摘要
目的探究miR-135b靶向糖原合成酶激酶3B(glycogen synthase kinase 3B,GSK3B)对地塞米松诱导的成骨细胞(MC3T3-E1)凋亡的影响。方法CCK8检测地塞米松对MC3T3-E1细胞活力的影响,筛选最适地塞米松浓度进行后续实验。流式细胞术检测细胞凋亡,RT-qPCR检测miR-135b和GSK3B表达,荧光素酶报告实验验证miR-135b和GSK3B靶向作用关系。细胞实验分为对照组(Control)、地塞米松组(Dex)、阴性对照mimics组(mimics NC)、miR-135b组、miR-135b+糖原合成酶激酶3B组(miR-135b+pc-GSK3B)。蛋白印迹检测蛋白表达。结果与对照组比较,Dex组细胞凋亡增加,并且miR-135b表达下降,GSK3B表达升高,荧光素酶报告实验显示,miR-135b和GSK3B存在靶向作用关系。与Dex组比较,miR-135b组细胞凋亡减少,同时GSK3B、Bax、cleaved caspase-3和cleaved caspase-9蛋白水平下降,Bcl-2蛋白水平升高,与miR-135b组比较,miR-135b+pc-GSK3B组细胞凋亡增加,同时GSK3B、Bax、cleaved caspase-3和cleaved caspase-9蛋白水平升高,Bcl-2蛋白水平降低。结论miR-135b可通过靶向作用于GSK3B,抑制地塞米松诱导的成骨细胞凋亡,这一作用与其对Bax/Bcl-2表达和caspase-3/9活化的调控有关。
Objective To investigate the effect of miR-135b targeting glycogen synthase kinase 3B(GSK3B)on dexamethasone-induced apoptosis of osteoblasts.Methods CCK8 was used to detect the effect of dexamethasone on the viability of MC3T3-E1 cells.The optimal concentration was selected for subsequent experiments.Flow cytometry was used to detect apoptosis.RT-PCR was used to detect the expression of miR-135b and GSK3B.The effect of luciferase reporting experiments verified the relationship between miR-135b and GSK3B targeting.The cells were divided into control group,dexamethasone group(Dex),negative control mimics group(mimics NC),miR-135b group,and miR-135b+pc-GSK3B group.The protein expression was detected with Western blotting.Results Compared to those in the Control group,apoptosis increased,the expression of miR-135b decreased,and the expression of GSK3B increased in the Dex group.The luciferase report experiment showed that there was a targeting relationship between miR-135b and GSK3B.Compared to those in the Dex group,the apoptosis of miR-135b group reduced,the levels of GSK3B,Bax,cleaved caspase-3 and cleaved caspase-9 protein decreased,and Bcl-2 protein level increased.Compared to those in the miR-135b group,the apoptosis of the miR-135b+pc-GSK3B group increased,the levels of GSK3B,Bax,cleaved caspase-3 and cleaved caspase-9 increased,and the level of Bcl-2 decreased.Conclusion miR-135b inhibits dexamethasone-induced apoptosis of osteoblasts by targeting GSK3B.This effect is related to the regulation of Bax/Bcl-2 expression and caspase-3/9 activation.
作者
肖建生
张鹏
赵洪洲
钟俊青
XIAO Jiansheng;ZHANG Peng;ZHAO Hongzhou;ZHONG Junqing(Graduate School of Tianjin Medical University,Tianjin 300070;Department of Orthopedics,Tianjin Hospital,Tianjin 361000,China)
出处
《中国骨质疏松杂志》
CAS
CSCD
北大核心
2021年第11期1588-1593,共6页
Chinese Journal of Osteoporosis
基金
天津市科技计划项目(12ZCDSY)。
