hUC-MSCs来源的外泌体miR-1260a影响PCOS患者颗粒细胞凋亡的研究

The effect of exosomal miR-1260a derived from human umbilical mesenchymal stem cells on apoptosis of granulosa cells in PCOS patients

目的探究人脐带间充质干细胞(hUC-MSCs)来源的外泌体miR-1260a靶向CASP8对多囊卵巢综合征(PCOS)患者颗粒细胞凋亡的影响。方法取健康足月胎儿的脐带进行组织块贴壁法分离培养获得hUC-MSCs,采用流式细胞仪检测其标志性蛋白CD73和CD90的表达。收集hUC-MSCs培养上清液,提取hUC-MSCs外泌体,透射电镜观察其形态,Western blot检测外泌体膜表面标志物热休克蛋白70(HSP70)和肿瘤易感基因101(TSG101)的表达。PKH67标记外泌体,采用激光共聚焦显微镜观察PCOS颗粒细胞对外泌体的内化作用。Western blot检测hUCMSCs外泌体对PCOS颗粒细胞凋亡的影响。利用GSE69909临床样本进行生物信息学分析,得到hUC-MSCs外泌体中异常高表达的miRNAs,并通过实时荧光定量PCR进行验证。合成miR-1260a模拟物及其对照转染至KGN细胞中,流式细胞术检测颗粒细胞凋亡率。利用Target Scan Human 7.2数据库进行miR-1260a靶基因的预测,采用双荧光素酶报告基因系统检测miR-1260a对靶基因的调控作用。qPCR检测PCOS颗粒细胞摄取hUC-MSCs外泌体前后miR-1260a及CASP8的表达情况。结果经鉴定所培养的细胞符合hUC-MSCs的特征,hUC-MSCs外泌体为直径30~150 nm的圆形或椭圆形囊泡状结构,表达标志物蛋白HSP70和TSG101,并且可被PCOS颗粒细胞摄取。与对照组相比,hUC-MSCs上清液处理组及hUC-MSCs外泌体处理组Cleaved-Caspase-8、Cleaved-Caspase-3和Bax蛋白表达显著下调,抗凋亡蛋白Bcl-2表达显著上调(P<0.05)。经GSE69909数据库筛选及qPCR验证发现miR-1260a在hUC-MSCs外泌体中富集,miR-1260a过表达可显著抑制颗粒细胞的凋亡(P<0.05),经验证miR-1260a可直接靶向CASP8且PCOS颗粒细胞摄取hUC-MSCs外泌体后,颗粒细胞中miR-1260a的表达升高,CASP8的表达降低(P<0.05)。结论hUC-MSCs来源的外泌体miR-1260a通过靶向CASP8抑制PCOS颗粒细胞凋亡。

Objective To explore the effect of exosomal miR-1260a derived from human umbilical mesenchymal stem cells(hUC-MSCs)on apoptosis of granulosa cells(GCs)in polycystic ovary syndrome(PCOS)by targeting CASP8.Methods hUC-MSCs were obtained from the umbilical cord of healthy full-term fetus by tissue explants adherent method.The surface markers CD73 and CD90 were detected by flow cytometry.The hUC-MSCs-exosomes(exos)were extracted from the supernatant of hUC-MSCs,and the morphology was observed by transmission electron microscopy(TEM).The surface heat shock protein(HSP70)and tumor susceptibility gene 101(TSG101)were detected by Western blot assay.Confocal microscope was used to observe whether PKH67 labeled hUC-MSCs-exos could be absorbed by GCs.Western blot assay was used to assess the protective effect of hUC-MSCs-exos on PCOS GCs.Clinical samples of GSE69909 were used for bioinformatics analysis.The differentially expressed miRNAs in hUC-MSCs-exos were obtained and verified by qPCR.miR-1260a mimics and negative control(NC)were synthesized and then transfected into KGN cells.Subsequently,cell apoptosis was measured by flow cytometry.Target genes of miR-1260a were predicted using Target Scan Human 7.2 database and verified by a dual-luciferase reporter gene assay.Expression levels of miR-1260a and CASP8 before and afteruptake of hUC-MSCs-exos by PCOS GCs were detected by qPCR.Results The isolated cells accorded with thecharacteristics of hUC-MSCs.hUC-MSCs-exos were circular or elliptical membranous vesicle with diameter ranged from30-150 nm and expressed the typical exosome markers HSP70 and TSG101.Confocal microscopy results showed that hUCMSCs-exos could be absorbed by GCs.Compared with the control group,the expression of Cleaved-Caspase-8,Cleaved-Caspase-3 and Bax were significantly down-regulated and the expression of Bcl-2 was significantly up-regulated in thehUC-MSCs supernatant treated and the hUC-MSCs-exos treated groups(P<0.05).After GSE69909 database screening andqPCR verification,it was found that miR-1260a was enriched in hUC-MSCs-exos,and the overexpression of miR-1260asignificantly inhibited the apoptosis of KGN cells(P<0.05).A dual-luciferase reporter gene assay showed that CASP8 was adirect target of miR-1260a.Moreover,the uptake of hUC-MSCs-exos by PCOS GCs increased the expression of miR-1260aand decreased the expression of CASP8(P<0.05).Conclusion Our study reveals that exosomal miR-1260a derived fromhUC-MSCs inhibits apoptosis of GCs in PCOS by targeting CASP8.