miR-295抑制高糖诱导的小鼠成骨细胞系MC3T3-E1的凋亡

miR-295 inhibits high glucose-inducedapoptosis of mouse osteoblast cell line MC3T3-E1

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。

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 proteinsβ-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,cellsurvival rate and the protein expression levels ofβ-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 ofβ-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.