miR-295抑制高糖诱导的小鼠成骨细胞系MC3T3-E1的凋亡

摘要/Abstract

摘要： 目的探讨微小RNA(miR)-295对高糖诱导的小鼠成骨细胞系MC3T3-E1凋亡的影响及其机制。方法将MC3T3-E1细胞分为对照(Ctrl)组、高糖(HG)组(以22 mmol/L葡萄糖诱导培养)、(HG+miR-control)组(转染miR-control后高糖诱导)和(HG+miR-295)组(转染miR-295模拟物后高糖诱导);实时荧光定量PCR(RT-qPCR)检测MC3T3-E1细胞中miR-295表达水平;噻唑蓝(MTT)法检测MC3T3-E1细胞存活率;流式细胞测量术检测MC3T3-E1细胞凋亡率;免疫印迹法检测Wnt/β-catenin通路相关蛋白β连环蛋白(β-catenin)、Dickkopf同源物1(DKK1)、c-Myc和B淋巴细胞瘤-2基因(Bcl-2)蛋白表达;双荧光素酶报告基因实验检测miR-295和DKK1的靶向关系。结果与对照组比较,HG组MC3T3-E1细胞中miR-295表达水平、细胞存活率和β-catenin、c-Myc及Bcl-2蛋白表达水平均明显降低(P＜0.05),而细胞凋亡率和DKK1蛋白表达水平均明显升高(P＜0.05);与(HG+miR-control)组比较,(HG+miR-295)组MC3T3-E1细胞中miR-295表达水平、细胞存活率和β-catenin、c-Myc、Bcl-2蛋白表达水平均明显升高(P＜0.05),而细胞凋亡率和DKK1蛋白表达水平均明显降低(P＜0.05)。Dkk1可能是miR-295的靶基因。结论 miR-295可能通过下调DKK1表达激活Wnt/β-catenin通路来抑制高糖诱导的成骨细胞系MC3T3-E1的凋亡。

关键词: 成骨细胞, 高糖, miR-295, 凋亡, Wnt/β连环蛋白通路

Abstract: Objective To investigate the effect of microRNA (miR)-295 on high glucose-induced apoptosis of mouse osteoblast cell line(MC3T3-E1) and underlying the mechanism. Methods MC3T3-E1 cells cultured in vitro were divided into three groups: control (Ctrl) group, high glucose (HG) group (induced by 22 mmol/L glucose), (HG + miR-control) group (induced by high glucose after miR-control transfection) and (HG+miR-295) group (induced by high glucose after transfection of miR-295 mimic). The expression of miR-295 in MC3T3-E1 cells was detected by real-time fluorescent quantitative PCR, the survival rate of MC3T3-E1 cells was detected by methyl thiazolyl tetrazolium (MTT) assay, the apoptosis rate of MC3T3-E1 cells was detected by flow cytometry, the protein expression of Wnt/β-catenin pathway related proteins β-catenin, Dickkopf homolog 1 (DKK1), c-Myc and B-cell lymphoma-2 (Bcl-2) were detected by Western blot, double luciferase reporter gene assay was used to detect the targeting relationship between miR-295 and DKK1. Results Compared with those in the Ctrl group, the expression levels of miR-295, cell survival rate and the protein expression levels of β-catenin, c-Myc and Bcl-2 in MC3T3-E1 cells in HG group were significantly lower, the apoptosis rate and protein expression level of DKK1 were significantly higher (P＜0.05); Compared with those in (HG + miR-control) group, the expression level of miR-295 (P＜0.05), cell survival rate and the protein expression levels of β-catenin, c-Myc and Bcl-2 in MC3T3-E1 cells in (HG + miR-295) group were significantly higher (P＜0.05), the apoptosis rate and protein expression level of DKK1 were significantly lower(P＜0.05). Dkk1 might be the target gene of miR-295. Conclusions miR-295 may inhibit high glucose-induced MC3T3-E1 cell apoptosis by down-regulating DKK1 expression and activating Wnt/β-catenin pathway.

Key words: osteoblast, high glucose, miR-295, apoptosis, Wnt/β-catenin pathway

中图分类号:

R587.2

曲野, 刘立柱, 徐瑞敏, 吴开弟, 代伟宏. miR-295抑制高糖诱导的小鼠成骨细胞系MC3T3-E1的凋亡[J]. 基础医学与临床, 2021, 41(11): 1588-1593.

QU Ye, LIU Li-zhu, XU Rui-min, WU Kai-di, DAI Wei-hong. miR-295 inhibits high glucose-induced apoptosis of mouse osteoblast cell line MC3T3-E1[J]. Basic & Clinical Medicine, 2021, 41(11): 1588-1593.

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