细胞自噬及其micrornas调控分子的筛选与验证-生物医学工程学专业论文.docxVIP

LC3-II 表达增多更为显著。（2）筛选出与自噬相关的microRNAs为miR-142-5p，经生物实验验证miR-142-5p对雷帕霉素诱导的MCF7 细胞自噬中LC3表达无明显影响；（3）筛选与卵巢癌细胞自噬有关的4 个microRNAs 中，其中一个microRNA在雷帕霉素诱导SKOV-3 细胞情况下，瞬时转染microRNAs 模拟物或microRNAs 抑制剂，Western Blotting结果中microRNAs 模拟物可降低LC3 蛋白的表达水平，反之，microRNAs 抑制剂可使LC3 蛋白表达水平升高。荧光显微镜下观察到，microRNAs模拟物使细胞中GFP-LC3 点状聚集物减少，而microRNA抑制剂物却增加了GFP-LC3 点状聚集物的聚集。而且其余3 个microRNAs 模拟物对SKOV-3 细胞中LC3表达无显著变化；（4）Western Blotting结果显示SH-SY5Y 细胞的LC3-II 蛋白表达回转组高于对照组回转+氯化铵组显著高于回转组和对照组，荧光显微镜下观察到GFP-LC3 点状聚集物随之增多。结论雷帕霉素可以诱导SKOV-3 细胞和MCF7 细胞产生自噬效应，miR-142-5p 对MCF7 细胞的自噬无明显作用。而筛选出的与卵巢癌细胞自噬有关的4个microRNAs中亦有一个microRNAs对SKOV-3细胞自噬有影响，其余3个microRNAs对SKOV-3细胞自噬无统计学意义。自噬相关microRNAs分子的筛选鉴定，可以促进自噬分子机制的认识。模拟微重力可诱导SH-SY5Y细胞中自噬体增多，故自噬对神经细胞产生的影响可能为航天医学的研究提供一个新的线索。关键词：自噬，microRNAs，模拟微重力，回转，微管相关蛋白1轻链3InductionofAutophagyand the ScreeningandValidationof Autophagy-related MicroRNAsAbstractBackground andObjectiveAutophagyisaprocessofself-degradationandrecyclingofcellularconstituents. Autophagyexistsineukaryoticcells,whichisacommonphenomenon.Autophagyhas essentialrolesinsurvival,development,homeostasisandcontributestoavarietyofdiseases. MicroRNAs(miRNAs)areendogenous,non-protein-encodingsmallsingle-strandedRNAs. MicroRNAscantargetmRNAsfordegradationorinhibittheirtranslationthroughsequence complementary.LotsofreportshaveshownthatmicroRNAsareinvolvedinvariousbiologic processes,therefore,microRNAsplayimportantrolesinthedevelopmentandprogressionof manydiseases,includingcancer,neurodegenerativediseases,infections,aging,heartdisease, etc.RecentstudieshaveobservedthatmicroRNAscanregulateAtgproteinsexpressionsin theformationofautophagosome.Inthisstudy,potentialautophagy-relatedmicroRNAswere screenedthroughaseriesofbioinformaticsmethods,andthentheywerefurtherverifiedby biologicalexperiments.Inaddition,theeffectsofautophagyontheneuronalcellsunder simulatedmicrogravityhavenotbeenreported.Toexplorewhetherautophagywereinvolved intheeffectsofmicrogravityontheneuronalcells,theexpressionofautophagy-relatedgene LC3inSH-SY5Ycellsunder