文章摘要

MiRNA-326抑制人椎间盘退变髓核细胞活力和凋亡的机制

作者: 1高 笛, 1刘 殿鹏
1 安阳市第六人民医院骨科,河南 安阳 455000
通讯: 高 笛 Email: gaodi197708@163.com
DOI: 10.3978/j.issn.2095-6959.2020.04.002

摘要

目的:探讨过表达miRNA-326对椎间盘退变(intervertebral disc degeneration,IDD)髓核(nucleus pulposus,NP)细胞凋亡的影响及其作用机制。方法:构建miRNA-326慢病毒表达载体,在293T细胞中获得重组慢病毒,经感染NP细胞得到稳定过表达细胞系GV369-miRNA-326-NP,同时设置空载体GV369-NP组与空白组。荧光显微镜观察慢病毒载体的标签蛋白[绿色荧光蛋白(green fluorescent protein,GFP)]的表达,实时荧光定量PCR(real-time quantitative polymerase chain reaction,RT-qPCR)方法检测miRNA-326的表达,流式细胞术检测细胞凋亡,荧光素酶报告基因分析验证miRNA-326与FasL的靶向关系,蛋白质印迹法检测细胞中凋亡相关蛋白FADD,caspase-3,Bcl-2及Bax的表达,使用试剂盒检测细胞线粒体膜电势的变化情况。结果:荧光显微镜下示,经慢病毒感染的过表达细胞系和空载体细胞系均出现绿色荧光,而空白组未见绿色荧光。与空白组相比,GV369-miRNA-326-NP组中miRNA-326的表达水平、Bcl-2表达水平和线粒体膜电位明显升高,而细胞凋亡率,FADD,caspase-3和Bax的表达水平明显下降,差异均有统计学意义(P<0.05);GV369-NP组与空白组相比,差异无统计学意义(P>0.05)。荧光素酶报告基因分析证实miRNA-326与FasL存在靶向关系。结论:MiRNA-326可抑制IDD NP细胞发生凋亡,既可通过靶向性调控外源性FasL/Fas通路参与caspase-3和FADD介导的细胞凋亡,也可通过线粒体途径对细胞凋亡发挥作用。
关键词: miRNA-326;椎间盘退变髓核细胞;细胞凋亡;实时荧光定量PCR;流式细胞术

Mechanism of miRNA-326 inhibiting the activity and apoptosis of human disc degeneration nucleus pulposus cells

Authors: 1GAO Di, 1LIU Dianpeng
1 Department of Orthopedics, Anyang Sixth People’s Hospital, Anyang Henan 455000, China

CorrespondingAuthor:GAO Di Email: gaodi197708@163.com

Abstract

Objective: To investigate the effect and mechanism of miRNA-326 overexpression on apoptosis of degenerative nucleus pulposus (NP) cells in intervertebral disc. Methods: MiRNA-326 lentivirus expression vector was constructed and recombinant lentivirus was obtained in 293T cells. After infection with NP cells, the stable overexpressed cell line GV369-miRNA-326-NP was obtained, at the same time, the empty vector GV369-NP group and the blank group were set up. The expression of lentivirus label protein GFP was observed by fluorescence microscope. The expression of miRNA-326 was detected by real-time quantitative polymerase chain reaction (RT-qPCR). Apoptosis of cells was detected by flow cytometry. The targeting relationship between miRNA-326 and FasL was verified by Luciferase reporter gene analysis and the expression of apoptosis-related proteins (FADD, caspase-3, Bcl-2 and Bax) was detected by Western bloting. The change of cell mitochondrial membrane potential was detected with the kit. Results: Under fluorescence microscope, green fluorescence was observed in the lentivirus infected overexpressed cell lines and empty carrier cell lines, but not in the blank cell group. Compared with the blank group, the expression levels of miRNA-326, Bcl-2 and mitochondrial membrane potential in the GV369-miRNA-326-NP group were significantly increased, while the expression levels of apoptosis rate, FADD, caspase-3 and Bax were significantly decreased, with statistically significant differences (P<0.05). There was no statistically significant difference between the GV369-NP group and the blank group (P>0.05). Luciferase reporter gene analysis confirmed a targeted relationship between miRNA-326 and FasL. Conclusion: MiRNA-326 can inhibit the apoptosis of degenerative nucleus pulposus cells in intervertebral disc. It can not only participate in the apoptosis mediated by caspase-3 and FADD through the targeted regulation of exogenous FasL/Fas pathway, but also play a role in the apoptosis through mitochondrial pathway.
Keywords: miRNA-326; intervertebral disc degeneration of nucleus pulposus cells; cell apoptosis; real-time quantitative polymerase chain reaction; flow cytometry