miR-9-5p调控雄激素受体的表达促进卵巢颗粒细胞增殖  

miR-9-5p promotes ovarian granulosa cell proliferation in patients with polycystic ovary syndrome by regulating androgen receptor expression

作　　者：王芳 杨丽华[1] 余柯达[1] 何健英[1] 邹立波[1] WANG Fang;YANG Li-hua;YU Ke-da;HE Jian-ying;ZOU Li-bo(Reproductive Center,Jinhua People’s Hospital,Jinhua 321000)

机构地区：[1]金华市人民医院生殖中心,金华321000

出　　处：《生殖医学杂志》2021年第2期236-242,共7页Journal of Reproductive Medicine

基　　金：金华市科技计划项目(2018-4-050)。

摘　　要：目的分析微小RNA-9-5p(miR-9-5p)在多囊卵巢综合征(PCOS)中的诊断价值,并探讨对卵巢颗粒细胞(KGN)增殖的作用机制。方法采用荧光定量PCR(qRT-PCR)技术分析miR-9-5p在50例PCOS患者(实验组)及50例健康女性(对照组)血清中的表达特征。采用受试者工作特征曲线(ROC)评估miR-9-5p在诊断PCOS中的曲线下面积(AUC)、特异度及敏感度。培养KGN细胞并分为空白组、阴性转染组及转染组,分别采用MTT及流式细胞技术检测干扰miR-9-5p后KGN增殖和细胞周期的变化。利用双荧光素酶报告基因实验检测miR-9-5p对雄激素受体(AR)的直接调节作用。采用western blot技术分析干扰miR-9-5p后KGN中AR蛋白表达水平的变化。结果与对照组相比,miR-9-5p在PCOS患者血清中的表达显著升高(P<0.05)。ROC显示,miR-9-5p在诊断PCOS中AUC为0.76,敏感性为65.43%,特异性为74.09%。与空白及阴性转染组相比,转染拮抗剂后KGN中miR-9-5p表达水平显著降低(P<0.05)。干扰miR-9-5p显著降低KGN增殖(P<0.05),并抑制细胞周期G1到S期的转变,表现为G0/G1期细胞比例增加,而S期细胞比例减少。双荧光素酶报告基因实验显示,AR是miR-9-5p的靶基因,miR-9-5p可直接与AR mRNA 3’-非编码区互补结合而降低KGN中AR蛋白的表达水平。结论miR-9-5p在PCOS患者血清中表达上调,并可能是一种新的PCOS诊断标志物。在机制上,miR-9-5p通过直接调控AR而促进KGN增殖。Objective:To analyze the diagnostic value of microRNA-9-5p(miR-9-5p)in polycystic ovary syndrome(PCOS),and explore its effect and mechanism on the proliferation of ovarian granulosa-like tumor cell line(KGN).Methods:The expression of miR-9-5p in serum was analyzed by real-time PCR(qRT-PCR)for 50 PCOS patients(experimental group)and 50 healthy female controls(control group).The area under the curve(AUC),specificity and sensitivity of serum miR-9-5p in the diagnosis of PCOS were assessed by using receiver operating characteristic curve(ROC).KGN cells were cultured and divided into blank group,negative transfection group and transfection group.MTT and flow cytometry were used to detect the changes of KGN cells proliferation and cell cycle after mir-9-5p interference.The double luciferase reporter gene assay was used to detect the direct regulation of the androgen receptor(AR)by miR-9-5p.The expression of AR protein in KGN after miR-9-5p interference was analyzed by Western blot.Results:The expression of miR-9-5p was significantly elevated in serum of PCOS patients compared with healthy female controls.The ROC showed that miR-9-5p had an AUC of 0.76 in the diagnosis of PCOS with a sensitivity of 65.43%and a specificity of 74.09%.The expression level of miR-9-5p in KGN was significantly reduced after transfection of the antagomir compared to the blank and negative transfection groups(P<0.05).Knockdown of miR-9-5p significantly reduced KGN proliferation and inhibited cell cycle G1 to S phase transition,showing an increase in the proportion of cells in G0/G1 phase and a decrease in the proportion of cells in S phase.Double luciferase reporter gene assay showed that AR was a target gene of miR-9-5p,and miR-9-5p directly bound to the 3’-untranslated region(3′-UTR)of AR messenger RNA(mRNA)to inhibit the expression level of AR protein in KGN.Conclusions:Serum miR-9-5p is up-regulated in the PCOS patients,and may become a novel diagnostic biomarker for PCOS.Mechanically,miR-9-5p promotes KGN proliferation by targeting AR

关 键 词：多囊卵巢综合征 miR-9-5p 颗粒细胞 增殖 雄激素受体 

分 类 号：R711.75[医药卫生—妇产科学]