微小RNA-19a通过第一凋亡信号及其配体信号通路促进骨关节炎软骨细胞增殖的机制
Mechanism of microRNA-19a promoting the proliferation of osteoarthritis chondrocytes through first apoptosis signal/first apoptosis signal ligand signaling pathway
吴博宇 1肖文杰 1刘红翔 1朱颖 1朱思源 1范志海1
作者信息
- 1. 苏州大学附属第二医院骨科,苏州 215000
- 折叠
摘要
目的 探讨微小RNA-19a(miR-19a)通过调控第一凋亡信号/第一凋亡信号配体(Fas/FasL)通路对骨关节炎(OA)软骨细胞增殖和凋亡的影响.方法 体外培养人正常软骨细胞及OA软骨细胞,实时荧光定量聚合酶链反应(RT-qPCR)法检测细胞中miR-19a表达.将OA软骨细胞分为对照组、miR-19a阴性对照(NC)组、miR-19a模拟物(mimics)组、miR-19a mimics+空载质粒(pcDNA)组和miR-19a mimics+Fas过表达质粒(Fas)组.RT-qPCR法检测各组细胞miR-19a、第一凋亡信号(Fas)、Fas配体(FasL)的mRNA表达;噻唑蓝(MTT)法检测各组OA软骨细胞增殖;流式细胞术分析细胞凋亡;免疫印迹法测定增殖、凋亡相关蛋白及Fas、FasL蛋白表达;双荧光素酶实验测定miR-19a与Fas的相互作用,以t检验和单因素方差分析(ANOVA)进行组间比较.结果 OA软骨细胞组miR-19a的表达水平显著低于正常软骨细胞组(0.45±0.06比1.01±0.19,t=2.95,P＜0.05).增加 miR-19a 表达的 miR-19a mimics 组 Fas 和 FasL 表达显著低于 miR-19a NC 组(Fas:0.31±0.05 比 0.93±0.09,t=15.78,P＜0.05;FasL:0.37±0.04 比 0.87±0.10,t=11.43,P＜0.05),miR-19a mimics 组细胞增殖显著高于 miR-19a NC 组(A 值:0.66±0.07 比 0.45±0.05,t=8.70,P＜0.05),miR-19a mimics 组细胞凋亡率显著低于 miR-19a NC 组[(8.57±1.65)％比(22.45±2.66)％,t=12.02,P＜0.05].结论 miR-19a可通过靶向抑制Fas/FasL信号通路,促进OA软骨细胞增殖,抑制细胞凋亡.
Abstract
Objective To investigate the effects of microRNA-19a(miR-19a)on the proliferation and apoptosis of osteoarthritis(OA)chondrocytes by regulating the first apoptosis signal/first apoptosis signal ligand(Fas/FasL)pathway.Methods Chondrocytes were isolated from specimens of total knee arthroplas-ty,and both normal human chondrocytes and osteoarthritis(OA)chondrocytes were cultured in vitro.The expression of miR-19a was detected by real-time fluorescent quantitative polymerase chain reaction(RT-qPCR).OA chondrocytes were divided into control group,miR-19a negative control(NC)group,miR-19a mimic(mimics)group,miR-19a mimics+empty plasmid(pcDNA)group and miR-19a mimics+Fas overexpression plasmid(Fas)group.RT-qPCR method was used to detect the expression of miR-19a,Fas,FasL mRNA in each group of cells.Thiazole blue(MTT)method was used to detect the proliferation of OA chondrocytes in each group.Flow cytometry was used to detect cell apoptosis.Western blotting was used to detect the expression of cell proliferation,apoptosis-related proteins and Fas,FasL protein in each group.The dual luciferase reporter gene experiment was used to verify the targeting relationship between miR-19a and Fas.Data were analyzed using SPSS 22.0 software,and inter-group comparisons were con-ducted using t-tests and one-way analysis of variance(ANOVA),with a significance level set at P＜0.05.Results The expression level of miR-19a in the OA chondrocyte group was significantly lower than in the normal chondrocyte group(0.45±0.06 vs.1.01±0.19,t=2.95,P＜0.05).In the miR-19a mimics group,the expression levels of Fas and FasL were significantly lower than in the miR-19a NC group(Fas:0.31±0.05 vs.0.93±0.09,t=15.78,P＜0.05;FasL:0.37±0.04 vs.0.87±0.10,t=11.43,P＜0.05).The cell proliferation in the miR-19a mimics group was significantly higher than in the miR-19a NC group(A value:0.66±0.07 vs.0.45±0.05,t=8.70,P＜0.05),and the apoptosis rate in the miR-19a mimics group was significantly lower than in the miR-19a NC group[(8.57±1.65)％vs.(22.45±2.66)％,t=12.02,P＜0.05].Conclusion MiR-19a can promote the proliferation of OA chondrocytes and inhibit cell apoptosis by targeting the inhibition of Fas/FasL signaling pathway.
关键词
微小RNA/第一凋亡信号/第一凋亡信号配体通路/骨关节炎/软骨细胞/增殖/凋亡Key words
MicroRNA/First apoptosis signal/first apoptosis signal ligand pathway/Osteoar-thritis/Chondrocytes/Proliferation/Apoptosis引用本文复制引用
基金项目
江苏省科技计划项目(BK20191168)
出版年
2024
NETL
NSTL
万方数据