绵羊miR-200b对卵泡颗粒细胞周期和凋亡的影响

Effects of Ovine miR-200b on Cell Cycle and Apoptosis of Follicular Granulosa Cells

旨在揭示miR-200b在绵羊卵泡颗粒细胞的作用。本研究利用在线工具(TargetScan、miRTarBase及miRDB)预测miR-200b的靶基因,并利用KOBAS对其进行GO和KEGG通路富集分析。将分离培养的绵羊卵泡颗粒细胞分为4组,分别转染miR-200b mimic、mimic NC、miR-200b inhibitor和inhibitor NC,每组6个重复。采用CCK8法分别检测转染24、48和72 h颗粒细胞的存活率;采用荧光定量PCR检测miR-200b、CDK 4、CDK 6、CCND 1、CCND 2、Bax和Bcl-2基因的表达水平。利用3种在线工具预测到25个miR-200b的共同靶基因,GO和KEGG通路富集分析发现这些基因富集在细胞的增殖分化、细胞周期及生殖发育过程;转染miR-200b mimic、miR-200b inhibitor和NC后,颗粒细胞存活率随时间延长呈“V”型趋势降低,且48 h达最低(P<0.01);miR-200b inhibitor与inhibitor NC组相比,miR-200b表达量无显著差异(P>0.05);与mimic NC相比,miR-200b mimic组极显著促进miR-200b表达(P<0.001),显著下调CDK 4(P<0.01)、CDK 6(P<0.001)和CCND 1(P<0.001)、CCND 2(P<0.05)和Bcl-2(P<0.01)基因表达水平,Bax表达并无显著变化(P>0.05),且极显著下调Bcl-2与Bax比值(P<0.001)。综上所述,miR-200b抑制绵羊卵泡颗粒细胞细胞周期和增殖并促进其凋亡。

The aim of study was to reveal the effect of ovine miR-200b on the follicular granulosa cells.The predicted target genes of miR-200b by 3 online tools(TargetScan,miRTarBase and miRDB)were analyzed with KOBAS for GO and KEGG pathway enrichment.The isolated and cultured sheep follicular granulosa cells were divided into 4 groups transfected with miR-200b mimic,mimic NC,miR-200b inhibitor and inhibitor NC,respectively,with 6 replicates in each group.CCK8 was used to detect the proliferation of granulosa cells at 24,48 and 72 h after transfection.The expression level of miR-200b,CDK 4,CDK 6,CCND 1,CCND 2,Bax and Bcl-2 genes were measured by fluorescence quantitative PCR.A total of 25 common target genes of miR-200b were predicted by 3 online tools.These predicted target genes were enriched in cell proliferation and differentiation,cell cycle and reproductive development by GO and KEGG enrichment analysis.The survival rate of granulosa cell with time was a“V”decreased trendency and reached the lowest at 48 h(P<0.01)after transfection.There were no significant difference in miR-200b expression(P>0.05)between miR-200b inhibitor and inhibitor NC groups.Compared with mimic NC group,miR-200b mimic group significantly increased the expression of miR-200b(P<0.001),significantly down-regulated the expression of CDK 4(P<0.01),CDK 6(P<0.001),CCND 1(P<0.001),CCND 2(P<0.05)and Bcl-2(P<0.01),while the expression of Bax did not significantly change(P>0.05),and extremely significantly decreased the ratio of Bcl-2 to Bax(P<0.001).In summary,miR-200b can inhibit the cell cycle and proliferation,and promote the apoptosis of follicular granulosa cells in sheep.