miR-22通过靶向MTDH抑制胶质瘤细胞的生长

miR-22通过靶向MTDH抑制胶质瘤细胞的生长李荣国;王剑;杨少陵【摘要】背景与目的：miR-22miR-22控MTDH表达抑制胶质瘤细胞生长，从而进一步揭示miR-22的抑瘤机制。方法：运用实时定量PCR(quantitativereal-timepolymerasechainreaction，qRT-PCR7517miR-22MTDH3’UTR-荧光素酶报告载体，通过荧光素酶报告检测miR-22MTDH3’UTRmiR-22mimicsU251MTDHsiRNA(WesternblotMTDHMTTmiR-22U251qRT-PCR，miR-2275miR-22能特异性地与MTDH3’UTRWesternblot，miR-22MTDHMTDHMTTmiR-22MTDHU251(P<0.05)。结论：miR-22MTDH长。%Backgroundandpurpose:miR-22hasbeenreportedtobedown-regulatedingastriccancer,lungcancer,colorectalcancer,andbreastcancer.However,itsexpressioningliomawasstillpoorlyknown.ThisstudyaimedtoexplicitwhethermiR-22suppressescellproliferationbytargetingMTDH,thustorevealmolecularmechanismthatmiR-22functionsasatumorsuppressoringlioma.Methods:Quantitativereal-timepolymerasechainreaction(qRT-PCR)wasconductedfordetectingtheexpressionofmiR-22ingliomasandnormalbraintissues.MTDH3’UTR-luciferasevectorwasconstructedanddual-luciferasereportergeneassaywasemployedtoexaminetheeffectofmiR-22onluciferaseactivity.U251cellsweretransfectedwithmiR-22mimics,andMTDHsiRNAasforpostivecontrol,thenWesternblotwasperformedtodetecttheexpressionsofMTDHprotein.TheproliferationabilityofU251cellswasevaluatedbyMTTassay.Results:miR-22wasdown-regulatedingliomatissues.GliomapatientswithrelativelyhighexpressionofmiR-22showedlowermortalitycomparedwithlowexpressionofmiR-22byusingKaplan-Meiersurvivalcurves.WedemonstratedmiR-22couldbindtothe3’untranslatedregion(UTR)ofMTDHandinhibitedtheluciferaseactivity.WesternblotshowedthattheexpressionofMTDHproteinwasinhibitedbyrestoredmiR-22orsiRMTDHinU251cells.OverexpressionofmiR-22orsiRMTDHinhibitedtheproliferationofU251cells.Conclusion:miR-22suppressescellproliferationbytargetingMTDHinglioma.【期刊名称】《中国癌症杂志》【年(卷),期】2014(000)006【总页数】5页(P401-405)【关键词】miR-22;胶质瘤;异粘蛋白;生长【作者】李荣国;王剑;杨少陵【作者单位】南华大学附属第一医院急诊科，湖南衡阳421001;南华大学附属第一医院急诊科，湖南衡阳421001;南华大学附属第一医院急诊科，湖南衡阳421001【正文语种】中文【中图分类】R739.41miRNAs(miR22ntRNAmRNA3’非编码区结合从而在转录后水平负性调控基因的表达［1］。miRmiRmiR，miRmiR-22MTDHreal-timepolymerasechainreaction，qRT-PCR)、蛋白质印迹法(WesternblotMTTmiR-22病例资料收集2009年1月—2012年12月南华大学附属第一医院收治的确诊为胶质瘤患者75例。其中男性49例，女性26例，中位年龄39(9～58)岁；其中病理分级Ⅰ级15例、Ⅱ级19例、Ⅲ级34例、Ⅳ级7例。病理诊断结果均经2名以上病理科医师确认。以17例正常大脑组织为对照组，均来自开颅手术的脑外伤患者。