miRNA-302b Suppresses Human Hepatocellular Carcinoma by … · Cell Cycle and Senescence miRNA-302b...

Cell Cycle and Senescence

miRNA-302b Suppresses Human Hepatocellular Carcinomaby Targeting AKT2

Lumin Wang1, Jiayi Yao1, Xiaogang Zhang2, Bo Guo1, Xiaofeng Le6, Mark Cubberly7, Zongfang Li4,Kejun Nan3, Tusheng Song1, and Chen Huang1,5

AbstractmiRNAs (miR) play a critical role in human cancers, including hepatocellular carcinoma. Although miR-302b

has been suggested to function as a tumor repressor in other cancers, its role in hepatocellular carcinoma is unknown.This study investigated the expression and functional role of miR-302b in human hepatocellular carcinoma. Theexpression level of miR-302b is dramatically decreased in clinical hepatocellular carcinoma specimens, as comparedwith their respective nonneoplastic counterparts, and in hepatocellular carcinoma cell lines. Overexpression ofmiR-302b suppressed hepatocellular carcinoma cell proliferation andG1–S transition in vitro, whereas inhibition ofmiR-302b promoted hepatocellular carcinoma cell proliferation and G1–S transition. Using a luciferase reporterassay, AKT2 was determined to be a direct target of miR-302b. Subsequent investigation revealed that miR-302bexpression was inversely correlated with AKT2 expression in hepatocellular carcinoma tissue samples. Importantly,silencing AKT2 recapitulated the cellular and molecular effects seen upon miR-302b overexpression, whichincluded inhibiting hepatocellular carcinoma cell proliferation, suppressing G1 regulators (Cyclin A, Cyclin D1,CDK2) and increasing p27Kip1 phosphorylation at Ser10. Restoration of AKT2 counteracted the effects of miR-302b expression. Moreover, miR-302b was able to repress tumor growth of hepatocellular carcinoma cells in vivo.

Implications: Taken together, miR-302b inhibits HCC cell proliferation and growth in vitro and in vivo bytargeting AKT2. Mol Cancer Res; 12(2); 190–202. �2013 AACR.

IntroductionHepatocellular carcinoma is the sixth most prevalent

cancer and the third most frequent cause of cancer mortalityworldwide. It is thought to develop in a multistep processrequiring the accumulation of several structural and genomicalterations and affecting many different pathways (1). It iswell documented that a defect in cell-cycle control is anessential step in the development and progression of humancancer. A number of oncogenes and tumor suppressorsinvolved in cell-cycle regulation are often aberrant in hepa-

tocellular carcinoma, thereby promoting cancer cell prolif-eration (2).Cell-cycle–related genes such as cyclin D1, cyclin E, cyclin

A, p53, p14, p16, p19, c-Jun, and Pten have been implicatedin hepatocarcinogenesis. Among these, AKT2 involvementhas been investigated thoroughly in the induction andprogression of hepatocellular carcinoma both in cancer cellproliferation and migration (3, 4). AKT2, a key downstreameffector of the phosphatidylinositol 3-kinase (PI3K) signal-ing pathway, modulates the function of numerous substratesrelated to cell-cycle progression at the G1–S and G2–Mtransitions, either by direct phosphorylation of the targetproteins themselves or, indirectly, by regulating proteinexpression levels (5). In the G1–S phase, AKT2 controlsthe transcription and/or stabilization of c-Myc and cyclinD1, whereas it also suppresses the expression of multiplenegative cell-cycle regulators such as p21Cip1, p27Kip1,and p15INK4b by phosphorylation of their substrates,respectively (6, 7). In addition, AKT binds to and phosphor-ylates Mdm2 and MdmX at Ser166, Ser186, and Ser367 toenhance protein stability and facilitate the function of theheterocomplex of Mdm2-MdmX; which is to induce p53ubiquitination, therefore bypassing the G1 checkpoint(8, 9). In the G2–M phase, AKT2 is necessary for efficiententry intomitosis during unperturbed cell cycles bymechan-isms such as phosphorylation of CDK1 and CDK2 andcytoplasmic accumulation of CDC25B (10, 11). Thus,phosphorylation by AKT regulates compartmentalization

Authors' Affiliations: 1Department of Genetics and Molecular Biology,Xi'an Jiaotong University, School of Medicine, Departments of 2Hepato-biliary Surgery and 3Oncology, First Affiliated Hospital, Xi'an JiaotongUniversity School of Medicine; 4Engineering Research Center of Biother-apy and Translational Medicine of Shaanxi Province; 5Key Laboratory ofEnvironmentally and Genetically Associated Diseases at Xi'an, JiaotongUniversity, Ministry of Education, Xi'an, China; 6Department of Experimen-tal Therapeutics, The University of Texas, M.D. Anderson Cancer Center,Houston, Texas; and 7Division of Cardiology, David Geffen School ofMedicine, UCLA, Los Angeles, California

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

L. Wang and J. Yao contribute equally to this work.

CorrespondingAuthor:ChenHuang, Department of Genetics andMolec-ular Biology, Xi'an Jiaotong, University School ofMedicine, 76 Yan TaWestRoad, Xi'an, Shaanxi 710061, China. Phone: 86-29-82655077; Fax: 86-29-82655077; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-13-0411

�2013 American Association for Cancer Research.

