微小RNA-214靶向调控PTEN对成骨细胞MC3T3-E1增殖、分化、矿化的影响及机制研究

Effects of miR-214 on proliferation,differentiation and mineralization of osteoblast MC3T3-E1 by targeting PTEN and its mechanism

目的:探讨微小RNA-214(miR-214)靶向调控第10号染色体丢失性磷酸酶及张力蛋白同源基因(PTEN)对成骨细胞MC3T3-E1增殖、分化、矿化的影响及机制。方法:将成骨细胞MC3T3-E1分为miR-124 mimic组(转染miR-124 mimic)、miR-124 NC组(转染miR-124 NC)、pcDNA3.1-PTEN组(转染pcDNA3.1-PTEN)、miR-124 mimic+pcDNA3.1-PTEN组(转染miR-124 mimic+pcDNA3.1-PTEN)。采用在线生信预测miR-124和PTEN靶向结合位点,通过双荧光素酶报告基因进行验证。采用实时荧光定量聚合酶链式反应(RT-qPCR)检测细胞miR-124和PTEN mRNA表达。采用CCK-8法检测细胞增殖。采用碱性磷酸酶活性测定检测细胞分化能力。采用茜素红染色和骨钙素水平测定检测细胞的矿化。采用Werstern blot检测PTEN、Ⅰ型胶原蛋白(ColⅠ)、骨桥蛋白(OPN)、磷酸化磷脂酰肌醇3-激酶(p-PI3K)、磷酸化蛋白激酶B(p-AKT)水平。结果:与miR-124 NC组比较,miR-124 mimic组野生型PTEN的荧光素酶活性降低(均P<0.05)。与miR-124 NC组比较,miR-124 mimic组miR-124及p-PI3K、p-AKT蛋白表达升高,PTEN mRNA和蛋白、细胞增殖率、碱性磷酸酶活性、ColⅠ和OPN蛋白、细胞矿化率和骨钙素水平降低,而pcDNA3.1-PTEN组则相反(均P<0.05)。与miR-124 mimic组比较,miR-124 mimic+pcDNA3.1-PTEN组p-PI3K、p-AKT蛋白表达降低,PTEN mRNA和蛋白、细胞增殖率、碱性磷酸酶活性、ColⅠ和OPN蛋白、细胞矿化率和骨钙素水平升高(均P<0.05)。结论:miR-214通过靶向下调PTEN蛋白表达激活PI3K/AKT信号通路,从而调控成骨细胞MC3T3-E1增殖、分化和矿化。

Objective:To investigate the effects of miR-214 on proliferation,differentiation and mineralization of osteoblast MC3T3-E1 by targeting PTEN and its mechanism.Methods:MC3T3-E1 cells were divided into miR-124 mimic group(transfected with miR-124 mimic),miR-124 NC group(transfected with miR-124 NC),pcDNA3.1-PTEN group(transfected with pcDNA3.1-PTEN)and miR-124 mimic+pcDNA3.1-PTEN group(transfected with miR-124 mimic+pcDNA3.1-PTEN).The target binding sites of miR-124 and PTEN were predicted by online bioinformatics and verified by dual luciferase reporter gene.The expression of miR-124 and PTEN mRNA was detected by RT-qPCR.Cell proliferation was detected by CCK-8 assay.Alkaline phosphatase activity assay was used to detect the differentiation ability of cells.The mineralization of the cells was detected by alizarin red staining and osteocalcin level determination.The levels of PTEN,ColⅠ,OPN,p-PI3K,and p-AKT were detected by Werstern blot.Results:Compared with miR-124 NC group,the luciferase activity of wild-type PTEN in miR-124 mimic group decreased(all P<0.05).Compared with miR-124 NC group,the expression of miR-124,p-PI3K and p-AKT protein in miR-124 mimic group increased,while PTEN mRNA and protein,cell proliferation rate,alkaline phosphatase activity,ColⅠand OPN protein,cell mineralization rate and osteocalcin level decreased,while the pcDNA3.1-PTEN group had the opposite effect(all P<0.05).Compared with miR-124 mimic group,the expressions of p-PI3K and p-AKT protein in miR-124 mimic+pcDNA3.1-PTEN group were decreased,whike PTEN mRNA and protein,cell proliferation rate,alkaline phosphatase activity,ColⅠand OPN protein,cell mineralization rate and osteocalcin level were increased(all P<0.05).Conclusion:MiR-214 regulates the proliferation,differentiation and mineralization of MC3T3-E1 cells by down-regulating PTEN protein expression and activating PI3K/AKT signaling pathway.