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DOI:http://dx.doi.org/10.7314/APJCP.2013.14.1.387 Let-7c Inhibits NSCLC Cell Proliferation by Targeting HOXA1

Asian Pacific J Cancer Prev, 14 (1), 387-392

Introduction

MicroRNAs (miRNAs) represent a class of endogenous, single-strand and highly conserved non-coding small RNAs. They post-transcriptionally regulate protein expression by directly binding the 3’-UTR of target genes and thus play critical roles in numerous biological processes, including development, apoptosis, life span and metabolism (Bartel, 2004). Let-7was the secondmiRNA tobe identified afterlin-4 in Caenorhabditis elegans (Pasquinelli et al., 2000). To date, thirteen members of the let-7 family have been identifiedinthehumangenome(Pasquinellietal.,2000;Roush et al., 2008). Previous studies have demonstrated that let-7 is underexpressed in various cancers and is related to the initiation, progression and prognosis of cancer (Takamizawa et al., 2004;Barh et al., 2010).Furthermore, restoration of its expression prevented tumor growth by targeting various oncogenes, including Ras, C-mycandHMGA2(Johnsonetal.,2007;Mayretal.,2007;Kumaretal.,2008;Kimetal.,2009). Let-7c is one member of the let-7 family and maps to21q11-21,aregionfrequentlydeletedinlungcancer(Yamada et al., 2008). Let-7c is poorly expressed in various cancers, including lung, liver and prostate (Navarro et al., 2009;Shimizuetal.,2010;Nadimintyetal.,2012).Directevidence for the potential of let-7c as a tumor suppressor

Institute of Clinical Pharmacology, Hunan Key Laboratory of Pharmacogenetics, Central South University, Changsha, China *For correspondence: hhzhou01@gmail.com

Abstract

Objective: The aim of the present study was to explore mechanisms by which let-7c suppresses NSCLC cell proliferation. Methods: The expression level of let-7c was quantified by qRT-PCR. A549 and H1299 cells were transfected with let-7c mimics to restore the expression of let-7c. The effects of let-7c were then assessed by cell proliferation, colony formation and cell cycle assay. Mouse experiments were used to confirm the effect of let-7c on tumorigenicity in vivo. Luciferase reporter assays and Western blotting were performed to identify target genes for let-7c. Results: HOXA1 was identified as a novel target of let-7c. MTS, colony formation and flow cytometry assays demonstrated that forced expression of let-7c inhibited NSCLC cell proliferation by inducing G1 arrest in vitro, consistent with inhibitory effects induced by knockdown of HOXA1. Mouse experiments demonstrated that let-7c expression suppressed tumorigenesis. Furthermore, we found that let-7c could regulate the expression of HOXA1 downstream effectors CCND1, CDC25A and CDK2. Conclusions: Collectively, these results demonstrate let-7c inhibits NSCLC cell proliferation and tumorigenesis by partial direct targeting of the HOXA1 pathway, which suggests that restoration of let-7c expression may thus offer a potential therapeutic intervention strategy for NSCLC. Keywords: Let-7c-NSCLC-HOXA1-G1arrest

RESEARCHARTICLE

Let-7c Inhibits NSCLC Cell Proliferation by Targeting HOXA1Min Zhan, Qiang Qu, Guo Wang, Ying-Zi Liu, Sheng-Lan Tan, Xiao-Ya Lou, Jing Yu, Hong-Hao Zhou*

was demonstrated by functional studies in several cancer cell lines. For example, the ectopic expression of let-7c promoted the granulocytic differentiation of AML cell linesandprimaryblastsbytargetingPBX2.Restorationof let-7c also inhibited cell proliferation, clonogenicity and the anchorage-independent growth of PCa cells in vitro and led to decreased metastasis in colorectal cancerbytargetingMMP11andPBX3(Hanetal.,2012;Nadiminty et al., 2012; Pelosi et al., 2012). In lungcancer, let-7c inhibited growth and cell cycle progression in vitro (Johnson et al., 2007;Esquela-Kerscher et al.,2008). However, mechanisms in which let-7c inhibits proliferationandtumorigenesisofNSCLCremainlargeunknown.In thecurrentstudy,HOXA1wasidentifiedasanoveltarget of let-7c. Furthermore, we demonstrated that let-7c inhibitedNSCLCcellproliferationandtumorigenesisbydirectlytargetingthe3’UTRofHOXA1mRNA,whichsubsequentlyreducedtheexpressionofCCND1,CDC25AandCDK2. Materials and Methods