MolecularCancer

Research

Mol Cancer Res; 12(2) February 2014190

on April 3, 2020. © 2014 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Published OnlineFirst December 13, 2013; DOI: 10.1158/1541-7786.MCR-13-0411

http://mcr.aacrjournals.org/

of multiple substrates involved in cell-cycle progression inhepatocellular carcinoma proliferation.MicroRNAs (miRNA) are a class of non-protein-coding,

endogenous, small RNAs that regulate gene expression bytranslational repression,mRNA cleavage, and decay initiatedby miRNA-guided rapid deadenylation (12). More than thepast 10 years, miRNAs are particularly important in nearlyall cancer development studies in that they can be targets ofgenomic lesions, controlled by classic tumor signals, andthey themselves present as a class of oncogenes or tumorsuppressors (13). Importantly, miRNAs have been demon-strated to have essential roles in hepatocellular carcinomaprogression by affecting cell proliferation and metastasis bytargeting tumor-related gene (14–16). The present studiesproved that miR-122, miR-26, and miR-223 suppressedtumor proliferation, whereasmiR-130b,miR-221,miR-222promoted tumor development in hepatocellular carcinoma(17–21). Ever since the adoption of the miRNA arraytechnique, many more precursors and mature miRNAs havebeen found to be aberrantly regulated in hepatocellularcarcinoma progression; among which, the miR-302 clusterhas garnered much attention (15). The miR-302 cluster,which consists of miR-302a, -302a�, -302b, -302b�, -302c,-302c�, -302d, -367, and -367�, was first found to befunctionally correlated with self-renewal and proliferationproperties in the stemness maintenance of embryonic stemcells (ESC; refs. 22 and 23). Further studies confirmed thatthe ESC-specific transcription factors Oct4, Sox2, Nanog,and Rex-1 had binding sites on the miR-302 promoter, thusregulating its expression (24–26). In addition,miR-302-367has been identified to posttranscriptionally regulateCYCLIND1 and CDK4, therefore affecting cell-cycle pro-gression (25). More recently, miR-302 has been reported tobe a new tumor marker used to predict the malignantbehavior of different types of tumor. Lin and colleaguesdemonstrated the tumor suppressive activity of miR-302 inhuman pluripotent stem cells by both the cyclin E-Cdk2 andcyclin D-Cdk4/6 pathways in the G1–S cell-cycle transition.More importantly, miR-302was found to target Bmi-1, thuspromoting the tumor suppressor functions of p16Ink4a andp14/p19Arf directed against Cdk4/6–mediated cell prolif-eration (27). Furthermore, tumor-related miRNA studiesproved the potential tumor-suppressor role for miR-302b inattenuating cell growth, promoting cell apoptosis, andinducing cell metastasis in tumorigenesis of gastric tissue,liver metastasis, and primary colorectal cancer tissues (28,29). In this study, we found that miR-302b was frequentlydownregulated in hepatocellular carcinoma tissues and celllines. Overexpression of miR-302b inhibited hepatocellularcarcinoma proliferation dramatically both in vitro and invivo. Furthermore, miR-302b controlled the hepatocellularcarcinoma cell cycle at the G1–S phase by directly targetingAKT2 and subsequently interrupted the AKT2-related cell-cycle pathway. Silencing of AKT2 produced similar cellularand molecular effects as miR-302b overexpression, whereasrestoration of AKT2 counteracted the effects of miR-302bexpression. These findings illustrated the tumor suppressorrole of miR-302b in the control of the G1–S cell cycle and

therefore suppressed hepatocellular carcinoma progression,which may be a beneficial strategy for future cancer therapy.

Materials and MethodsCell line and tissue specimensHuman liver cancer cell line SMMC-7721, Bel-7404,

Huh7, and normal liver cell HL-7702 cells were maintainedin 1640 medium (1640; PAA Laboratories GmbH) supple-mented with 10% FBS (PAA Laboratories GmbH). Normalliver tissues were collected from patients undergoing resec-tion of hepatic hemangiomas. Twenty-seven paired hepato-cellular carcinoma and adjacent nontumor liver tissues werecollected from patients undergoing resection of hepatocel-lular carcinomaat thehepatobiliary surgerydepartmentof theFirst Affiliated Hospital (Xi'an Jiaotong University, Xi'an,China). The relevant characteristics of the studied subjectswere shown in Table 1. No local or systemic treatment hadbeen conducted before operation. Tissue samples wereimmediately snap frozen in liquid nitrogen until RNAextraction. Both tumor and nontumor tissues were histolog-ically confirmed. Informed consent was obtained from eachpatient and was approved by the Institute Research EthicsCommittee at Cancer Center (Xi'an Jiaotong University).

RNA extraction, cDNA synthesis, and quantitative real-time PCRTotal RNAwas extracted fromprepared liver samples with

TRizol reagent (Invitrogen) and cDNA was synthesizedaccording to the manufacturer's protocol (MBI Fermentas).Quantitative real-time PCR (qRT-PCR) was performedusing a Maxima SYBR Green qPCR Master Mixes (MBIFermentas), and PCR-specific amplification was conductedin the IQ5 Optical System real-time PCR machine. Therelative expression of genes (miR-302b, U6, Akt2, b-actin)was calculated with the 2-(DDCt) method (30). The primersused are listed here (qRT-PCR, miR-302b-F 50-ATC-CAGTGCGTGTCGTG-30, miR-302b-R 50-TGCTTAA-GTGCTTCCATGTT-30; AKT2-F 50-CTCACACACAG-TCACCGAGAGC-30, AKT2-R 50-TGGGTCTGGAAG-GCATACTT-30; U6-F 50-TGCGGGTGCTCGCTTCG-GCAGC-30, U6-R 50-CCAGTGCAGGGTCCGAGGT-30; b-actin-F 50-CGTGACATTAAGGAGAAGCTG-30,b-actin-R 50-CTAGAAGCATTTGCGGTGGAC-30).

Construction of expression plasmidsHuman miR-302b precursor (pri-miR-302b) was sub-

cloned into pSilencerTM4.1-CMV vector (Ambion, Gene-works) according to the manufacturer's instructions: miR-302b-F 50-AATTCGCTCCCTTCAACTTTAACATGG-AAGTGCTTTCTGTGACTTTAAAAGTAAGTGCTT-CCATGTTTTAGTAGGAGTA-30; R 50-AGCTTACT-CCTACTAAAACATGGAAGCACTTACTTTTAAAGT-CACAGAAAGCACTTCCATGTTAAAGTTGAAGGG-AGCG-30. Primers contained 50 EcoRI and 30 HindIIIrestriction sites to facilitate cloning into the vector. We alsocommercially synthesized a 20-O-methyl-modified antisenseoligonucleotide of miR-302b (ASO-miR-302b) as an inhib-itor of miR-302b (BGI). GV141 vector (GeneChem) was

miR-302b Targets AKT2 in Hepatocellular Carcinoma

www.aacrjournals.org Mol Cancer Res; 12(2) February 2014 191




used to construct vector of re-expression AKT2. The cDNAof AKT2was chemically synthesized and cloned intoGV141vector between the XhoI and KPnI sites.

