miR-467g下调Runx-2表达抑制成骨前体细胞的分化和矿化

MiR-467g suppresses the osteogenic differentiation and mineralization of mouse preosteoblasts via down-regulation of Runx-2 expression

背景:最新的研究发现miR-467g能抑制骨质再生,但其具体的作用和作用机制尚不明确。目的:探索miR-467g对小鼠成骨前体细胞成骨分化和矿化的影响及其机制。方法:体外分离、培养C57小鼠成骨前体细胞,通过Western-blot、实时定量PCR、碱性磷酸酶染色、茜素红染色等实验探索小鼠成骨前体细胞诱导分化过程中,成骨分化指标Runx-2、Osterix、Osteocalcin表达变化情况及碱性磷酸酶活性和矿化能力,以及miR-467g和它潜在靶基因Runx-2表达水平的变化;通过脂质体转染构建miR-467g高表达的小鼠成骨前体细胞,研究miR-467g对小鼠成骨前体细胞成骨分化的影响。利用荧光素酶实验检测miR-467g对Runx-2的靶向作用。结果与结论:(1)分离培养的小鼠成骨前体细胞具有良好的体外增殖与分化能力;(2)miR-467g表达水平随小鼠成骨前体细胞诱导分化时间的增加而降低;(3)miR-467g能够抑制小鼠成骨前体细胞成骨分化和矿化;(4)荧光素酶实验证实mi R-467g直接靶向Runx-2;(5)结果提示:miR-467g通过下调Runx-2抑制小鼠成骨前体细胞成骨分化和矿化。

BACKGROUND： Preliminary studies have found mi R-467 g inhibits bone regeneration, however, there is little information about the underlying mechanism. OBJECTIVE： To explore the effect of mi R-467 g on osteogenic differentiation and mineralization of mouse preosteoblasts and the underlying mechanism. METHODS： C57 mouse preosteoblasts were isolated and cultured in vitro. The expression levels of Runx-2, Osterix, and Osteocalcin, as well as alkaline phosphatase and mineralization activities were determined by western blot, real-time PCR, alkaline phosphatase and Alizarin red staining, respectively. mi R-467 g-overexpressed preosteoblasts were constructed to investigate the effect of mi R-467 g on osteogenic differentiation and mineralization of preosteoblasts by lipofection transfection. Dual luciferase reporter assay was used to identify whether the 3＇UTR of Runx-2 m RNA was a binding target of mi R-467 g. RESULTS AND CONCLUSION： The primary mouse preosteoblasts had a good osteogenic proliferation and differentiation ability in vitro. Expression level of mi R-467 g was decreased with the increase in osteogenic induction time. MiR-467 g suppressed the osteogenic differentiation and mineralization of mouse preosteoblasts. Luciferase assay confirmed that mi R-467 g targeted Runx-2 directly. In summary, mi R-467 g can suppress the osteogenic differentiation and mineralization of mouse preosteoblasts via down-regulation of Runx-2 expression.