【摘要】 目的:探讨miRNA-200c的表达对卵巢癌细胞侵袭能力的影响,并初步分析其可能的作用机制。方法:应用Lipofectamine 2000瞬时上调ES-2细胞中miR-200c的表达,并通过荧光定量PCR验证;Transwll侵袭小室检测细胞侵袭能力的变化;生物信息学的方法预测miR-200c的靶基因,同时荧光素酶报告基因实验验证E盒结合锌指蛋白2(zinc finger E-box bingding homeobox 2,ZEB2)是miR-200c的靶基因;蛋白印迹法检测E-钙黏蛋白和波形蛋白的表达水平。结果:pre-miR-200c转染ES-2细胞后,转染组miR-200c的表达(4.82±1.73)较对照组(1.00±0.16)明显升高,t=-67.28,P<0.001;上调miR-200c后,转染组(27.70±1.19)与对照组(57.30±2.01)相比,细胞的侵袭能力明显减弱,t=11.17,P<0.001;蛋白印迹法的结果显示,转染组ZEB2的表达水平为0.38±0.015,较对照组的0.66±0.024明显下降,t=94.67,P<0.001。采用生物信息学的方法预测ZEB2是miR-200c的靶基因;且荧光素酶报告实验结果证实,共转染野生型荧光素酶质粒和pre-miR-200c,与其他各组相比,荧光素酶活性明显降低,差异有统计学意义,P<0.001;转染pre-miR-200c后,转染组E-钙黏蛋白的表达(0.75±0.023)较对照组(0.32±0.014)明显增加,t=-97.77,P<0.001,波形蛋白的表达为0.28±0.010,较对照组0.67±0.129明显降低,t=71.64,P<0.001。结论:miR-200c能通过靶向ZEB2抑制人卵巢癌细胞的侵袭能力,其异常表达与卵巢癌细胞的生物学行为密切相关。更多还原

【Abstract】 OBJECTIVE:To explore the effect of miRNA-200c(miR-200c)on the invasion ability of ovarian cancer cells and to analysis its possible mechanism.METHODS:Transit transfection with Lipofectamine 2000was used to up-regulate the expression of miR-200cin ES-2cells and real-time quantitative PCR was used to identify the effect.Transwell assay was used to evaluate the ES-2cells invasion ability.Apply bioinformatics analysis to predict miR-200ctarget genes and luciferase reporter assay was used to determine whether ZEB2was the direct target of miR-200c.The expression of E-cad and vimentin were detected by western blot.RESULTS:The miR-200cexpression of ES-2cells was significantly up-regulated after pre-miR-200ctransfection when compared with control group(t=-67.28,P<0.001).Pre-miR-200ctransfection group can significantly decrease the invasion ability of ES-2cell compared with control group(t=11.17,P<0.001).The difference of the expression of ZEB2protein in pre-miR-200ctransfection group(0.38±0.015)was statistically significant(t=94.67,P<0.001)when compared with control group(0.66±0.024).ZEB2is the prediction target gene of miR-200cby the method of bioinformatics.The luciferase activity decreased significantly in cotransfection of the wild-type luciferase plasmid and pre-miR-200cgroup when compared with other groups(P<0.001).The expression of E-cadherin protein was significantly increased(t=-97.77,P<0.001)and the expression of vimentin protein(t=71.64,P<0.001)was decreased after pre-miR-200ctransfection.CONCULUSION:The miR-200cup-regulation can inhibit ovarian cancer cell invasion by targeting ZEB2,and abnormal expression of miR-200cis closely relevant to the biological effect of ES-2cells.更多还原