主要材料miR-22mimics及scramble购自Ambion公司。MTDHsiRNA质粒购自Santacruz公司。MTDH3’UTR的各种报告基因(Wild-MTDH-3’UTRvector，Mut-MTDH-3’UTRvector)由复能基因公司构建，双报告基因载体购自复能基因公司购买。双荧光素酶活性检测试剂盒购自Promega公司。MTDH抗体和β-actin抗体购自CST公司。TRIzol和LipofectamineTM2000转染试剂购自美国Invitrogen公司。RPMI-1640培养基和小牛血清购自Gibco公司。MTT粉购自美国Sigma公司。qRT-PCRRNARNAcDNA。PCR20μLTaqDNA(5U/μL)0.2μL，2×SYBR10μL，miRNA2.0μL，miR-PCR(5μmol/L0.4μL，灭菌蒸馏水7.4μL。循环体系为：95℃3min，9512s，6235s，7230s，35β-actinmiR-222-ΔΔCTWesternblotmiR-22mimicsMTDHsiRNAU25148hSDSPVDF5MTDHβ-actin，430min1h，TBST30min，然后加ECL发光剂，X荧光素酶活性检测将荧光素酶报告载体与miR-22mimics或scramblemimics共转染U251细胞。48hPromega操作，在单光子检测仪检测细胞荧光素酶的活性。计算相对荧光素酶活性=萤火虫荧光素酶活性值/海肾荧光素酶活性值。MTT消化转染细胞，接种于96孔板中，每孔3000个细胞，每组设5个复孔，于37℃、CO2体积分数为5%的培养箱中培养。在未接种细胞的孔中加入RPMI-1640中作调零孔。接种72h后，每孔加20μLMTT液，37℃温育4h，用酶标仪测定570nm波长吸光度值(A570)。实验重复3次。统计学处理采用SPSS13.0软件进行统计学分析。所有结果均以表示，两组间比较采用t检验，P＜0.05为差异有统计学意义。miR-22RNA，U6snRNAqRT-PCRmiR-221miR-2212-△△CtmiR-22751.047±0.121，17miR-2212.263±0.142，差异有统计学意义(P＜0.0011)。miR-22miR-22miR-22754626(34.67%75423Kaplan-Meier存曲线分析发现，在随访的患者中，46例miR-22低表达组中死亡28例(60.9%)，26miR-228(30.8%)，miR-22miR-22P＜0.052miR-22生存时间越长，预后越好。miR-22MTDH3’UTR运用生物信息学方法，通过在线预测软件(TargetscanandMiranda)预测发现miR-22MTDH3AMTDHmiR-22miR-22mimics(Wt-miR-22/MTDH)或突变型(Mut-miR-22/MTDH)重组质粒。单光子检测发现，miR-22mimicsWtmiR-22/MTDH56P＜0.05，图3B)。miR-22MTDHmiR-22MTDHmiR-22mimicsU251miR-22-scrambleMTDHsiRNA48hWesternblotmiR-22siRNAMTDH4)。结果提示，miR-22MTDHmiR-22miR-22-mimics、miR-22-scrambleMTDHsiRNA(阳性U25148hMTTMTDHsiRNAmiR-22mimicsU25148h明显减慢，与miR-22scramble对照组细胞相比，差异均有统计学意义(P＜0.05，5miR-22MTDHU251细胞的增殖。恶性胶质瘤是起源于胶质细胞的恶性肿瘤，也是脑肿瘤中最常见的类型。胶质瘤约80%，尽管目前治疗方案已有改进，但胶质瘤的死亡率仍较高，诊断后平均生存期为12～15个月［13-14］。胶质瘤主要的治疗方式以外科根治术加放化疗辅助治疗为主，放化疗辅助治疗的目的是阻滞细胞周期的演进以及促进肿瘤细胞的调亡［15］。然而，恶性胶质瘤的早期复发率高，其主要原因是肿瘤细胞对放化疗抵抗所致。因此亟待发现新的治疗靶点。miRNAHeLamiR-22行了研究。Tang［3］miR-22Ling［4］研究显示，miR-22ErbB3A549H1299miR-22miR-22［16］研究表明，miR-22HDAC4Kaplan-MeierLi［5］miR-22TIAM1HCT-116本研究通过qRT-PCR检测发现miR-22在胶质瘤组织中表达下调，且Kaplan-Meier生存分析发现，miR-22表达越低的胶质瘤患者其生存时间越短，预后越差。反之，miR-22表达越高的胶质瘤患者其生存时间越长，预后越好。我们通过荧光素酶报告载体和Westernblot证实MTDH是miR-22直接调控的靶基因。MTDH最初在胎儿胶质细胞瘤中发现，是一个HIV诱导的基因，并且作为癌基因参与多种肿瘤的发生、发展。研究证实MTDH在乳腺癌、肝癌、结肠癌、膀胱癌、以及子宫内膜癌中表达上调，并且通过PI3K-AKT、NF-κB、MAPK以及Wnt/β-catenin信号途径参与肿瘤发生的多种生物学过程［9-12,17］。