Analysis of clonogenicity in vitro and tumorigenicityin vivoFor clonogenicity analysis, 24 hours after transfection,

HL-7702, SMMC-7721, Huh7, and Bel-7404 cells wereresuspended and seeded onto 12-well plates at density of2,000 cells/well, incubated 2 weeks later, colonies werestained with 0.5% crystal violet.All experimental procedures involving animals were in

accordancewith theGuide for theCare andUseofLaboratoryAnimals and were performed according to the institutionalethical guidelines for animal experiment. Viable miR-302band negative control (NC)–transfected SMMC-7721 viablecells were suspended in 100 mL PBS and then injectedsubcutaneously into either side of the posterior flank of thesame femalenudemouseat4 to5weeksof age.Tumorgrowthwas examined every 3 days for 4 weeks. Tumor volume (V)was monitored by measuring the length (L) and width (W)with calipers and calculatedwith the formula (L�W2)�0.5.

miRNA and RNA interferencepri-miR-302b, the ASO-miR-302b control vector or

oligonucleotides, ASO-NC, siRNAs duplexes targetinghumanAKT2were synthesized and purified by BGI. SiRNAduplexes with nonspecific sequences were used as siRNAnegative control. RNA oligonucleotides were transfected byusing Lipofectamine RNAi-MAX (Invitrogen) and mediumwas replaced 6 hours after transfection. A final concentrationof 100 nmol/L miRNA or siRNA was used unless indicated.RNA transfection efficiency is approximately 70% to 80%and the overexpression of miRNA or siRNA persists for atleast 48 hours. Lipofectamine 2000 (Invitrogen) was usedfor transfection of plasmid alone or together with RNAoligonucleotides. All oligonucleotide sequences are listedhere (ASO-NC 50-TGACTGTACTGAACTCGACTG-30. ASO-MiR-302b 50-TGATTTTGTACCTTCTGGAA-TG-30. siRNA-ctrl-F 50-AATTCTCCGAACGTGTCAC-GT-30, siRNA-ctrl-R 50-ACGTGACACGTTCGGAGAA-TT-30. siAKT2-F 50-AAGGATGAAGTCGCTCACACA-30 siAKT2-R 50-TGTGTGAGCGACTTCATCCTT-30).

MTT assay for cell proliferationCells were washed with warm 1640 and MTT (Sigma)

working solution was added into wells. Cells were incubatedat 37�C for 4 hours, and then solubilized the converted dyewith acidic isopropanol (0.04 M HCl in absolute isopropa-nol). Absorbance of the converted dye was measured at awavelength of 490 nm with FLUOstar OPTIMA (BMG).

Cell-cycle analysisCells were harvested by trypsinization and 1 � 106 cells

were used for analysis. The cells were washed in PBS andfixed in ice-cold ethanol overnight at 4�C. The cells werethen washed in PBS and incubated in 1mL staining solution(20 mg/mL propidium iodide and 10 U/mL RNaseA) for 30

minutes at room temperature. The cells were examined byfluorescence-activated cell sorting (FACS) using a flowcytometer (FACSort; Becton), and the cell-cycle populationswere determined by ModFit software.

Dual-luciferase reporter gene assayLuciferase reporter gene assay was performed using the

Dual-Luciferase Reporter Assay System (Promega) accord-ing to the manufacturer's instructions. Cells of 90% con-fluence were seeded in 96-well plates. For AKT2 30-untrans-lated region (UTR) luciferase reporter assay, wild-type ormutant reporter constructs (termed WT or Mut) werecotransfected into SMMC-7721 cells in 96-well plates with100 nmol/L miR-302b or 100 nmol/L miR-NC and Renillaplasmid by using lipofectamine 2000 (Invitrogen). Reportergene assays were performed 24 hours posttransfection usingthe Dual-Luciferase Assay System (Promega). Firefly lucif-erase activity was normalized for transfection efficiency usingthe corresponding Renilla luciferase activity. All experimentswere performed at least 3 times.

In vivo bioluminescence imagingAt 4 weeks after injection, mice from themiR-302b group

(n ¼ 4) and control group (n ¼ 4) were subjected to in vivobioluminescence imaging (31, 32). Briefly, the animals,anesthetized by isoflurane as described previously (33), wereintraperitoneally injected with D-luciferin (Biotium) in aconcentration of 150 mg/kg, and 20 minutes later, weresubjected to the in vivo bioluminescence imaging using thesystem of IVIS Spectrum.

Western blot analysisCell protein lysates were separated in 10% SDS-PAGE

transferred to polyvinylidene difluoride membranes(Roche), then detected with rabbit polyclonal antibodyspecific for AKT2 (Bioworld Biotechnology) and commer-cial ECLKit (Pierce). Protein loading was estimated by usinghuman anti-b-actin monoclonal antibody (BioworldBiotechnology).

ImmunohistochemistryImmunohistochemistry (IHC) was performed according

to the methods described previously (34). The sections werepretreated with microwave, blocked, and incubated usingpolyclonal rabbit anti-human AKT2 (Bioworld Technolo-gy). Staining intensity was assessed by Leica Q550 imageanalysis system.

