Abstract: Objective Effects of miR-494 on osteoblast differentiation and matrix mineralization and related mechanisms. Method mouse MC3T3-E1 osteoblasts were randomly divided into control group, miR-494 over-expression group and miR-494 inhibition group. The control group was not treated, and miR-494 over-expression group and miR-494 inhibition group were transfected with miR-494 MICs transfection reagent and miR-494 inhibitors transfection reagent respectively. ALP activity was detected by alkaline phosphatase ( ALP ) kit, and Osteocalcin ( OC ) activity was detected by osteocalcin radioimmunoassay kit. VonKossa staining was used to detect the number of calcified nodules in each group. The mRNA expression levels of miR-494 and phosphatase tensin homolog gene ( PTEN) were detected by qRT- PCR, the protein expression levels of phosphatidylinositol 3-kinase ( PI3K ) / Aktpathway were detected by Western blot, and the targeting relationship between miR-494 and PTEN was verified by double Luciferase Report experiment. Result Dual luciferase experiments confirmed that PTEN was the target gene of miR-494. miR-494 mRNA and p-Akt protein in osteoblasts with miR-494 over-expression were significantly higher than those in the control group, while PTEN mRNA, ALP and OC activity and calcified nodule number were significantly lower than those in the control group; miR-494 inhibited osteoblast miR-494 mRNA and p-Akt protein were significantly lower than those in the control group and miR-494 over-expression group, while PTEN mRNA level, ALP and OC activity and calcified nodule number were significantly higher than those in the control group and miR-494 over-expression group ( P < 0. 05 ) . Conclusion miR-494 can effectively inhibit osteoblast differentiation and matrix mineralization, and its mechanism may be related to the regulation of Akt pathway by miR-494 mediated target gene PTEN. 

Key words: miR-494, PTEN;osteoblast differentiation, matrix mineralization, mechanism

摘要： :目的 探讨 miR-494 对成骨细胞分化及基质矿化的影响及相关机制。 方法 选择小鼠 MC3T3-E1 成骨细胞 系,随机分为对照组、miR-494 过表达组及 miR-494 抑制组。 对照组不给予处理,miR-494 过表达组及 miR-494 抑制 组分别加入 miR-494 mimics 转染试剂、miR-494 inhibitors 转染试剂进行细胞转染。 采用碱性磷酸酶( ALP) 试剂盒 检测各组细胞 ALP 活性,采用骨钙素放射免疫分析试剂盒检测各组细胞骨钙素( OC) 活性。 Vonkossa 液染色检测 各组细胞钙化结节数。 qRT-PCR 检测 miR-494、磷酸酶张力蛋白同源物基因( PTEN) mRNA 表达水平,Western blot 检测细胞磷脂酰肌醇 3-激酶( PI3K) / Akt 通路蛋白表达水平,双荧光素酶报告实验验证 miR-494 与 PTEN 的靶向关 系。 结果 双荧光素酶实验证实 PTEN 是 miR-494 的作用靶基因。 miR-494 过表达组成骨细胞 miR-494 mRNA、pAkt 蛋白明显高于对照组,PTEN mRNA、ALP 和 OC 活性、钙化结节数水平明显低于对照组;miR-494 抑制组成骨细 胞 miR-494 mRNA、p-Akt 蛋白明显低于对照组、miR-494 过表达组,PTEN mRNA 水平、ALP 和 OC 活性、钙化结节数 明显高于对照组、miR-494 过表达组(P<0. 05) 。 结论 miR-494 可有效抑制成骨细胞分化及基质矿化过程,其机制 可能与 miR-494 介导靶基因 PTEN 调控 Akt 通路有关。

关键词: miR-494, PTEN, 成骨细胞分化, 基质矿化, 机制