Du Jing, Lin Xiufeng, Wu Riran, Gao Zixuan, Du Yan, Liao Yuechan, Quan Song
Reproductive Medical Center, Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical University Guangzhou, China.
Reproductive Medical Center, Boai Hospital of Zhongshan Zhongshan, Guangdong Province, China.
Am J Transl Res. 2020 Jul 15;12(7):3660-3673. eCollection 2020.
Polycystic ovary syndrome (PCOS) is associated with alteration of Apelin signaling in ovarian granulosa cells (GCs). However, the molecular mechanisms regulating Apelin expression remain poorly understood. This study aims to investigate the role of miR-424 in modulating Apelin expression and GC functions.
miRNA expression in GCs was altered by transfection with specific miR-424 mimics and inhibitors. Apelin level was determined by ELISA. miR-424 and mRNA expression were analyzed by quantitative RT-PCR. Protein abundance was measured by western blotting. Genomic sequence targeted by miR-424 was validated by dual-luciferase reporter assay. Apelin gene was overexpressed by transfection of LV-003 vector carrying its cDNA. GC proliferation was analyzed by MTS method, and its cell cycle progression and apoptosis were measured by flow cytometry.
Apelin concentration was increased in serum and follicular fluid from PCOS patients, accompanied by upregulated APJ (Apelin receptor) expression and suppressed miR-424 expression in GCs. miR-424 mimics suppressed Apelin and APJ expression in KGN cells by targeting 3' UTR of Apelin and APJ, whereas miR-424 inhibitors had the opposite effects. miR-424 inhibited KGN cell proliferation and cell cycle progression by down-regulating Cyclin-D/E expression. Moreover, miR-424 promoted KGN cell apoptosis by increasing truncated Caspase-3 level. The regulation of KGN cell proliferation and apoptosis by miR-424 was mediated by directly suppressing Apelin gene expression, instead of inhibiting Apelin peptide activity.
miR-424 suppresses proliferation and promotes apoptosis of human ovarian granulosa cells by directly targeting and inhibiting Apelin and APJ expression.
多囊卵巢综合征（PCOS）与卵巢颗粒细胞（GCs）中Apelin信号通路的改变有关。然而，调节Apelin表达的分子机制仍知之甚少。本研究旨在探讨miR-424在调节Apelin表达和GC功能中的作用。
通过转染特异性miR-424模拟物和抑制剂来改变GCs中的miRNA表达。采用ELISA法测定Apelin水平。通过定量RT-PCR分析miR-424和mRNA表达。采用蛋白质印迹法检测蛋白质丰度。通过双荧光素酶报告基因检测验证miR-424靶向的基因组序列。通过转染携带Apelin cDNA的LV-003载体来过表达Apelin基因。采用MTS法分析GC增殖，通过流式细胞术检测其细胞周期进程和凋亡情况。
PCOS患者血清和卵泡液中Apelin浓度升高，同时GCs中Apelin受体（APJ）表达上调，miR-424表达受到抑制。miR-424模拟物通过靶向Apelin和APJ的3'UTR抑制KGN细胞中Apelin和APJ的表达，而miR-424抑制剂则产生相反的效果。miR-424通过下调细胞周期蛋白D/E的表达抑制KGN细胞增殖和细胞周期进程。此外，miR-424通过增加截短的半胱天冬酶-3水平促进KGN细胞凋亡。miR-424对KGN细胞增殖和凋亡的调节是通过直接抑制Apelin基因表达介导的，而非抑制Apelin肽的活性。
miR-424通过直接靶向并抑制Apelin和APJ的表达来抑制人卵巢颗粒细胞的增殖并促进其凋亡。