Cell culture A549, H1299, 16HBE and HEK293 cells werepurchased from American Type Culture Collection (ATCC)andmaintainedinRPMI-1640supplementedwith

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10%fetalbovineserum,100U/mlpenicillinand100μg/mLstreptomycinat37℃with5%CO2.

miRNA and siRNA transfection Let-7c mimics (let-7c), small interfering RNA targetingHOXA1 (si-HOXA1) and negative control(NC)were purchased fromGenePharma (Shanghai,China).Cellswere transfectedwith100nM let-7c, si-HOXA1orNCusingLipofectamine2000 (Invitrogen,Carlsbad,CA,USA) according to themanufacturer’sinstruction. The target sequence of si-HOXA1 is5’-GTTGTGGTCCAAGCTATGG-3’ according to apreviousreport(Zhuetal.,2005).

Total RNA extraction and quantitative RT-PCR (qRT-PCR) Total RNA, including miRNA and mRNA, was extracted from cells or tissue usingTRIzol® reagent (Invitrogen),accordingtothemanufacturer’sinstructions. Let-7c expressionwas quantified using amiRNA-specificTaqmanMiRNAAssaykit(AppliedBiosystems,FosterCity,CA,USA).U6smallnuclearRNAwasusedas an internal control. To detect mRNA expression levels, total RNA was reverse-transcribedusingtheFirst-StrandcDNASynthesiskit (Fermentas). mRNA levels were determined by qRT-PCRusingSYBRgreenI(Invitrogen).β-actinwasusedas an internal control. All primer sequences are obtained frompreviousreports(Suzukietal.,2001;Jemaletal.,2007;Wangetal.,2010;Fengetal.,2011). AllqRT-PCRtestswereperformedusinganABI7500SequenceDetectionSystem.Therelativeexpressionfoldchange of mRNA and miRNAs were calculated using the 2-rrCt Method.

Lentiviral vector and generation of stable Cell Lines A let-7c lentivirus was purchased from Genepharma. This vector consisted of the pre-hsa-let-7c sequence cloned into theGLVH1/GFP (LV-let-7c) plasmid.AGLVH1/GFP plasmid expressing a scrambled RNAwasusedasacontrol(LV-NC).StableH1299celllinesexpressing let-7c or NC were generated by transfecting with LV-let-7c or LV-NC, respectively.

MTS assay Cellswereseededinto96-wellplates(3×103 viable cells/well)andincubatedfor24h,48h,72hand96h.At the indicated time points, cell viability was measured using theCellTiter96® Aqueous Non-Radioactive Cell ProliferationAssay(MTS)kit(Promega,Madison,WI,USA),accordingtothemanufacturer’sinstruction.

Colony formation assay Cellswerecollectedandseededinto10cm2 culture plates(500cellsperforeachplates)forcolonyformation.Mediawerereplacedevery3-4days,andafter12-14daysofincubation,colonieswerestainedinwith0.1%crystalvioletsolution(preparedin20%ethanol).

Flow cytometry for cell cycle analysis Cellswereseededinto6-wellplatesataconcentrationof2×105cells/wellandallowedtoadhereovernight.Cells

wereserum-starvedfor24hinRPMIsupplementedwith0.5%FBSandthenrestoredtoRPMIwith10%FBSforan additional 24 h. The cells were then collected for cell cycleprofileanalysisbyflowcytometry,accordingtothepreviousreport(Yinetal.,2012).

Western Blot CellsortissuewerelysedinRIPAbufferwith1mMPMSF(Sigma,St.Louis,MO,USA).TheBCAProteinAssayKit(Pierce,Rockford,Italy)wasusedtodetectthetotalproteinconcentration.Westernblotwasperformedas previously described (Simoncini et al., 2000).Thefollowing primary antibodies were used: rabbit polyclonal antibodiesagainstHOXA1,CCND1,CDC25AorCDK2andamousemonoclonalantibodyagainstβ-actin(allfromCellSignalingTechnology,Beverly,USA).

