子宫内膜基质细胞体外蜕膜化过程中的微小RNA差异表达谱及其对细胞周期的调控

MicroRNA expression and its role in the cell cycle regulation in decidualized endometrial stromal cells in vitro

目的 研究在子宫内膜基质细胞（ ESC）体外蜕膜化过程中的微小RNA （miRNA）差异表达谱及其细胞周期调控.方法 培养原代ESC,构建ESC体外蜕膜化模型（蜕膜化组）,并进行形态学及催乳素水平验证,鉴定其构建成功；以未经蜕膜化处理的ECS作为对照（对照组）.采用miRNA微阵列芯片技术筛选两组ESC中miRNA差异表达谱,利用实时荧光定量PCR技术对miRNA微阵列芯片检测结果进行验证.转染miR-222抑制物后,流式细胞仪检测两组体外蜕膜化ESC的细胞周期比例.结果 （1） miRNA微阵列芯片技术筛选结果显示,蜕膜化组与对照组中有49个ESCmiRNA表达水平具有显著差异,其中16个miRNA显著上调,33个miRNA显著下调.实时荧光PCR技术检测显示,hsa-miR-27b、30c、143、101、181b、29b、30d、507、23a、222、221的表达水平在蜕膜化组与对照组比较,差异均有统计学意义（P＜0.05）.（2）对照组转染阴性对照-6-羧基荧光素（NC-FAM）后S期细胞比例为（6.2±0.7）％,明显低于转染前的（10.9±0.8）％（P＜0.05）；而G0/G1期细胞[（77.5±1.3）％]较转染前[（73.0±1.6）％]有升高趋势,但差异无统计学意义（P＞0.05）.蜕膜化组转染后S期细胞比例[（3.3±0.6）％]较转染前[（7.8±0.9）％]明显减少（P＜0.05）；G0/G1期细胞比例[ （80.7±1.6）％]较转染前[（74.9±1.1）％]有升高趋势,但差异无统计学意义（P＞0.05）.对照组和蜕膜化组G2/M期细胞比例转染前、后比较,差异均无统计学意义（P均＞0.05）.结论 miRNA可能通过调控细胞周期进程参与ESC体外蜕膜化过程.

Objective To study microRNA （miRNA） expression and role of cell cycle regulation in decidualized endometrial stormal cells （ESC） in vitro.Methods ESC was induced decasualization in vitro and matched with non-decidualized cells as controls.The expression repertoire of miRNA was measured by microarray chip and was validated by real-time PCR.Flow cytometry was used to identify ESC cycle during decidual reaction in vitro and after miRNA222 inhibitor was transfected into it.Results （1） Between decidualized and undecidualized stromal cells,there were 49 miRNAs significantly different expression by microarray chip,including 16 miRNA up-regulation and 33 miRNA down-regulation.hsa-miR-27b,30c,143,101,181 b,29b,30d,507,23 a,222,221 exhibited significantly differential expression between decicualized and undecidualized stromal cells by real-time PCR （P 〈0.05）.（2） After miRNA222 inhibitor （NC-FAM） transfection to decidual ESC,ESC were cultured by FBS medium for 24 hours,the rate of transfection was 70%.ESC were transfected with miRNA 222 inhibitor and cultured for 48 hours,the percentage of ESC at Sphase of （6.2 ± 0.7 ） % were significantly lower than （ 10.9 ± 0.8 ） % in control group （ P 〈 0.05 ） ; the percentage of ESC at G0/G1 phase increased at transfection group [ （77.5 ± 1.3 ） % vs.（73.0 ± 1.6） % at control group ],but there was no significant difference （P 〉 0.05 ）.Decasualization ESC were transfected with miRNA 222 inhibitor and cultured for 48 h,the percentage of ESC at S-phase was （ 3.3 ± 0.6） % in transfection group,which were significantly lower than （7.8 ± 0.9 ） % in control group （ P 〈 0.05 ）.The percentage of ESC at G0/G1 phase was （ 80.7 ± 1.6 ） % in transfection group and （ 74.9 ± 1.1 ） %.In control group,which did not reached statistical difference （ P 〉 0.05）.Conclusion miRNA was involved in ESC decidual process in vitro by regulating cell cycle.