Immunofluorescence microscopyTo determine the effect of miR-302b on the protein level

of AKT2/Ki-67, we also performed immunofluorescencestaining using the AKT2 (Abcam) or Ki-67 antibody (Milli-pore). After 48 hours, the transfected SMMC-7721, Huh7,and Bel-7404 cell lines were fixed with 4% formaldehyde for20 minutes, then incubated with 0.5% Triton X-100.Rabbit/mouse anti-AKT2/Ki-67 antibody was used forimmunofluorescence staining. After washed 3 times withPBS, the cells were incubated with a goat anti-rabbit/mouse

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antibody (Millipore), andmeasured by immunofluorescencemicroscopy.

Statistical analysisData are expressed as the mean � SEM from at least 3

separate experiments. Unless otherwise noted, the differ-ences between groups were analyzed using Student t testwhen only 2 groups. All tests performed were two-sided.Differences were considered statistically significant at P <0.05. All statistical analysis was performed using SPSS13.0software (SPSS Inc.). The linear correlation coefficient(Pearson r) was calculated to estimate the correlationbetween miR-302b values and AKT2 levels in the matchedhepatocellular carcinoma tumor specimens.

ResultsmiR-302b is frequently reduced in human hepatocellularcarcinoma tissue samples and hepatocellular carcinomacell linesTo explore the role of miR-302b in liver cancer, we

analyzed the expression of miR-302b in 27 paired hepato-cellular carcinoma and adjacent noncancerous liver tissues by

real-time PCR. Compared with their peritumor counter-parts, significant downregulation of miR-302b was observedin 74% (20/27) of hepatocellular carcinoma samples (Fig.1A). Next, we found that miR-302b was downregulated inexamined hepatocellular carcinoma cells compared withnormal hepatocytes (Fig. 1B). This observation was consis-tent with the results of the expression of miR-302b inhepatocellular carcinoma tissues at RNA levels. Meanwhile,we examined the correlation of 302b levels with grading andtumor stage and found that the expression of 302b wasdownregulated in 16 (84%) of poorly differentiated tumortissues (Supplementary Table S1) or in 11 (55%) of thetumor stage III (Supplementary Table S2), thus indicatingthat the miR-302b may act as a tumor suppressor in thehepatocellular carcinoma.

miR-302b decreases SMMC-7721/Bel-7404 cellsgrowth, induces G1–S arrest in vitromiR-302b, a highly conserved sequence across species, is

located in intron 8 of the Larp7 gene on chromosome 3,sharing the same seed sequence with miR-302a/c/d (25).To explore the tumor suppressor role of miR-302b in

Table 1. Background data among 27 patients with hepatocellular carcinoma

Case Sex Age (y)Hepatitishistory

Livercirrhosis Size (cm)

Histologicgrade

TNMstage

1 Male 65 Yes Yes 8.5 Poor IV2 Male 36 Yes Yes 2.5 Moderate I3 Male 58 Yes Yes 6.5 Poor IV4 Male 56 Yes Yes 5.5 Well II5 Male 72 Yes Yes 8.0 Poor III6 Male 51 No No 3.5 Well I7 Male 41 Yes Yes 6.5 Poor III8 Male 44 No Yes 4.0 Poor II9 Female 61 Yes Yes 7.5 Well III10 Female 66 Yes Yes 3.5 Poor II11 Male 40 Yes Yes 9.5 Poor III12 Male 21 No No 2.5 Moderate I13 Male 47 No Yes 5.5 Well II14 Female 58 Yes Yes 6.5 Poor III15 Male 58 Yes Yes 7.5 Poor III16 Male 46 Yes Yes 4.5 poor III17 Male 51 Yes Yes 12.0 Poor IV18 Male 42 Yes Yes 6.5 Moderate III19 Male 59 Yes Yes 4.5 Poor I20 Male 57 Yes Yes 5.0 Poor II21 Male 58 Yes Yes 10.5 Poor IV22 Male 35 No No 3.0 Well I23 Male 61 Yes Yes 10.5 Poor III24 Male 41 Yes Yes 8.5 Well III25 Female 47 Yes Yes 3.5 Poor III26 Female 31 Yes No 4.0 Well II27 Male 59 Yes Yes 7.0 Poor III

Abbreviations: HBV, hepatitis B virus; HCV, hepatitis C virus; TNM, tumor–node–metastasis staging system.






hepatocellular carcinoma, SMMC-7721/Bel-7404 cellswere transfected with miR-302b precursor or its negativecontrols. The efficiency of transfection wasmonitored with aGFP-labeled oligo and an average 80% efficiency wasobserved at a concentration of 100 nmol/L without obvioustoxicity (data not shown). qRT-PCR was performed toexamine the expression levels of miR-302b after transfectionof themiR-302b expression construct, or its negative control(empty vector). As expected, a 30-fold increase in theexpression of miR-302b was observed in the SMMC-7721 cells transfectedwith 100 nmol/L ofmiR-302b relativeto the cells transfected with 100 nmol/L of miR-302bnegative control and a 20-fold increase of the miR-302bexpression was shown in Bel-7404 cells (Fig. 2A). To studythe role of miR-302b in hepatocellular carcinoma cellproliferation, MTT assays were used. Results demonstratedthat transient overexpression of miR-302b inhibited theproliferation of SMMC-7721/Bel-7404 at 48 and 72 hoursafter transfection (Fig. 2B). To further examine the inhib-itory role of miR-302b in hepatocellular carcinoma cells, acolony formation assay was used after similar transienttransfection. Notably, miR-302b–transfected cells displayedfewer and smaller colonies compared with control NC-transfectants (Fig. 2C). Meanwhile, we confirmed thatmiR-302b–mediated repression of AKT2 or ki-67 by immu-nofluorescence staining. The red signal of AKT2 or greensignal ki-67 in the miR-302b–transfected SMMC-7721/Bel-7404 cells was visibly low compared with that of the cellsinfected with miR-ctrl (Fig. 2D), suggesting a growth-inhibitory role of miR-302b. Taken together, MTT, colonyformation, and immunofluorescence staining assays dem-onstrated thatmiR-302bwas able to inhibit the proliferationof hepatocellular carcinoma cells in vitro.To further investigate the mechanisms by which miR-

