MiR-296-3p调节多囊卵巢综合征大鼠颗粒细胞凋亡的作用机制

Mechanism of MiR-296-3p Regulating Granulosa Cell Apoptosis in Rats with Polycystic Ovary Syndrome

目的:探讨miR-296-3p对多囊卵巢综合征(polycystic ovary syndrome,PCOS)大鼠颗粒细胞凋亡的影响。方法:全自动化学发光免疫分析法检测PCOS大鼠血清雌二醇(estradiol,E2)、孕酮(progesterone,P)、睾酮(testosterone,T)水平;利用苏木素-伊红染色(hematoxylin-eosin staining,HE)检测卵巢病理学改变;TUNEL法检测卵巢细胞凋亡;qPCR检测卵巢miR-296-3p表达水平;将各质粒分别转染卵巢颗粒细胞(ovarian granulosa cells,GCs)后分为miR-NC、miR-296-3p mimic、miR-296-3p inhibitor、miR-296-3p mimic+Vector、miR-296-3p mimic+PRKCA和miR-296-3p mimic+PRKCA+NF-κB组,以未处理的GCs为对照组;Western blot法检测p38、p-p38及NF-κB的表达水平,利用StarBase在线预测和双荧光素酶报告实验验证miR-296-3p与PRKCA的靶向关系,流式细胞术检测细胞凋亡率。结果:与对照组相比,PCOS大鼠卵巢中E2、P、T水平升高,细胞凋亡比例增加,miR-296-3p表达升高;miR-296-3p与PRKCA具有靶向关系,在3'UTR区存在结合位点;与miR-NC组相比,miR-296-3p mimic组PRKCA mRNA表达水平显著下调,p-p38/p38和NF-κB表达上调,miR-296-3p inhibitor组PRKCA mRNA表达水平显著上调,p-p38/p38和NF-κB表达下调;与miR-NC组相比,miR-296-3p mimic组和miR-296-3p mimic+Vector组细胞凋亡比例显著上调;与miR-296-3p mimic组和miR-296-3p mimic+Vector组相比,miR-296-3p mimic+PRKCA组卵巢颗粒细胞凋亡比例显著下调;与miR-296-3p mimic+PRKCA组相比,miR-296-3p mimic+PRKCA+NF-κB组卵巢颗粒细胞凋亡比例显著上调。结论:miR-296-3p通过靶向PRKCA激活p38 MAPK/NF-κB信号通路介导多囊卵巢综合征大鼠颗粒细胞凋亡。

Objective:To investigate the effect of miR-296-3p on granulosa cell apoptosis in rats with polycystic ovary syndrome(PCOS).Methods:The levels of estradiol(E2),progesterone(P)and testosterone(T)in serum of PCOS rats were detected by automatic chemiluminescence immunoassay;HE staining was used to detect the pathological changes of the ovary;TUNEL was used to detect apoptosis in the ovary;The expression level of miR-296-3p and PRKCA in the ovary was detected by qPCR;Ovarian granulosa cells(GCs)were divided into miR-NC,miR-296-3p mimic,miR-296-3p inhibitor,miR-296-3p mimic+Vector,miR-296-3p mimic+PRKCA and miR-296-3p mimic+PRKCA+NF-κB groups after transfection of various plasmids,and untreated GCs were used as control group.The expression level of p38,p-p38 and NF-κB was detected by Western blot.StarBase website and double luciferase report test predicted and verified the targeting relationship between miR-296-3p and PRKCA,respectively.Flow cytometry was used to detect cell apoptosis rate.Results:Compared with the control group,the levels of E2,P and T in the ovaries of PCOS rats increased;The proportion of apoptosis increased;The expression of miR-296-3p increased.miR-296-3p has a targeting relationship with PRKCA,and there are binding sites in the 3'UTR region.Compared with miR-NC group,the expression level of PRKCA mRNA in miR-296-3p mimic group was significantly decreased,and p-p38/p38 and NF-κB expression was up-regulated.Compared with miR-NC group,the expression level of PRKCA mRNA in miR-296-3p inhibitor group was significantly increased,and p-p38/p38 and NF-κB expression was down-regulated.Compared with the Mir-NC group,the percentage of apoptosis of miR-296-3p mimic and miR-296-3p mimic+Vector groups was significantly up-regulated.Compared with miR-296-3p mimic and miR-29-3p mimic+Vector,the percentage of apoptosis of ovarian granulocyte cells in miR-296-3p mimic+PRKCA group was significantly down-regulated.Compared with miR-296-3p mimic+PRKCA+NF-κB group,the percentage of apoptosis of ovarian granulosa cells in miR-296-3p mimic+PRKCA+NF-κB group was significantly up-regulated.Conclusion:MiR-296-3p activates p38 MAPK/NF-κB signal pathway by targeting PRKCA and mediating granulosa cell apoptosis in rats with polycystic ovary syndrome.