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目的:研究microRNA-20a(miR-20a)对小鼠C3H/10T1/2细胞成骨分化作用的影响及其调控机制。方法:对贴壁培养的小鼠C3H/10T1/2细胞成骨诱导不同时间,ALP染色及应用qRT-PCR验证其诱导结果,同时观察MiR-20a在诱导过程中表达变化;细胞转染MiR-20a mimics,CKIP-1 siRNA,在荧光显微镜下观察转染效果,应用qRT-PCR测定过表达及干扰效率;qRT-PCR检测过表达MiR-20a及干扰CKIP-1对细胞成骨分化的影响及过表达MiR-20a对CKIP-1基因表达的影响。结果:成骨标记基因ALP、OSX、OCN、BSP随小鼠C3H/10T1/2细胞诱导过程逐渐升高,且成骨诱导分化过程中MiR-20a表达上调。过表达MiR-20a促进C3H/10T1/2细胞成骨分化,成骨标记基因碱性磷酸酶(ALP)、RUNX2、OSX、0CN、BSP显著高于对照组,并且抑制CKIP-1的表达;干扰CKIP-1能促进细胞成骨标记基因表达。结论:MiR-20a可能通过下调骨形成负调节因子CKIP-1的表达促进C3H/10T1/2细胞成骨分化。
Objective: To investigate the effect of microRNA-20a (miR-20a) on osteogenic differentiation of C3H / 10T1 / 2 cells and its regulatory mechanism. METHODS: Osteoblasts of adherent cultured mouse C3H / 10T1 / 2 cells were induced to osteoblast for different time, ALP staining and qRT-PCR were used to verify the induction of miR-20a. MiR-20a expression was also observed during induction. MiR- 20a mimics, CKIP-1 siRNA were used to observe the transfection effect under the fluorescence microscope. The overexpression of qRT-PCR and the interference efficiency were detected by qRT-PCR. The effect of MiR-20a and CKIP-1 overexpression on osteogenic differentiation was detected by qRT- Effect of MiR-20a Overexpression on CKIP-1 Gene Expression. RESULTS: Osteogenic marker genes ALP, OSX, OCN and BSP increased gradually with the induction of C3H / 10T1 / 2 cells and the expression of MiR-20a was up-regulated during osteogenic differentiation. Overexpression of MiR-20a promoted the osteogenic differentiation of C3H / 10T1 / 2 cells. The expressions of alkaline phosphatase (ALP), RUNX2, OSX, 0CN and BSP were significantly higher than those of the control and inhibited the expression of CKIP-1 CKIP-1 can promote the gene expression of osteoblast marker. Conclusion: MiR-20a may promote the osteogenic differentiation of C3H / 10T1 / 2 cells by down-regulating the expression of CKIP-1, a negative regulator of bone formation.