302b inhibits hepatocellular carcinoma cells proliferation,we adopted a serum starvation–stimulation strategy to clarifywhether miR-302b–induced inhibition of cell proliferationresulted from a blocked cell-cycle checkpoint. SMMC-

7721/Bel-7404 cells were transfected with a miR-302bexpression construct or an empty vector for 24 hours,followed by synchronization with serum deprivation for24 hours and then stimulation to enter S-phase by serumreaddition. Overexpression of miR-302b resulted in amarked accumulation of the G1-population in the humanhepatocellular carcinoma cell line SMMC-7721/Bel-7404(Fig. 2E), suggesting that miR-302b blocked the G1–Stransition.To examine the antiproliferative role of miR-302b in

human hepatoma cells, we eliminated endogenous miR-302b in SMMC-7721/Bel-7404 cells by using its inhibitor,ASO-miR-302b. Reduced endogenous expression of miR-302b in the SMMC-7721/Bel-7404 cells by ASO-miR-302b resulted in increased cell viability, colony formationpotential, and expression of AKT2 and Ki-67 (Fig. 2F–I).Further investigation of ASO-miR-302b on cell-cycle pro-gression of the SMMC-7721/Bel-7404 cells proved thatASO-transfected cells were more inclined to enter S-phase(Fig. 2J). These results suggested that the baseline expressionof miR-302b would initiate its antiproliferation potential atG1–S phase in hepatoma cells, thus preventing furthermalignancy progression.

miR-302b inhibits hepatocellular carcinoma cellproliferation via directly targeting AKT2To uncover the mechanisms by which miR-302b induced

G1–S arrest, we searched for the target genes of miR-302b.Among the predicted target genes of miR-302b in theTargetScan (http://www.targetscan.org/), DIANA (http://diana.cslab.ece.ntua.gr/microT/), and PicTar (http://pictar.mdc-berlin.de/) databases, we found that AKT2, but notAKT1 or AKT3. AKT2 was one of the top candidates and ofparticular interest because of its essential role in differentcancers (5). As shown in Fig. 3A, the region complementaryto the miR-302b seed region was found in the 30-UTR ofhuman AKT2. To validate whether AKT2 was the directtarget gene ofmiR-302b, aDual-Luciferase Reporter System

Figure 1. Dysregulated miR-302b inhepatocarcinoma tissues andcells. A, qRT-PCR was performedtoexaminemiR-302bexpression in27 paired human hepatocellularcarcinoma and adjacent nontumortissues. B, qRT-PCR was usedto analysis ofmiR-302b expressionin normal hepatocytes andhepatocellular carcinoma cells(��, P < 0.01; �, P < 0.05; Studentt test).

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containing wild-type or mutant 30-UTR of AKT2 was used.The luciferase assay showed that miR-302b significantly ledto the suppression of luciferase activity, indicating that miR-302b directly bound to its predicted binding site on AKT2.In contrast, miR-302b had no effect on the luciferase activityof amutant 30-UTR of AKT2 construct (Fig. 3B), indicating

that miR-302b specifically binds to the seed sequence at the30-UTR of AKT2. To test whether miR-302b expressionaffected endogenous AKT2 expression, we transfected themiR-302b expression construct, ASO-miR-302b, emptyvector, or ASO-NC into SMMC-7721 cells. We observeda decrease of AKT2 at both of the mRNA and protein levels

Figure 2. miR-302b decreasesSMMC-7721/Bel-7404 cellsproliferation, induces G1–S arrestin vitro. A, qRT-PCR analysis ofmiR-302b in SMMC-7721/Bel-7404 cells transfected with miR-302b overexpression construct,empty vector to be its control. B,the effects of miR-302b on SMMC-7721/Bel-7404 cells proliferationwere determined by MTT assayat 24, 48, and 72 hours. C,representative results of colonyformation of SMMC-7721/Bel-7404 after miR-302boverexpression. D, expression ofAKT2 or Ki-67 was verified byimmunofluorescence staining.Merged pictures are overlays ofboth AKT2 red signals or Ki-67green signals and nuclear stainingby DAPI (blue). E, cell cycledetermined in SMMC-7721/Bel-7404 cells 48 hours aftertransfection. F, qRT-PCR analysisof miR-302b in SMMC-7721/Bel-7404 cells transfected with ASO-miR-302b, ASO-NC as its control.G, MTT was used to assay theeffects of miR-302b on SMMC-7721/Bel-7404 cells. H, the growthof SMMC-7721/Bel-7404 cells wasdetected by colony formation. I, theexpression of AKT2 or Ki-67 wasdetected by immunofluorescencestaining. J, cell cycle wasdetermined in SMMC-7721/Bel-7404 cells transfected with ASO-miR-302b, or its control (�,P < 0.05;��, P < 0.01; Student t test).






after transfection with miR-302b, whereas knockdown ofmiR-302b enhanced AKT2 protein expression (Fig. 3C). Tofurther investigate the relationship between AKT2 and miR-302b, we also examined the expression of AKT2 and miR-302b in 27 paired samples of hepatocellular carcinoma andtheir adjacent normal tissues. We found that AKT2 expres-sion was higher in hepatocellular carcinoma tissues com-pared with their normal counterparts (Fig. 3D). MiR-302blevels were inversely correlated with AKT2 expression (Fig.3E). The expression of AKT2 was upregulated in 4 pairs oftissues and in hepatocellular carcinoma cell lines comparedwith their normal counterparts, separately (Fig. 3F). Takentogether, the above data suggests that miR-302b is able todirectly regulate AKT2 expression in hepatocellular carci-noma cells.

