长链非编码RNANORAD在肺癌中的表达及其对肺癌细胞增殖能力的影响.docx

长链非编码RNANORAD在肺癌中的表达及其对肺癌细胞增殖能力的影响王靖L杨利杰，宋政，王瑞宇，张钱璐，李俊大理大学临床医学院，云南省大理市671000【摘要】目的研究长链非编码RNADNA损伤诱导的非编码RNA(NoRAD)在肺癌中的表达及其对肺癌细胞增殖的影响。方法RNA-Seq分析肺癌组织中LnCRNA及miRNA的表达改变，根据表达量确定候选LnCRNA；RT-qPCR检测分析肺癌组织及肺癌细胞中候选NORAD的表达；免疫荧光检测分析肺癌组织中细胞增殖核抗原Ki-67的表达；通过直线回归分析肺癌组织中NORAD与Ki-67的表达相关性。生物在线软件联合miRNA测序结果，并通过荧光素酶检测分析NORAD与miR-26b-5p>miR-654-5p结合可能性。将A549细胞分成空白对照组(ContrO1)、si-NORAD组、miR-26b-5pmimics、miR-654-5pmimics、si-NORAD与miR-26b-5pmimics联合组、si-NORAD与miR-654-5pmimics联合组，利用脂质体法将si-NORAD组、miR-26b-5pmimics、miR-654-5pmimics分别或联合转染入A549细胞，24h后，利用EdU检测细胞增殖能力、ArmeXinV-FITC/PI双染检测细胞凋亡、蛋白印迹(WeStemblot)检测细胞凋亡相关蛋白(Bad、Bd-2、CIeaVedCaSPaSe-3)的表达。结果NoRAD在肺癌组织及肺癌细胞中的表达均显著升高(均P<0.01),其表达量与Ki-67的表达呈正相关(r=0.646,P<0.01)omiR-26b-5p>miR-654-5p在肺癌组织及细胞中的表达均显著降低(均PV0.01),生物在线软件及荧光素酶检测验证，NORAD与miR-26b-5p、miR-654-5p存在结合位点；在肺癌组织中，miR-26b-5p(r=-0.403,P=O.000)、miR-654-5p(r=-0.423,P=0.000)的表达与Ki-67的表达均呈负相关；miR-26b-5p(=-0.435,PV0.05)与miR-654-5p(r=-0.395,P<0.05)的表达与NoRAD表达均呈负相关。与ComroI组比较，si-NORAD可显著抑制A549细胞的增殖(PV0.01)、促进其凋亡(PVO.01),Bad及CleaVedCaSPaSe-3的表达均显著升高(均PVO.01),Bd2的表达显著降低(PV0.05)；与Si-NoRAD组比较,Si-NoRAD与miR-26b-5pmimics联合组、si-NORAD与miR-654-5pmimics联合组可进一步抑制细胞增殖(PV0.01),促进细胞凋亡(PVO.01),Bad及CIeaVedcaspase-3的表达均显著提高(均P<0.05),Bcl-2的表达显著降低(PV0.05)。结论肺癌中NORAD的表达显著上调，可能通过抑制miR-26b-5p.miR-654-5p的表达而促进肺癌细胞增殖，NORAD可能作为肺癌的诊断标志物。*基金项目：大理大学博士科研启动基金项目(NoiKYBS)通讯作者：王靖,E-mail：【关键词】肺癌；长链非编码RNA；细胞增殖；微小RNAExpressionoflongnon-codingRNANORADinlungcanceranditseffectonlungcancercellproliferation.WANGJing,YANGLi-jie,SONGZheng,WANGRui-yu,ZHANGYu-l,LlJun.ClinicMedicalCollege,DaliUniversity,Dali671000,Yunnan,CHINA(AbstractObjectiveToinvestigatetheexpressionoflongnon-codingRNADNAdamageinducednon-codingRNA(NORAD)inlungcanceranditseffectontheproliferationoflungcancercells.MethodsTheexpressionchangesofLncRNAandmiRNAinlungcancertissueswereanalyzedbyRNA-seq,andcandidateLncRNAwereidentifiedaccordingtotheexpressionlevels.RT-qPCRwasusedtodetecttheexpressionofcandidateNORADinlungcancertissuesandlungcancercells.ImmunofluorescenceassaywasusedtodetecttheexpressionofKi-67inIungcancertissues.ThecorrelationbetweenNORADandKi-67expressioninlungcancertissueswasanalyzedbylinearregression.TheresultsofmiRNAsequencingwerecombinedwithbiologicalonlinesoftware,andthepossibilityofNORADbindingtomiR-26b-5pandmiR-654-5pwasanalyzedbyIucifasedetection.A549cellsweredividedintoblankControlgroup(Control),si-NORADgroup,miR-26b-5pmimics,miR-654-5pmimics,si-NORADandmiR-26b-5pmimicscombinedgroup,si-NORADandmiR-654-5pinthecombinedmimicsgroup,thesi-NORADgroup,miR-26b-5pmimicsandmiR-654-5pmimicswererespectivelyorjointlytransfectedintoA549cellsbyliposomemethod.24hlater,CellproliferationwasdetectedbyEdU,apoptosiswasdetectedbyAnnexinV-FITC/PIdoublestaining,andapoptosisrelatedproteins(Bad,Bcl-2,Cleavedcaspase-3)expressionwasdetectedbyWesternblot.ResultsTheexpressionofNORADinlungcancertissuesandlungcancercellswassignificantlyincreased(allPVo.01),andtheexpressionlevelofNORADwaspositivelycorrelatedwiththeexpressionofKi-67(r=0.646,PVO.01).TheexpressionsofmiR-26b-5pandmiR-654-5pinIungcancertissuesandcellsweresignificantlydecreased(allP<0.01).BiologicalonlinesoftwareandIuciferasedetectionverifiedthatNORADhadbindingsiteswithmiR-26b-5pandmiR-654-5p.Inlungcancertissues,theexpressionofmiR-26b-5p(r=-0.403,P<0.01)andmiR-654-5pQ=-0.423,P<0,01)wasnegativelycorrelatedwiththeexpressionofKi-67.TheexpressionsofmiR-26b-5p(r=-0.435,PVo.05)andmiR-654-5p(r=-0.395,PVO.05)werenegativelycorrelatedwithNORAD.ComparedwiththeControlgroup,si-NORADsignificantlyinhibitedtheproliferationofA549cells(P<0.01)andpromotedapoptosis(PVo.01),andsignificantlyincreasedtheexpressionofBadandCleavedcaspase-3(allP<0,01),andsignificantlydecreasedtheexpressionofBcl-2(P<0.05).Comparedwithsi-NORAD,si-NORADcombinedwithmiR-26b-5pmimicsgroupandsi-NORADcombinedwithmi-654-5pmimicsgroupcouldfurtherinhibitcellproliferation(P<0.01)andpromotecellapoptosis(P<0.01).TheexpressionofBadandCleavedcaspase-3wassignificantlyincreased(allP<0.05),andtheexpressionofBcl-2wassignificantlydecreased(PVO.05).ConclusionTheexpressionofNORADissignificantlyup-regulatedinlungcancer,whichmaypromotetheproliferationofIungcancercellsbyinhibitingtheexpressionofmiR-26b-5pandmiR-654-5p.NORADmaybeusedasadiagnosticmarkeroflungcancer.(Keywords)lungcancer;longnon-codingRNA;cellproliferation;microRNA非编码RNA(non-codingRNA,ncRNA)主要包括长链非编码RNA(LongNoncodingRNAs,LnCRNAS)和微小RNA(miRNAs),LnCRNAS是一种长度超过200个核甘酸ncRNA,主要位于细胞核或者细胞质中山。LnCRNAS在细胞生长、发育、肿瘤发生及进展等多种细胞生物学过程中发挥关键作用。LncRNAs可通过信号分子(SignalS)、诱饵分子(decoys)、导向分子(guides)和支架分子(SCaffOkl)等途径调控靶基因的表达叫此外，LncRNAs可通过竞争内源性RNA(CeRNAs)的方式，作为miRNA“分子海绵”来抑制miRNA介导的靶标mRNA的降解阳1。例如，LncRNAPVTl抑制miR-619-5p的表达后，增加人尾肢同源蛋白2(PygoPUS2,Pygo2)和自噬相关基因14(autophagy-related14,ATG14)的表达，导致Wnt/p-catenin和自噬通路的激活，促进胰腺癌患者吉西他滨耐药。LncRNALCATl可作为CeRNA,通过“海绵化"miR-4715-5p上调Ras相关C3肉毒菌素物底物1(ras-relatedC3botulinumtoxin,Racl)活性，进而促进肺癌的进展向。LnCRNADRAIC在肺癌中的表达显著升高，与患者的预后密切相关，其可通过抑制miR-223-3p的表达，促进肺癌细胞的增殖、迁移、侵袭。LncRNA可作为包括肺癌在内的多种肿瘤的诊断标志物及候选治疗靶点网。1.ncRNADNA损伤诱导的非编码RNA(NORAD),也叫LINCoO65