 30901791, 81172055, and 81071735), Major projects ofNational Natural Science Foundation of China (No. 81090422), State KeyProgram of National Natural Science Foundation of China (U1201226),National Basic Research Program of China (973 Program, Nos.2010CB529402 and 2010CB529403), Guangdong Provincial Natural Sci-ence Foundation of China (No. S2012010009643), and Zhu Jiang Science &TechnologyNewStar Foundation inGuangzhou city (2012046), Science andTechnology Innovation Foundation of Guangdong Higher Education(CXZD1016), and Key Program of National Natural Science Foundation ofGuangdong, China (2010B031500012).

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 January 25, 2013; revised May 31, 2013; accepted June 20, 2013;published OnlineFirst July 11, 2013.

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Liao et al.





2013;19:4662-4672. Published OnlineFirst July 11, 2013.Clin Cancer Res Wen-Ting Liao, Ting-Ting Li, Zheng-Gen Wang, et al.

PHLPP2 and PHLPP1Human Colorectal Cancer by Repressing microRNA-224 Promotes Cell Proliferation and Tumor Growth in

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