Construction of 3’UTR reporter plasmids and luciferase reporter assays The 3’UTR of HOXA1was amplified by PCRusing the following primer sequences: Forward, 5’-TTCCAGAACCGCCGAATGA-3’; Reverse,5’-TCCCAGCCCAAGGAGATGC-3’. The 3’UTRsegment was inserted into the pGL3.0 control vector (Promega) using theXBAⅠ site.Amutated 3’-UTRfragmentofHOXA1wassynthesizedafterpointmutationswere made in several bases within the binding sites. Both constructswereconfirmedbysequencing. HEK293 cellswere plated into 24-well plates at aconcentrationof 5×104 cells/well and allowed to growovernight. 200 ng luciferase reporter plasmid and 20 ng ofpRL-TK(Promega)weretransfectedwithlet-7corNC.Twenty-four hours after transfection, luciferase activity was measured using the dual luciferase reporter assay system(Promega)onaSIRIUSluminometer(BertholdDetection System, Pforzheim, Germany). Fireflyluciferase activity was normalized to Renilla luciferase activity for each transfected well.

In vivo assays Male BALB/c nudemice aged 4–6weekswerepurchased from the Laboratory Animal Centre of Hunan experiment center. All procedures were performed according to internationally accepted ethical guidelines. LV-let-7c-H1299 orLV-NC-H1299 cells (2×106 cells/mice)wereinjectedsubcutaneouslyintotherightflanks.Afterthemiceweresacrificed,expressionlevelsoflet-7c,HOXA1,CCND1,CDC25AandCDK2weremeasuredbyqRT-PCRand/orWesternblot.

Statistical analysis Datawereexpressedasmean±SDofatleastthreeindependent experiments.Differenceswere analyzedbyStudent’s t-test anda two-tailedPvalue

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Figure 1. NSCLC Cells Lose Let-7c and Express High Level of HOXA1. (A)QRT-PCRanalysisoflet-7cinA549,H1299and16HBEcells.(B,C)ThemRNA(B)andprotein(C)expressionlevelsofHOXA1inA549,H1299and16HBEcellsdetected byqRT-PCRandWestern blot. *p

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let-7c inA549andH1299cells.Wefound that siRNAknockdownofHOXA1inA549andH1299cellsledtosignificantcellgrowthinhibitionandG1arrest,similartothatinducedbylet-7c(Figure3D-3H).TheseresultsindicatethatthereductionofHOXA1expressioncouldmimicthe inhibitoryeffectsof let-7conNSCLCcells,suggesting that targeting HOXA1may be a novelmechanism mediating the tumor suppressor function of let-7cinA549andH1299cells.

Let-7c reduces the expression of the HOXA1 downstream effectors CCND1, CDC25A and CDK2. Previous studies have confirmed that CCND1,CDC25AandCDK2areinvolvedinregulationofG1/Stransition(Jinnoetal.,1994;Aleemetal.,2005;Liuetal.,2008).CCND1hasbeenreportedtobeadownstreamtarget ofHOXA1 inA549 cells (Cho et al., 2012).Microarray profiles have demonstrated thatCDC25AandCDK2weremodulated byHOXA1 inMCF-10cells(Mohankumaretal.,2007).Wethereforesoughttodeterminewhether theHOXA1-mediated downstreameffectorswerealsoimpactedbylet-7c.WefoundthattheknockdownofHOXA1reducedtheexpressionofCCND1,CDC25AandCDK2inA549(Figure4A,4C)andH1299cells(Figure4B,4D),whichissimilartopreviousstudies(Mohankumaretal.,2007;Choetal.,2012).Furthermore,the ectopic expression of let-7c downregulated the mRNA andprotein levels ofCCND1,CDC25AandCDK2 inA549(Figure4A,4C)andH1299cells(Figure4B,4D).Collectively,thesefindingsdemonstratethatlet-7ccouldregulatetheexpressionofHOXA1downstreameffectorsCCND1,CDC25AandCDK2.

Let-7c suppresses tumorigenesis in vivo To further study the role of let-7c in tumorigenesis, we evaluated the effect of let-7c overexpression on tumor growthinvivo.LV-let-7c-H1299orLV-NC-H1299cellswereinjectedsubcutaneouslyintotherightdorsalflanksof nude mice.