Knockdown of AKT2 produces similar effects to that ofmiR-302b overexpression in hepatocellular carcinomacellsNext, we silenced AKT2 expression by RNAi method to

test whether AKT2 is involved in the antitumor effects ofmiR-302b. From mRNA and protein expression levels,AKT2 can be specifically knocked-down by siRNA inSMMC-7721 and Bel-7404 cells at 35% and 42%, sepa-rately (Fig. 4A and G). As shown in Fig. 4B–E, silencing ofAKT2 resulted in suppressed cell proliferation and inducedG1–S arrest, which was in line with the effects of miR-302boverexpression in SMMC-7721 cells. Similar antiprolifera-tion effects by AKT2 silencing was also evident in Bel-7404cells, which was in line with that of the miR-302b over-expression (Fig. 4G–K). Next, we analyzed protein levels of

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0.94 0.52 0.96 1.28

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Figure 3. miR-302b inhibits tumorproliferation via directly targetingAKT2 in hepatocellular carcinoma.A, conserved miR-302b seedregion sequence in the 30-UTR ofAKT2 among different species. B,luciferase assay. miR-302b wascotransfected with pLUC-AKT2vector or pLUC-AKT2 mut. C,mRNA and protein expression levelof AKT2 were measured by qRT-PCR andWestern blot analyses. D,qRT-PCR of AKT2 expression in 27paired primary hepatocellularcarcinoma tissues and theircorresponding nontumorous livers(��, P < 0.01). E, inverse correlationbetween miR-302b and AKT2expression in hepatocellularcarcinoma tissues. Statisticalanalysis was performed usingPearson's correlation coefficient(r ¼ �0.42; �, P < 0.05). F, Westernblot analysis of AKT2 expression innormal hepatocytes, hepatomacell lines, 4 pairs of hepatocellularcarcinoma tissues, and theircorresponding nontumorouslivers, with b-actin as an internalcontrol. The intensity for each bandwas quantified. The value undereach lane indicates the relativeexpression level of the AKT2.

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related G1 regulators after overexpressing miR-302b orsilencing AKT2 in SMMC-7721 and Bel-7404 cells byWestern blot analysis. As shown in Fig. 4F and L, both

miR-302b expression and AKT2 siRNA could reduce theexpression of CYCLINA, CYCLIND, and CDK2, whichare 3 essential regulators of the G1–S phase transition.

Figure 4. miR-302b inhibits cellproliferation through AKT2. A,qRT-PCR and Western blotanalyses were performed todetermine the expressionlevel of AKT2 after transfectionof siAKT2. B, MTT assay wasperformed to determine thegrowth of SMMC-7721 cellstreated with siAKT2. C, thegrowth of SMMC-7721 cells wasdetected by colony formationafter AKT2 knockdown.D, the expression of AKT2 orKi-67 was detected byimmunofluorescence staining.E, cell cycle determined inSMMC-7721 cells at 48 hoursafter transfection of siAKT2.F, analysis of the expressionof G1 regulators in SMMC-7721cells after transfected with miR-ctrl, miR-302b overexpressionconstruct, ASO-NC, ASO-miR-302b, siRNA-control, siAKT2.The intensity for each band wasquantified. The value undereach lane indicates the relativeexpression level of the regulators.G, the expression level of AKT2was verified by qRT-PCR andWestern blot analyses aftertransfection of siAKT2 in Bel-7404 cells. H, MTT assay wasperformed to determine thegrowth of Bel-7404 cells aftertransfection of siAKT2. I, thegrowth of Bel-7404 cells wasdetected by colony formationafter AKT2 knockdown.J, the expression of AKT2 orKi-67 was detected byimmunofluorescence staining.K, cell cycles were determined inBel-7404 cells at 48 hours aftertransfection of siAKT2 (�,P<0.05;��, P < 0.01; Student t test). L,analysis of cell cycles regulatorsexpression in miR-302b,ASo-miR-302b, and siAKT2transfected the Bel-7404 cells.






Notably, miR-302b expression and AKT2 knockdownclearly decreased CYCLIND1 phosphorylation at Thr286,which is regulated by the AKT2-glycogen synthase kinase-3b pathway (5). In addition, miR-302b expression andAKT2 knockdown increased the Ser10 phosphorylation ofp27Kip1. Moreover, reducing the expression of miR-302bby ASO-miR-302b had adverse results. These results dem-onstrate that miR-302b and its target gene AKT2 sharesimilar cellular and molecular effects in SMMC-7721 andBel-7404 cells.

Overexpression of AKT2 eliminated the effects of miR-302b on hepatocellular carcinoma cellsTo further demonstrate that the miR-302b exhibited

tumor suppressor function through AKT2, we constructedAKT2 overexpression vector, which was cotransfected withmiR-ctrl or miR-302b into SMMC-7721 or Bel-7404 cells.Overexpression of AKT2 in SMMC-7721 or Bel-7404cells rescued AKT2 expression level reduced by miR-302b(Fig. 5A). Moreover, after cotransfected with the miR-302b

and AKT2 vector, we found that the overexpression ofAKT2counterbalanced the tumor suppressor effect ofmiR-302b inhepatocellular carcinoma cells at cell proliferation (Fig. 5B).To investigate the effect of overexpression of AKT2 on cell-cycle progression of the SMMC-7721/Bel-7404 cells, cell-cycle assay was used. From the Fig. 5C, we found thatoverexpression of AKT2 was in cline to re-enter S-phase inboth SMMC-7721 and Bel-7404 cells. Meanwhile, com-pared with cotransfected with miR-302b and AKT2-ctrl,cotransfected with miR-302b and AKT2 could be able tore-enter S-phase in hepatocellular carcinoma cells also.Furthermore, cell-cycles regulators expression levels weretexted by Western blot analysis after cotransfected withmiR-ctrl and AKT2 or cotransfected with miR-302b andAKT2 in hepatocellular carcinoma cells. The expression ofCYCLINA,CYCLIND1, andCDK2were upregulated aftertransfection of AKT2. Meanwhile, compared with cotrans-fected with miR-302b and AKT2-ctrl, the expression ofregulators was unregulated after cotransfected with miR-302b and AKT2 (Fig. 5D). These results further suggest that

Figure 5. AKT2 rescues miR-302b–induced cellular phenotypes in hepatocellular carcinoma cells. A, cells were cotransfected with the AKT2 and miR-ctrl orcontrol vector and miR-302b. The expression levels of AKT2 were verified by qRT-PCR and Western blot analyses. B, MTT assay was performed todetermine the growth of SMMC-7721/Bel-7404 cells after cotransfected with AKT2 and miR-ctrl or control vector and miR-302b (�, P < 0.05; ��, P < 0.01;Student t test). C, cell cycle was determined in SMMC-7721/Bel-7404 cells at 48 hours. D, the expression of cell-cycle regulators were assayed byWestern blot analysis. The intensity for each band was quantified. The value under each lane indicates the relative expression level of the regulators.

