长链非编码RNA A-30-P01019163对卵巢颗粒细胞βB2晶体蛋白表达的调控及对颗粒细胞增殖和凋亡的影响

LncRNA A-30-P01019163 regulatesβB2 crystallin gene expression in ovarian granulosa cells and its effects on cell proliferation and apoptosis

目的:探讨lncRNA A-30-P01019163对卵巢颗粒细胞βB2晶体蛋白(βB2 crystallin,CryBB2)表达的调控作用,以及其对卵巢颗粒细胞增殖、凋亡的影响。方法:构建小鼠卵巢颗粒细胞过表达lncRNA A-30-P01019163体内模型,同时基于人卵巢颗粒细胞系KGN构建过表达或干扰lncRNA A-30-P01019163稳定细胞株。采用qRT-PCR和Western印迹法检测lncRNA A-30-P01019163过表达或干扰后颗粒细胞中CryBB2的表达水平,采用EdU法检测细胞增殖,采用TUNEL法和流式细胞术检测细胞凋亡。结果:随着小鼠周龄增长,卵巢颗粒细胞lncRNA A-30-P01019163表达逐渐增加(P<0.05)。过表达lncRNA A-30-P01019163后,小鼠卵巢颗粒细胞中CryBB2 mRNA和蛋白表达均增加(P<0.01)。对卵巢颗粒细胞系KGN过表达lncRNA A-30-P01019163后,其CryBB2 mRNA和蛋白表达均增加(P<0.05);颗粒细胞增殖加快,细胞周期相关蛋白D1及增殖细胞核抗原蛋白表达均增加(P<0.01);颗粒细胞凋亡增加,凋亡相关蛋白半胱氨酸蛋白酶3(caspase-3)切割体及多聚ADP-核糖聚合酶(poly ADP-ribose polymerase,PARP)切割体表达均增加(P<0.05)。而干扰卵巢颗粒细胞系KGN中lncRNA A-30-P01019163后,CryBB2 mRNA和蛋白表达均下调(P<0.01);颗粒细胞增殖减慢,细胞周期相关蛋白D1及增殖细胞核抗原蛋白表达均降低(P<0.01);颗粒细胞凋亡减少,caspase-3及PARP切割体表达均降低(P<0.05)。结论:小鼠卵巢颗粒细胞中lncRNA A-30-P01019163表达随卵巢发育逐渐增加,参与调控CryBB2表达,并与颗粒细胞增殖、凋亡密切相关。

Objective:To explore the regulatory effect of lncRNA A-30-P01019163 on the expression of ovarian granulocytesβB2 crystallin(CryBB2)and its effects on ovarian granulocyte proliferation and apoptosis.Methods:The model of over-expression lncRNA A-30-P01019163 was constructed,and a stable cell line based on human ovarian granulosa cell line KGN with of overexpression or interference lncRNA A-30-P01019163 was established.The expression level of CryBB2 in after expression or interference was detected by qRT-PCR and protein blotting method,and the cell proliferation was detected by EdU,and apoptosis was detected by TUNEL and flow cytometric analysis.Results:As the mice age increasesd,the expression of lncRNA A-30-P01019163 in the granulocytes gradually increasesd(P<0.05).After overexpression of lncRNA A-30-P01019163 in KGN cells,the CryBB2 mRNA and protein expressions increased(P<0.05);granulocyte proliferation accelerated,cell cycle-related protein D1 and proliferating cell nuclear antigen expressions increased(P<0.01);the apoptosis of granulocytes increased,and cleaved caspase-3 and poly ADP-ribose polymerase(PARP)expressions were all increased(P<0.05).After interfering lncRNA A-30-P01019163 in KGN cells,CryBB2 mRNA and protein expressions were reduced(P<0.01);cell proliferation slowed down,cell cycle-related protein D1 and proliferating cell nuclear antigen expressions decreased(P<0.01);cell apoptosis decreased,cleaved caspase-3 and PARP expressions decreased(P<0.05).Conclusions:The expression of lncRNA A-30-P01019163 increased along with ovarian granulosa cell development and is involved in the regulation of CryBB2 protein expression.LncRNA A-30-P01019163 is closely related to proliferation and apoptosis of ovarian granulosa cells.