微RNA-705对MC3T3-E1细胞成骨分化能力的影响

目的：观察微RNA（miR）-705对小鼠胚胎成骨细胞前体细胞（MC3T3-E1）成骨分化能力的影响。方法：将转染了miR-705模拟物、抑制物、对照物的MC3T3-E1细胞加入成骨诱导液，在其成骨诱导7 d时采用碱性磷酸酶（ALP）染色检测ALP的活性；在成骨诱导14 d时，用RT-PCR和蛋白质印迹技术检测Runt相关转录因子2（Runx2）和骨钙素（OCN）的mRNA及蛋白表达水平；在成骨诱导21 d时，以茜素红染色观察钙结节形成情况并作定量分析。结果：成骨诱导7 d时，模拟物组ALP活性降低，而抑制物组ALP活性升高（均P<0.05）；成骨诱导14 d时，模拟物组Runx2和OCN mRNA及蛋白表达水平较对照物组降低，而抑制物组则相反（均P<0.05）；成骨诱导21 d时，模拟物组钙结节最小，结节形成量下降，抑制物组钙结节较大且形成量较多（均P<0.05）。结论：miR-705对MC3T3-E1细胞成骨分化过程具有抑制作用。
Abstract: Objective: To investigate the effect of miR-705 on osteogenic differentiation of mouse embryo osteoblast precursor (MC3T3-E1) cells. Methods: miR-705 mimics, inhibitors and negative control were transfected into MC3T3-E1 cells. Alkaline phosphates (ALP) staining were performed and quantified after 7 days of osteogenic medium induction. The mRNA and protein expression levels of runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) were detected by real-time RT-PCR and Western blot after 14 days of osteogenic induction. Alizarin red staining was performed and quantified in MC3T3-E1 cells after 21 days of osteogenic induction. Results: After 7 days of osteogenic induction, ALP staining showed that overexpression of miR-705 significantly reduced ALP activity, whereas knockdown of miR-705 increased ALP activity (all P<0.05). Consistently, after 14 days of osteogenic induction, mRNA and protein expressions of Runx2 and OCN were suppressed by overexpression of miR-705, whereas they were promoted by knockdown of miR-705 (all P<0.05). After 21 days of osteogenic induction, alizarin red staining showed that overexpression of miR-705 significantly reduced the formation of mineralized node, the opposite results were found in miR-705 knockdown group (all P<0.05). Conclusion: miR-705 can inhibit the osteogenic differentiation of MC3T3-E1 cells. Key words: 3T3 cells/drug effects Osteoblasts/metabolism MicroRNAs/therapeutic use Cell transdifferentiation