Wang et al.





miR-302b exhibit tumor suppressor role by direct targetingat AKT2.

miR-302b inhibits hepatocellular carcinoma tumorgrowth in vivoTo further confirm the growth inhibitory function of

miR-302b in hepatocellular carcinoma, we tested theeffects of miR-302b on tumor growth in an in vivoxenograft model. miR-302b and miR-control–transfectedSMMC-7721 cells were injected subcutaneously intoeither posterior flank of the same nude mice. The micewere followed for observation of xenograft growth for 4weeks. As shown in Fig. 5A, the tumors from mice thatwere intratumorally injected with synthetic miR-302bwere significantly smaller than those in control mice onday 15 after the first injection. On day 30, the averagevolume of miR-302b–treated tumors was much smallerthan that in control tumors (Fig. 6A and B). The averagetumor weights for the control- and the miR-302b groupson day 30 were 0.25 and 0.04 g, respectively (Fig. 6C).Furthermore, the expression levels of miR-302b andAKT2 in the tumor tissues were examined by qRT-PCRand Western blot analyses. Consistent with the in vitrodata, the in vivo data showed that the expression of miR-302b was increased and the expression of the AKT2protein was reduced in miR-302–treated tumors (Fig.6D). Immunohistochemical analysis also demonstrateddecreased proliferation potential and AKT2 levels in thetumor tissue treated with miR-302b (Fig. 6E and F).

These data indicated that miR-302b expression is capableof inhibiting tumor growth and AKT2 expression levels invivo.

DiscussionIn the past 10 years, dysregulation of miRNAs has been

shown to be a common event that can control cell prolif-eration in hepatocellular carcinoma development and pro-gression (15, 16, 29). In this study, we have shown thatmiR-302b was frequently downregulated in both hepatocellularcarcinoma tissues and cell lines. Enforced expression of miR-302b inhibited hepatocellular carcinoma cells proliferationand cell viability by blocking the G1–S transition in vitro.Moreover, gain- and loss-of-function of miR-302b andcotransfected with miR-302b and AKT2 studies revealedthat miR-302b suppressed cell proliferation by directlytargeting AKT2-related cell-cycle signaling. Furthermore,tumor formation assays demonstrated that miR-302b–tar-geting AKT2 was responsible for suppression of tumorgrowth in vivo. Therefore, our data provided a more com-prehensive understanding of the tumor suppressor role ofmiR-302b during hepatocellular carcinoma development.In a previous miRNA microarray study, we found that

miR-302b was downregulated in clinical hepatocellularcarcinoma samples. After we examined the expression ofmiR-302b in hepatocellular carcinoma cell lines and tissuesin this research, we further confirmed the previous result thatmiR-302b was frequently suppressed in hepatocellular car-cinoma, suggesting that miR-302b might be a novel tumor

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Figure 6. miR-302b inhibitshepatocellular carcinomaprogression in vivo. A, small animalimaging analysis was used toassess the tumor volume in situduring the tumor development atthe fourth week. A, the flanks wereinjected with NC-transfected (leftflank) and miR-302b–transfectedcells (right flank) in 4 nudemice, respectively. The grossmorphology of tumors(lower portion) showed themorphology of mice injected withmiR-302b and miR-ctrl. B, tumorgrowth curves. C, tumor weight. D,the expression levels of miR-302bwere detected by qRT-PCRanalysis in the tumor tissues fromthe animal. E, IHC staining ofKi-67/AKT2 in the tumor tissuefrom miR-ctrl and miR-302binjected mice. F, the expressionlevels of AKT2 were detected byWestern blot analysis in tissuesfrom the animal.






suppresser. Notably, miR-302b is located at chromosome4q25, a region that is controlled by altered levels of tran-scriptional factors such as NANOG and OCT3/4, but notDNA copy number alteration. Of particular interest, over-expression of OCT4, amaster regulator of stemness and self-renewal in embryonic stem cells, has been reported to beevidence of a higher grade of malignant cancers, includingbreast cancer, bladder cancer, and hepatocellular carcinomacancer cells (35–37). Although the role of stemness andreprogramming potential of miR-302b in ES cells has beenidentified, the underlying mechanism responsible fordecreased expression of miR-302b in hepatocellular carci-noma is still unknown. Thus, in this study we have evaluateda potential alteration in the expression of miR-302b, themain regulatory miRNA in hepatocellular carcinomatumor samples. We found that miR-302b was significantlydownregulated in liver cancer cell lines and hepatocellularcarcinoma tissues. From gain- and loss-of-function studies,miR-302b in liver cancer cells has been proven to decreasecell proliferation, clonogenicity, and also induced G1–Sarrest in vitro. Also, ectopic expression of miR-302b inSMMC-7721 and Bel-7404 cells inhibited proliferation ofliver tumors in vivo. Therefore, our data provided a morecomprehensive understanding of the tumor suppressor roleof miR-302b during hepatocellular carcinoma development.AKT2 is a homolog of the v-akt oncogene, a protein serine/