本研究还发现在U251细胞中过表达miR-22或干扰MTDH后能明显抑制U251细胞的增殖，表明miR-22可能通过下调MTDH的表达抑制胶质瘤细胞的增殖。综上所述，miR-22在胶质瘤细胞中表达下调，并通过直接靶向调控MTDH的表达发挥抑瘤效应，miR-22/MTDH轴的证实为胶质瘤的治疗提供了新的治疗靶点。【相关文献】［1］AMBROSV.microRNAs:tinyregulatorswithgreatpotential［J］.Cell,2001,107(7):823-826.［2］TANGH,DENGM,TANGY,etal.miR-200bandmiR-200casprognosticfactorsandmediatorsofgastriccancercellprogression［J］.ClinCancerRes,2013,19(20):5602-5612.［3］TANGH,KONGY,GUOJ,etal.DiallyldisulfidesuppressesproliferationandinducesapoptosisinhumangastriccancerthroughWnt-1signalingpathwaybyup-regulationofmiR-200bandmiR-22［J］.CancerLett,2013,340:72-81.［4］LINGB,WANGGX,LONGG,etal.TumorsuppressormiR-22suppresseslungcancercellprogressionthroughposttranscriptionalregulationofErbB3［J］.JCancerResClinOncol,2012,138(8):1355-1361.［5］LIB,SONGY,LIUTJ,etal.miRNA-22suppressescoloncancercellmigrationandinvasionbyinhibitingtheexpressionofT-celllymphomainvasionandmetastasis1andmatrixmetalloproteinases2and9［J］.OncolRep,2013,29(5):1932-1938.［6］XIONGJ,YUD,WEIN,etal.Anestrogenreceptoralphasuppressor,microRNA-22,isdown-regulatedinestrogenreceptoralpha-positivehumanbreastcancercelllinesandclinicalsamples［J］.FEBSJ,2010,277(7):1684-1694.［7］SONGSJ,ITOK,ALAU,etal.TheoncogenicmicroRNAmiR-22targetstheTET2tumorsuppressortopromotehematopoieticstemcellself-renewalandtransformation［J］.CellStemCell,2013,13(1):87-101.［8］POLISENOL,SALMENAL,RICCARDIL,etal.IdentificationofthemiR-106b25microRNAclusterasaproto-oncogenicPTEN-targetingintronthatcooperateswithitshostgeneMCM7intransformation［J］.SciSignal,2010,3(117):29.［9］TOKUNAGAE,NAKASHIMAY,YAMASHITAN,etal.Overexpressionofmetadherin/MTDHisassociatedwithanaggressivephenotypeandapoorprognosisininvasivebreastcancer［J］.BreastCancer,2014,21(3):341-349.［10］ZHUK,DAIZ,PANQ,etal.Metadherinpromoteshepatocellularcarcinomametastasisthroughinductionofepithelial-mesenchymaltransition［J］.ClinCancerRes,2011,17(23):7294-7302.［11］WANGN,DUX,ZANGL,etal.PrognosticimpactofMetadherin-SND1interactionincoloncancer［J］.MolBiolRep,2012,39(12):10497-10504.［12］ZHOUJ,LIJ,WANGZ,etal.Metadherinisanovelprognosticmarkerforbladdercancerprogressionandoverallpatientsurvival［J］.AsiaPacJClinOncol,2012,8(3):e42-e48.［13］SRINIVASANS,PATRICI