miR-23a-3p通过调控靶基因Gja1参与调控大鼠卵泡闭锁及颗粒细胞凋亡

miR-23a-3p regulates follicular atresia and granulosa cell apoptosis through target gene Gja1

目的探讨miR-23a-3p在大鼠卵泡闭锁及卵巢颗粒细胞凋亡过程中的作用机制。方法分别在SD大鼠卵巢囊中注射miR-23a-3p模拟物/抑制剂,转染72 h后,采用HE染色法检测大鼠卵巢组织形态变化及闭锁卵泡数变化情况;采用生物信息学及荧光素酶报告系统预测并验证miR-23a-3p的靶基因及其结合位点;体外分离培养大鼠卵巢颗粒细胞,并分别转染miR-23a-3p模拟物及抑制剂,采用荧光定量PCR(RT-PCR)法检测转染72 h后颗粒细胞中miR-23a-3p及其靶基因缝隙连接蛋白43(Gja1)的表达情况;Gja1干扰慢病毒转染大鼠颗粒细胞,转染72 h后,采用RT-PCR和Western blot检测大鼠卵巢颗粒细胞中Gja1在mRNA和蛋白水平的表达情况;用流式细胞术检测转染miR-23a-3p模拟物/抑制剂、Gja1干扰慢病毒后卵巢颗粒细胞的凋亡情况。结果HE染色结果显示,大鼠卵巢囊注射miR-23a-3p模拟物后卵巢中的闭锁卵泡数量显著增高于模拟物对照组(P<0.05);双荧光素酶报告实验发现,转染miR-23a-3p模拟物组显著降低了荧光素酶活性(P<0.05);RT-PCR结果显示,转染miR-23a-3p模拟物后卵巢颗粒细胞中miR-23a-3p的表达显著增加,Gja1则显著降低(P<0.05),而转染miR-23a-3p抑制剂后对内源性miR-23a-3p和Gja1的表达无显著影响(P>0.05);用Gja1慢病毒干扰载体/对照载体分别转染大鼠卵巢颗粒细胞72 h后,RT-PCR和Western blot结果显示,Gja1干扰组Gja1在mRNA水平及蛋白水平均显著低于对照组(P<0.05);Western blot检测结果显示,转染miR-23a-3p模拟物后卵巢颗粒细胞中Gja1的表达显著降低(P<0.05);流式细胞术检测发现,转染miR-23a-3p模拟物后显著促进了卵巢颗粒细胞凋亡,下调Gja1后卵巢颗粒细胞凋亡率显著增加(P<0.05)。结论miR-23a-3p通过调控靶基因Gja1参与调节大鼠卵泡闭锁及卵巢颗粒细胞的凋亡。

Objective:To explore the effects of miR-23a-3p in ovarian follicular atresia and granulosa cell apoptosis in rats.Methods:miR-23a-3p simulant or inhibitor was respectively injected into the ovarian sac of SD rats.Seventy-two hours after transfection,HE staining was used to detect the changes of ovarian tissue morphology and number of atretic follicles.The target genes and binding sites of miR-23a-3p were predicted and verified by bioinformatics and luciferase reporting system.Rat ovarian granulosa cells were isolated and cultured in vitro and transfected with miR-23a-3p simulant and inhibitor respectively.The expression of miR-23a-3p and its target gene(Gja1)in granulosa cells was detected by fluorescence quantitative PCR(RT-PCR)72 hours after transfection.Rat granulosa cells were interfered with Gja1 interference lentivirus.The mRNA and protein expression of Gja1 in rat ovarian granulosa cells were detected by RT-PCR and Western blot 72 hours after transfection.The apoptosis of ovarian granulosa cells was detected by flow cytometry after transfection with miR-23a-3p simulant/inhibitor or Gja1 interference lentivirus.Results:He staining showed that the number of atretic follicles in the ovary of rats injected with miR-23a-3p simulant was significantly higher than that of the control group(P<0.05).The double luciferase report experiment showed that the luciferase activity was significantly reduced in miR-23a-3p simulant group(P<0.05).RT-PCR showed that the expression of miR-23a-3p increased significantly and Gja1 decreased significantly after transfection of miR-23a-3p simulant(P<0.05).Transfection of miR-23a-3p inhibitor had no significant effect on the expression levels of endogenous miR-23a-3p and Gja1(P>0.05).Western blot showed that after transfection with miR-23a-3p simulant,the expression of Gja1 in ovarian granulosa cells decreased significantly(P<0.05),and there was no significant change in the expression level of Gja1 in the inhibitor group(P>0.05).After rat ovarian granulosa cells were transfected with Gja1 lentivirus interference vector/control vector for 72 hours,the results of RT-PCR and Western blot showed that the mRNA and protein levels of Gja1 in Gja1 interference lentivirus group were significantly lower than those in control group(P<0.05).Flow cytometry showed that transfection of miR-23a-3p simulant significantly promoted the apoptosis of ovarian granulosa cells,and the apoptosis rate of ovarian granulosa cells increased significantly after down regulating Gja1(P<0.05).Conclusions:miR-23a-3p involves in ovarian follicle atresia and granulosa cell apoptosis through target Gja1.