threonine kinase prosurvival protein, which ismember of theAKT family of proteins (AKT1, 2, and 3) that are activatedby the PI3K pathway (11). The PI3K pathway is one of themost potent prosurvival pathways in cancer (38). Thederegulation of the AKT signaling pathway has been asso-ciated with numerous other cancers, including glioblastoma,breast, prostate, lung, and liver cancers (3, 5). Numerousinvestigators have reported correlations between tumor AKTactivity and various clinicopathologic parameters (39). Inparticular, AKT activation has been shown to correlate withadvanced disease and/or poor prognosis in some tumor types(40). In light of its importance in the regulation of cellproliferation, AKT2 is becoming increasingly recognized asan essential target for potential anticancer inhibitors topromote progression through normal, unperturbed cellcycles. This is achieved by acting on diverse downstreamfactors involved in controlling the G1–S and G2–M transi-tions (5). Here, we demonstrated that miR-302b suppressedAKT2 expression by binding directly to the 30-UTR ofAKT2, and an inverse correlation was observed betweenmiR-302b and AKT2 expression in hepatocellular carcino-ma tissues. We showed that AKT was upregulated in hepa-tocellular carcinoma tissues and that silencingAKT by RNAiinhibited tumor properties of liver cancer cells, similar to thatof miR-302b overexpression (Figs. 1 and 4). Meanwhile,overexpression of AKT2 could eliminate the effect of miR-302b on hepatocellular carcinoma cells (Fig. 5). Therefore,upregulation of AKT2 in hepatocellular carcinoma becauseof miR-302b deprivation may, at least partially, explain theantitumor effects of miR-302b involved in hepatocellularcarcinoma. In a word, the reported targets of miR-302b andour observations indicated that miR-302b might regulate

AKT2-related signaling pathways, and loss of miR-302bwould lead to the tumor progression in hepatocellularcarcinoma. There is an interesting thing, from the resultsof Figs. 2, 4, and 5, we found that AKT2 might not be theonly relevant miR-302b target for the observed phenotype.Subramanyam and colleagues demonstrated that embryonicstem cell-specific cell cycle-regulating (ESCC) miRNAscould have hundreds of target gene and play the roles frommany different functional modules, for instance, cell-cycleregulation, vesicular transition, epigenetic regulation, cellsignaling, and epithelial–mesenchymal transition. miR-302b as a number of ESCC miRNAs, it might target andregulate other cell cycles regulators, such as CDKN1A,RBL2, and CDC2L6 in the process of cell proliferation (41).AKT2 is essential for progression from G0–G1 to S-phase

by activating the positive regulator of G1–S transition,including cyclin D1, cyclin D2, cyclin E1, during cell-cycleprogression (31). Here, we showed that activated AKT2phosphorylates various substrates at serine/threonine resi-dues directly or indirectly by regulating the expression ofvarious substrates, including P27kip1, CDK2, CYCLIND1,and CYCLINA. In this research, miR-302b and siRNAtargeted at AKT2 could reduce both the expression levelsand phosphorylation of CYCLIND1 at threonine 286,suggesting a potent cell-cycle progression role of miR-302b. Moreover, miR-302b and si-AKT2 could not abolishthe expression levels of P27, whereas they could increase thephosphorylation of P27 at Ser10 (Fig. 4F and L). CDK2, anS-phase cyclin-dependent kinase, is the target for AKTduring the cell-cycle progression. In previous research,CDK2 was reported to be phosphorylated directly by AKT2at threonine 39 residues both in vitro and in vivo (11). Here,reduced expression levels of CDK2 were detected after miR-302b and si-AKT2, suggesting an indirect effect induced bythe elimination of AKT2. Our findings suggest that miR-302 may function as a cell-cycle suppressor by targetingAKT2 in hepatocellular carcinoma.The above results suggested that miR-302b could act as a

potent therapeutic intervention of hepatocellular carcinoma.Further animal studies indicated that miR-302b couldsuppress the growth of hepatocellular carcinoma xenograftsin nude mice and decrease the expression of AKT2 in treatedtumors (Fig. 6). The in vivo studies support our observationsthat miR-302b targets AKT2 and suppresses liver cancer cellgrowth in vitro. Further systemic administration of miR-302b delivered by cholesterol conjugated 20-O-methyl-mod-ified oligonucleotides, lentivirus vector (42), AAV vectors(43), or locked nucleic acid-modified oligonucleotides (44)could be performed for further determination of the anti-cancer potential of hepatocellular carcinoma.In summary, we investigated the roles ofmiR-302b and its

targeted gene, AKT2, in their control of the cell cycle andtheir potential implication in pathologic processes. Ourfindings suggests that miR-302b may be a novel tumorsuppressor that blocks the growth of hepatocellular carcino-ma cells through targeting AKT2-related cell-cycle progres-sion signaling pathways. Our findings highlight the func-tional association of miR-302b and their host genes, provide

Wang et al.





new insight into the regulatory network of the cell cycle, andopen the possibility for future therapeutic interventions.

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

Authors' ContributionsConception and design: L. Wang, J. Yao, X. Le, K. Nan, T. Song, C. HuangDevelopment of methodology: J. Yao, X. Zhang, K. NanAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): X. Zhang, B. GuoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics, compu-tational analysis): L. Wang, Z. LiWriting, review, and/or revision of the manuscript: L. Wang, J. Yao, X. Le,M. Cubberly, Z. Li, K. Nan, C. Huang

Administrative, technical, or material support (i.e., reporting or organizing data,constructing databases): Z. Li, K. NanStudy supervision: K. Nan, T. Song, C. Huang

Grant SupportThis work was supported by the Key Science and Technology Major Program of

Shaanxi Province, China (2010ZDKG-50), the National Natural Science Foundationof China (31100921), The Fundamental Research Funds for the Central Universities(08142006), and The Program for Changjiang Scholars and Innovative ResearchTeam in University (PCSIRT: 1171).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be herebymarked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

Received August 5, 2013; revised December 2, 2013; accepted December 2, 2013;published OnlineFirst December 13, 2013.

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2014;12:190-202. Published OnlineFirst December 13, 2013.Mol Cancer Res Lumin Wang, Jiayi Yao, Xiaogang Zhang, et al. Targeting AKT2miRNA-302b Suppresses Human Hepatocellular Carcinoma by

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