中国骨质疏松杂志

miR-26a-5p靶向PTEN基因影响成骨细胞分化及基质矿化

Effects of miR-26a-5p on osteoblasts differentiation and matrix mineralization by targeting PTEN

DOI：10.3969/j.issn.1006-7108.2022.08.007

中文关键词: 微小RNA-26a-5p 分化能力基质矿化第10染色体同源丢失性磷酸酶张力蛋白基因 Wnt/β-catenin信号通路

英文关键词:mir-26a-5p differentiation ability matrix mineralization PTEN Wnt/β-catenin signaling pathway

基金项目:基金项目：湖北省卫生健康委员会科研项目（WJ2019M038 ）

作者	单位
叶恒张卫华韩俊张建业蒋现永*	武汉科技大学附属汉阳医院骨一科

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中文摘要:

摘要：目的探究微小RNA-26a-5p（miR-26a-5p）对第10染色体同源丢失性磷酸酶张力蛋白基因（PTEN）的靶向作用及对成骨细胞分化及基质矿化的影响。方法通过在线软件预测miR-26a-5p与PTEN基因的结合位点，并经双荧光素酶报告基因检测验证。取生长状态良好的MC3T3-E1细胞，分为miR-26a-5p模拟物（mimic）组、miR-26a-5p mimic阴性对照（NC）组、pcDNA3.1-PTEN组、miR-26a-5p+pcDNA3.1-PTEN组，通过MTT法检测细胞增殖、碱性磷酸酶活性测定研究细胞分化情况、骨钙素定量检测分析和茜素红染色观察细胞基质矿化情况、Western blot检测成骨分化标志物和Wnt/β-连环蛋白（β-catenin）信号通路蛋白表达变化。结果与NC组比较，miR-26a-5p mimic组miR-26a-5p水平、细胞增殖率、碱性磷酸酶活性、Ⅰ型胶原（ColⅠ）、骨桥蛋白（OPN）、骨钙素、矿化率及β-catenin表达升高，PTEN mRNA及蛋白表达和糖原合成酶激酶-3b（Gsk-3b）表达降低（P＜0.05），pcDNA3.1-PTEN组PTEN mRNA及蛋白表达和Gsk-3b表达升高，细胞增殖率、碱性磷酸酶活性、ColⅠ、OPN、骨钙素、矿化率及β-catenin表达降低（P＜0.05）；与miR-26a-5p mimic组比较，miR-26a-5p+pcDNA3.1-PTEN组PTEN mRNA及蛋白表达和Gsk-3b表达升高，细胞增殖率、碱性磷酸酶活性、ColⅠ、OPN、骨钙素、矿化率及β-catenin表达降低（P＜0.05）。结论 miR-26a-5p可靶向下调PTEN表达，调控Wnt/β-catenin信号通路，促进成骨细胞增殖、分化及基质矿化。

英文摘要:

Abstract: Objective To explore the targeted effect of microRNA-26a-5p (miR-26a-5p) on phosphatase and tensin homology deleted on chromosome ten (PTEN) and its influences on osteoblasts differentiation and matrix mineralization. Methods The binding site of miR-26a-5p and PTEN was predicted by online software, and which was verified by dual luciferase reporter gene assay. MC3T3-E1 cells in good growth condition were selected and divided into miR-26a-5p mimic (mimic) group, miR-26a-5p mimic negative control (NC) group, pcDNA3.1-PTEN group and miR-26a-5p+ pcDNA3.1-PTEN group. The cells proliferation was detected by MTT. The cells differentiation was studied by alkaline phosphatase activity assay. The matrix mineralization observed by quantitative detection of osteocalcin and alizarin red staining. The changes in expressions of osteoblasts differentiation markers and Wnt/β-catenin (β-catenin) signaling pathway proteins were detected by Western blot. Results Compared with NC group, miR-26a-5p level, cells proliferation rate, activity of alkaline phosphatase, type I collagen (Col I), osteopontin (OPN), osteocalcin, mineralization rate and β-catenin were increased in miR-26a-5p mimic group, while expressions of PTEN mRNA and protein, and glycogen synthase kinase-3b (Gsk-3b) were decreased (P<0.05). Compared with NC group, expressions of PTEN mRNA and protein, and Gsk-3b were increased in pcDNA3.1-PTEN group, while cells proliferation rates, activity of alkaline phosphatase, Col I, OPN, osteocalcin, mineralization rate and β-catenin were decreased (P<0.05). Compared with miR-26a-5p mimic group, expressions of PTEN mRNA and protein, and Gsk-3b were increased in miR-26a-5p+pcDNA3.1-PTEN group, while cells proliferation rates, activity of alkaline phosphatase, Col I, OPN, osteocalcin, mineralization rate and β-catenin were decreased (P<0.05). Conclusion The miR-26a-5p can promote osteoblasts proliferation, differentiation and matrix mineralization by down-regulating PTEN expression and regulating Wnt/β-catenin signaling pathways.

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