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

doi:10.11843/j.issn.0366-6964.2017.08.021

摘要/Abstract

摘要：

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（3~7 mm）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48 h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Cyclin B1 mRNA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-2 mRNA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。

Abstract:

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.

中图分类号:

S826.2

李婷, 马爱团, 张英杰, 刘月琴, 段春辉. 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[J]. 畜牧兽医学报, 2017, 48(8): 1551-1556.

LI Ting, MA Ai-tuan, ZHANG Ying-jie, LIU Yue-qin, DUAN Chun-hui. Effects of Silencing INHα Gene by RNAi on Cycle,Apoptosis-Related Genes in Sheep Granulosa Cells[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(8): 1551-1556.

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