Thirty days after injection, the average tumor weight was also significantly reduced in theLV-let-7c-H1299group,comparedwiththeLV-NC-H1299group(Figure5B).Moreover,wemeasured the expressionof let-7c,HOXA1,CCND1,CDC25A andCDK2 in harvestedtumor tissues.Similar to the in vitro, the let-7c levelsweresignificantlyupregulated(Figure5C),andHOXA1,CCND1,CDC25A andCDK2 levelswere decreased(Figure5D,5E)intumorsderivedfromLV-let-7c-H1299cells,ascomparedtoLV-NC-H1299cells. Discussion

Inthepresentstudy,HOXA1wasidentifiedasanoveltargetoflet-7c.Ectopicexpressionoflet-7csignificantlysuppressedNSCLC cell proliferation and inducedG1arrest in vitro, which is consistent with the previous study(Johnsonetal.,2007).Wealsodemonstratedthatthe overexpression of let-7c could inhibit tumorigenesis invivo.Furthermore,wedemonstratedthatHOXA1anditsdownstreameffectors,CCND1,CDC25AandCDK2,contributedtotheinhibitoryeffectoflet-7conNSCLCcells. Admittedly, we could not exclude other additional factors within the complicated network regulating cell proliferation and tumorigenesis that we observed because a single miRNA could target multiple messenger RNAs (Bartel, 2004).

HOXA1isamemberofthehomeodomain-containingtranscription factor family, which plays a pivotal role during development (Chen et al., 2003). Previous studies havesuggestedthatHOXA1isoverexpressedinvariouscancers, including breast, liver, oral and lung cancer (Chariotetal.,1996;Abeetal.,2006;Bituetal.,2012;Zhaetal.,2012).TheforcedexpressionofHOXA1promotedoncogenictransformationbymodulatingthep44/42MAPkinasepathwayandbcl-2inMCF-10cells(Mohankumaret al., 2007).TheknockdownofHOXA1 reduced cellgrowthinbladderandlungcancercells(Choetal.,2012).Furthermore,highHOXA1expressionwascorrelatedwithpoor prognosis in oral squamous cell and hepatocellular carcinomas.On the other side,HOXA1was verifiedas a target ofmiR-10a, and the reduction ofHOXA1expression promoted invasiveness of pancreatic cancer

Figure 4. Figure 4. Let-7c Reduces the Expression of CCND1, CDC25A and CDK2. (A, B) QRT-PCR analysis ofCCND1,CDC25AandCDK2mRNAexpression inA549(A)andH1299(B)cellstransfectedwithlet-7c,si-HOXA1orNCfor48h.(C,D)WesternblotdemonstratingproteinlevelsofCCND1,CDC25AandCDK2inA549(C)andH1299(D)cellstransfectedwithlet-7c,si-HOXA1orNCfor48h.*p

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(Ohuchidaetal.,2012).Therefore,thefunctionofHOXA1is likely complicated, functioning as a proto-oncogene or oncogene, depending on specific conditions and indifferenttypesofcancer.Inthepresentstudy,HOXA1is found to be unregulated inA549 andH1299 cellscomparedto16HBEcells.Additionally,theknockdownofHOXA1inhibitedgrowthofcellsandinducedG1arrest.These results demonstrate that, similar to previous studies, HOXA1playsaroleasaproto-oncogeneinNSCLCcells(Abeetal.,2006;Choetal.,2012).

Let-7c has been shown to modulate the expression of CCND1atboththemRNAandproteinlevels,althoughthe study did not confirmwhether let-7c could directmodulate CCND1 through the 3’UTR of itsmRNA(Gongetal.,2011).CCND1hasbeenverifiedasadirecttarget of let-7b (Schultz et al., 2008). CDC25Awasreported to be a direct target of let-7 (Johnson et al., 2007). Bioinformatic analysis predicts that let-7c could targetCCND1 andCDC25A.We speculated that let-7ccouldalsomodulateCCND1andCDC25Adirectlythrough the 3’UTRsof theirmRNA transcripts. In thepresent study, we demonstrated that both the ectopic expressionof let-7candknockdownofHOXA1bysi-HOXA1downregulatedCCND1,CDC25AandCDK2.Taken together, we proposed that let-7c could modulate CCND1andCDC25A,notonlythroughknockdownofHOXA1indirectlybutalsobydirectlyinteractingwiththeir3’UTRs.FurtherexperimentsareneededtoconfirmwhetherCCND1andCDC25Aaredirecttargetsoflet-7c.

Insummary,thecurrentstudyprovides,forthefirsttime, an important link between let-7c-mediated tumor growthinhibition,G1-phasecellcyclearrestofNSCLCcells and the downregulation of HOXA1/CCND1,CDC25AandCDK2.Restoration of let-7c expressionmay thus provide a potential therapeutic intervention strategyforNSCLC.

Acknowledgements

The author(s) declare that they have no competing interests. This work was supported by the National Natural ScienceFoundationofChina (No.81072706) and863Project(No.2012AA02A517).

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