Fu Xiafei, He Yuanli, Wang Xuefeng, Peng Dongxian, Chen Xiaoying, Li Xinran, Wang Qing
Department of Obstetrics and Gynecology, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong, People's Republic of China.
Stem Cell Res Ther. 2017 Aug 14;8(1):187. doi: 10.1186/s13287-017-0641-z.
Chemotherapy-induced premature ovarian failure (POF) is a severe complication affecting tumor patients at a childbearing age. Mesenchymal stem cells (MSCs) can partially restore the ovarian structure and function damaged by chemotherapy. miR-21 is a microRNA that can regulate cell apoptosis. This study discusses the repair effect and mechanism of MSCs overexpressing miR-21 on chemotherapy-induced POF.
Rat MSCs and granulosa cells (GCs) were isolated in vitro. MSCs were transfected with miR-21 lentiviral vector (LV-miR-21) to obtain MSCs stably expressing miR-21 (miR-21-MSCs). The microenvironment of an ovary receiving chemotherapy was mimicked by adding phosphamide mustard (PM) into the cellular culture medium. The apoptosis rate and the mRNA and protein expression of target genes PTEN and PDCD4 were detected in MSCs. Apoptosis was induced by adding PM into the culture medium for GCs, which were cocultured with miR-21-MSCs. The apoptosis rate and the mRNA and protein expression of PTEN and PDCD4 were detected. The chemotherapy-induced POF model was built into rats by intraperitoneal cyclophosphamide injection. miR-21-MSCs were transplanted into the bilateral ovary. The rats were sacrificed at 15, 30, 45, and 60 days after the last injection. The ovarian weights, follicle count, estrous cycle, and sex hormone levels (estradiol (E2) and follicle-stimulating hormone (FSH)) were detected. Apoptosis of GCs was determined by TUNEL assay. The miR-21 and mRNA and protein expression of PTEN and PDCD4 were determined.
The apoptosis decreased in MSCs transfected with miR-21. The mRNA and protein expression of target genes PTEN and PDCD4 was downregulated. GCs cocultured with miR-21-MSCs showed a decreased apoptosis, an upregulation of miR-21, and a downregulation of PTEN and PDCD4. Following the injection of miR-21-MSCs, the ovarian weight and follicle counts increased; E levels increased while FSH levels decreased, with less severe apoptosis of GCs. The miR-21 expression in the ovaries was upregulated, while the mRNA expression and protein expression of PTEN and PDCD4 were downregulated.
Overexpression of miR-21 in MSCs promoted efficacy against chemotherapy-induced POF and its improvement of the repair effect was related to the inhibition of GC apoptosis by targeting PTEN and PDCD4.
化疗诱导的卵巢早衰（POF）是影响育龄期肿瘤患者的一种严重并发症。间充质干细胞（MSCs）可部分恢复化疗损伤的卵巢结构和功能。miR-21是一种可调节细胞凋亡的微小RNA。本研究探讨过表达miR-21的MSCs对化疗诱导的POF的修复作用及机制。
体外分离大鼠MSCs和颗粒细胞（GCs）。用miR-21慢病毒载体（LV-miR-21）转染MSCs，以获得稳定表达miR-21的MSCs（miR-21-MSCs）。通过向细胞培养基中添加磷酰胺芥（PM）模拟接受化疗的卵巢微环境。检测MSCs中的凋亡率以及靶基因PTEN和PDCD4的mRNA和蛋白表达。向GCs的培养基中添加PM诱导凋亡，将其与miR-21-MSCs共培养。检测凋亡率以及PTEN和PDCD4的mRNA和蛋白表达。通过腹腔注射环磷酰胺建立大鼠化疗诱导的POF模型。将miR-21-MSCs移植到双侧卵巢。在最后一次注射后15、30、45和60天处死大鼠。检测卵巢重量、卵泡计数、动情周期和性激素水平（雌二醇（E2）和卵泡刺激素（FSH））。通过TUNEL法检测GCs的凋亡情况。检测miR-21以及PTEN和PDCD4的mRNA和蛋白表达。
转染miR-21的MSCs凋亡减少。靶基因PTEN和PDCD4的mRNA和蛋白表达下调。与miR-21-MSCs共培养的GCs凋亡减少，miR-21上调，PTEN和PDCD4下调。注射miR-21-MSCs后，卵巢重量和卵泡计数增加；E水平升高而FSH水平降低，GCs的凋亡减轻。卵巢中miR-21表达上调，而PTEN和PDCD4的mRNA表达和蛋白表达下调。
MSCs中miR-21的过表达增强了对化疗诱导的POF的疗效，其修复效果的改善与通过靶向PTEN和PDCD4抑制GCs凋亡有关。
