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

doi:10.11958/20221070

天津医药 ›› 2023, Vol. 51 ›› Issue (4): 337-343.doi: 10.11958/20221070

• 细胞与分子生物学 • 下一篇

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

赵元元(), 吴小华()

石家庄市第四医院（河北医科大学附属妇产医院）生殖中心，河北省母胎医学重点实验室，石家庄市生殖健康与不孕不育研究所（邮编050011）

收稿日期:2022-07-06 修回日期:2022-10-21 出版日期:2023-04-15 发布日期:2023-04-20
通讯作者: 吴小华 E-mail:summerzhaoyy@163.com;wuxiaohua1965@163.com
作者简介:赵元元（1988），女，主管检验师，主要从事生殖内分泌方面研究。E-mail：summerzhaoyy@163.com
基金资助:
河北省自然科学基金面上资助项目(H2019106051);河北省自然科学基金青年资助项目(H2022106020);石家庄市科学技术研究与发展计划项目(211460513)

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

ZHAO Yuanyuan(), WU Xiaohua()

Center for Reproductive Medicine, the Fourth Hospital of Shijiazhuang (Gynecology and Obstetrics Hospital Affiliated to Hebei Medical University); Key Laboratory of Maternal and Fetal Medicine of Hebei Province; the Institute of Reproductive Health and Infertility, Shijiazhuang 050011, China

Received:2022-07-06 Revised:2022-10-21 Published:2023-04-15 Online:2023-04-20
Contact: WU Xiaohua E-mail:summerzhaoyy@163.com;wuxiaohua1965@163.com

赵元元, 吴小华. hUC-MSCs来源的外泌体miR-1260a影响PCOS患者颗粒细胞凋亡的研究[J]. 天津医药, 2023, 51(4): 337-343.

ZHAO Yuanyuan, WU Xiaohua. The effect of exosomal miR-1260a derived from human umbilical mesenchymal stem cells on apoptosis of granulosa cells in PCOS patients[J]. Tianjin Medical Journal, 2023, 51(4): 337-343.

摘要/Abstract

摘要：

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hUC-MSCs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。

关键词: 多囊卵巢综合征, 脐带, 间质干细胞, 外泌体, 粒层细胞, 细胞凋亡, 微RNAs, miR-1260a

Abstract:

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 after uptake of hUC-MSCs-exos by PCOS GCs were detected by qPCR. Results The isolated cells accorded with the characteristics of hUC-MSCs. hUC-MSCs-exos were circular or elliptical membranous vesicle with diameter ranged from 30-150 nm and expressed the typical exosome markers HSP70 and TSG101. Confocal microscopy results showed that hUC-MSCs-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 the hUC-MSCs supernatant treated and the hUC-MSCs-exos treated groups (P<0.05). After GSE69909 database screening and qPCR verification, it was found that miR-1260a was enriched in hUC-MSCs-exos, and the overexpression of miR-1260a significantly inhibited the apoptosis of KGN cells (P<0.05). A dual-luciferase reporter gene assay showed that CASP8 was a direct target of miR-1260a. Moreover, the uptake of hUC-MSCs-exos by PCOS GCs increased the expression of miR-1260a and decreased the expression of CASP8 (P<0.05). Conclusion Our study reveals that exosomal miR-1260a derived from hUC-MSCs inhibits apoptosis of GCs in PCOS by targeting CASP8.

Key words: polycystic ovary syndrome, umbilical cord, mesenchymal stem cells, exosomes, granulosa cells, apoptosis, microRNAs, miR-1260a

中图分类号:

R711.75

图/表 9

表1 qPCR引物序列

Tab.1 Primer sequence of qPCR

基因名称	引物序列（5'→3'）	产物大小（bp）
miR-1260a	上游：AGATCCCACCTCTGCC 下游：AGTGCAGGGTCCGAGGTATT	64
U6	上游：TGGAACGCTTCACGAATTTGCG 下游：GGAACGATACAGAGAAGATTAGC	68
CASP8	上游：GCTAAATTCCACACTACC 下游：CTTGGGTTCAAGCAATC	114
β-actin	上游：CAGAGCAAGAGAGGCATCC 下游：CTGGGGTGTTGAAGGTCTC	217

图1 hUC-MSCs的形态特征及表面抗原标志物鉴定 A：细胞培养至第3代，形态学观察（×100）；B：流式细胞术检测hUC-MSCs的表面标志物。

Fig.1 Morphological observation and identification of hUC-MSCs and surface antigen markers

图2 hUC-MSC外泌体的鉴定及被PCOS GCs摄取的情况 A：透射电镜图；B：Western blot检测HSP70、TSG101表达情况；C：免疫荧光检测PCOS GCs对外泌体的摄取情况，PKH67标记外泌体，DAPI标记细胞核（×200）。

Fig.2 Identification of exosomes derived from hUC-MSCs and the uptake of exosomes by pGCs

表2 各组处理后PCOS颗粒细胞凋亡蛋白的表达水平比较（n=3，$\bar{x}±s$）

Tab.2 Comparison of expression levels of Cleaved-Caspase-8, Cleaved-Caspase-3, Bax and Bcl-2 between the three groups of PCOS GCs

组别	Cleaved- Caspase-8	Cleaved- Caspase-3	Bax	Bcl-2
PBS对照组	1.26±0.15	2.19±0.13	1.64±0.04	0.29±0.11
hUC-MSCs上清液组	0.64±0.08^a	1.42±0.20^a	1.14±0.08^a	0.69±0.28^a
hUC-MSCs外泌体组	0.53±0.07^a	1.07±0.01^a	0.67±0.10^a	1.72±0.34^a
F	40.872^＊＊	51.686^＊＊	114.260^＊＊	23.807^＊＊

图3 hUC-MSCs-exos对PCOS颗粒细胞中Cleaved-Caspase-8、Cleaved-Caspase-3、Bax和Bcl-2蛋白表达水平的影响 A：PBS对照组；B：hUC-MSCs上清液处理组；C：hUC-MSCs外泌体处理组。

Fig.3 Effects of hUC-MSCs-exos on protein expression levels of Cleaved-Caspase-8, Cleaved-Caspase-3, Bax and Bcl-2 in PCOS granulosa cells

图4 miR-1260a在hUC-MSCs-exos中富集 A：利用GEO数据库GSE69909，分析miR-1260a在hUC-MSC-exos中的表达；B：qPCR检测miR-1260a的表达，F=122.902，P<0.01，a与PCOS GCs比较，P<0.05。

Fig.4 miR-1260a enriched in hUC-MSCs-exos

图5 miR-1260a抑制KGN颗粒细胞的凋亡 A：流式细胞凋亡图；B：凋亡率统计图。

Fig.5 miR-1260a inhibited cell apoptosis of KGN cells

图6 CASP8与miR-1260a靶向关系的预测与验证

Fig.6 Prediction and validation of the targeting relationship between CASP8 and miR-1260a

图7 miR-1260a和CASP8在PCOS颗粒细胞中的表达

Fig.7 Expression levels of miR-1260a and CASP8 in PCOS GCs

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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

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