miR-483-5p靶向Timp2调控破骨细胞的生成

miR⁃483⁃5p regulates osteoclast generation by targeting Timp2

目的:miR‐483‐5p可促进破骨细胞分化和骨破坏,本研究探讨miR‐483‐5p是否通过靶向作用于Timp2调控破骨细胞的生成。方法:采用miRNAs靶基因预测软件TargetScan8.0对miR‐483‐5p的靶基因进行预测,并构建野生型和突变型3'UTR质粒,通过双荧光素酶报告基因验证靶基因是否与miR‐483‐5p存在靶向调控关系。用免疫印迹方法检测miR‐483‐5p的mimics或inhibitor转染细胞后靶蛋白表达水平的相应变化。用靶基因siRNA或靶蛋白慢病毒转染或感染细胞,RANKL诱导后分别进行TRAP染色和q‐PCR检测。结果:运用生物信息学软件预测miR‐483‐5p的靶基因,发现具有调控破骨细胞作用的Timp2基因,且双荧光素酶报告基因检测系统发现miR‐483‐5p可以与预测的靶基因Timp2基因的3′‐UTR位点互补,两者之间存在靶向调控关系。在RAW264.7细胞中上调miR‐483‐5p水平可减少Timp2的表达;敲低细胞中的Timp2,诱导后较正常组破骨细胞数量显著增多,且破骨细胞特异性基因表达升高。将靶基因和miR‐483‐5p共转染入细胞后,与正常组相比,破骨细胞数显著减少,特异性基因表达降低。结论:Timp2作为miR‐483‐5p的下游靶点,参与并抑制了破骨细胞的生成。

Objective:To investigate whether miR‐483‐5p regulates osteoclast generation by targeting Timp2.miR‐483‐5p can promote osteoclast differentiation and bone destruction.Methods:Target genes of miR‐483‐5p were predicted by miRNAs target gene prediction software TargetScan8.0,and wild type and mutant 3'UTR plasmids were constructed.Dual luciferase reporter genes were used to verify whether target genes had a targeted regulatory relationship with miR‐483‐5p.Western blotting was used to detect the corresponding changes in the expression level of target protein after adjusting the level of miR‐483‐5p in cells.Cells were transfected or infected with target gene siRNA or target protein lentivirus,and TRAP staining and q‐PCR assays were performed.In addition,for osteoclast induction experiment,RAW264.7 cells were co‐transfected with ago‐miR‐483‐5p and target protein‐overexpressed lentiviruses q‐PCR and TRAP staining were performed respectively.Results:Bioinformatics software was used to predict the target gene of miR‐483‐5p,and the Timp2 gene was found to regulate osteoclasts,and the dual luciferase reporter detection system found that miR‐483‐5p could be associated with the 3‐UTR of the predicted target gene Timp2 gene.There are complementary loci and targeted regulatory relationship between them.Subsequently,we up‐regulated miR‐483‐5p in RAW264.7 cells to reduce the expression of Timp2.Compared with the normal group,the number of osteoclasts and the expression of osteoclast‐specific genes increased significantly after the induction of Timp2 in knockdown cells.After co‐transfection of tar‐get gene and miR‐483‐5p into cells,the number of osteoclasts and the expression of specific genes decreased significantly com‐pared with the normal group.Conclusion:Timp2 is a downstream target gene of miR‐483‐5p and is involved in and inhibits osteo‐clast generation.