RNA干扰INHα基因对绵羊颗粒细胞周期及凋亡相关基因表达的影响

Effects of Silencing INHαGene by RNAi on Cycle,Apoptosis-Related Genes in Sheep Granulosa Cells

旨在研究RNA干扰INHα基因对绵羊颗粒细胞周期及凋亡相关基因表达的影响,明确抑制素对绵羊颗粒细胞的局部调节作用,为今后提高动物的繁殖力奠定基础。选用1.0~1.5岁的小尾寒羊,从卵泡(3~7mm)中分离培养颗粒细胞,将siINHα和siNC(阴性对照)转染颗粒细胞,以未转染的细胞为空白对照(Control),利用脂质体介导法进行转染,荧光定量RT-PCR检测INHα基因的沉默效率、细胞周期和凋亡相关基因的变化,流式细胞仪检测细胞周期的分布变化。结果表明,siRNA转染绵羊颗粒细胞48h后,INHα基因的沉默效率达85%以上。与阴性对照相比,细胞周期相关基因Cyclin D1和CyclinB1mRNA的表达水平分别降低了0.2倍和0.7倍(P<0.05),而p21的mRNA表达水平升高了0.9倍(P<0.05);细胞凋亡通路中的促凋亡因子Bax、p53和Caspase3的mRNA表达量分别升高了1.8倍、3.6倍和0.4倍(P<0.05),而抗凋亡因子Bcl-2mRNA表达量不受影响,阴性对照与空白对照差异不显著(P>0.05)。与阴性对照相比,细胞周期的分布显著改变,干扰组G1细胞比例显著升高(P<0.05),S期细胞比例显著降低(P<0.05),而阴性对照与空白对照间无显著差异(P>0.05)。综上表明,INHα作为绵羊颗粒细胞中关键的调控因子,通过调控绵羊颗粒细胞的生长和凋亡参与调控绵羊卵泡排卵的过程。

The aim of this study was to investigate the effects of silencing the inhibin α-subunit （INHα） gene by RNAi on cell cycle and apoptosis-related genes in sheep granulosa cells （GCs）, explore the local regulation of INHα on sheep GCs, which can provide a foundation for improving the livestock＇s fecundity. siINHα and siNC （negative control） were transfected into GCs from antral follicles （3-7 mm） obtained from the ovaries of Thin-tailed Han sheep （1.0-1.5 years old）. Non-transfected cells were as blank control （Control）. The mRNA expression of INHα and the genes related to cell cycle and apoptosis were analyzed by quantitative real-time PCR （qRT-PCR）. The cell cycle progression was measured by flow cytometry. The results showed that siRNA was transfected into sheep GCs at 48 h with the silencing efficiency over 85%. Knockdown of INHα by siRNA significantly decreased the mRNA expressions of the cell cycle related genes CyclinD1 and Cyclin B1 by 0.2 and 0.7 times, respectively （P〈0.05）, but increased the mRNA expression of p21 by 0.9 times （P〈0.05）. Meanwhile, the mRNA expressions of pro-apoptotic genes Bax, p53 and Caspase3 were increased by 1.8, 3.6 and 0.4 times, respectively （P〈0.05）, but no significant changes of anti-apoptosis gene Bcl-2 compared with negative control. There was no significant difference between the control and negative control （P〉0.05）. INHα gene silencing altered the progression of cell cycle as well. The percentage of cell at S phase was decreased, while the percentage of cell at G1 phase was significantly increased compared with the negative control （P〈0.05）. No significant difference was observed between control and negative control （P〉0.05）. These results indicated that INHα was a key regulator in GCs and involved in the regulation of folliculogenesis by regulating the growth and apoptosis of GCs in sheep.