The Role Of MiR-142a-5p In Osteoblast Differentiation And The Mechanisms Involved

Objective: The regulation of microRNAs in directional differentiation of mesenchymal stem cells has become a hot research topic.miR-142a-5p is processed by transcription of miR-142 gene,It plays a key role in many physiological processes,such as embryogenesis,hematopoiesis,immune response,tumorigenesis,etc.However,its mechanism in the differentiation of mesenchymal stem cells into adipocytes or osteoblasts remains unclear.In this study,we aimed to investigate the biological function of miR-142a-5p in regulating osteoblast differentiation,and preliminarily elucidated the mechanism about osteogenic differentiation.Methods:1?The qRT-PCR was used to detect the tissue distribution of miR-142a-5p in C57BL/6J mice.2 ? miR-142a-5p agomir/antagomir were transfected into bone marrow stromal cell line ST2 or precursor osteoblast line MC3T3-E1 to increase the level of miR-142a-5p or inhibit miR-142a-5p,the effects of miR-142a-5p on osteogenic differentiation were investigated by ALP staining,qRT-PCR,Western Blotting.3?The miR-142a-5p overexpressing lentivirus was constructed and infected into mouse BMSCs.The effects of miR-142a-5p on osteogenic differentiation were investigated by ALP staining,qRT-PCR and Western Blotting.4?The target gene of miR-142a-5p was predicted by bioinformatics software,we constructed the target gene luciferase reporter vector and the miR-142a-5p expected binding site was subjected to site-directed mutagenesis to construct a mutation reporter plasmid.The luciferase activity of Nfia 3'UTR was detected by dual-luciferase reporter gene assay;Detection of protein expression changes of target gene after transfection of miR-142a-5p agomir and miR-142a-5p antagomir in ST2 cells;miR-142a-5p agomir and target gene overexpression plasmid were cotransfected to conduct cell rescue experiment.5 ? The cis-acting element prediction tool was used to analyze the promoter region of miR-142a-5p,then we constructed the miR-142a-5p promoter reporter vector,we next constructed a mutant miR-142a-5p promoter reporter vector by site-directed mutagenesis of the expected binding site of the upstream regulatoryfactor TCF7L2 in the reporter vector.Finally,the target gene is identified by using dual luciferase reporter gene technology;Overexpression of ?-catenin to detect the expression of miR-142a-5p;Chromatin immunoprecipitation technique(ChIP)was performed to investigate whether there is physical binding between ?-catenin and miR-142a-5p promoter region.Results: 1?miR-142a-5p was widely distributed in various tissues of C57BL/6J mice,and was highest expression in the bone,followed by spleen,colon and inguinal fat.2?Increasing the level of miR-142a-5p promoted the differentiation of bone marrow stromal cell line ST2 and pre-osteoblast cell line MC3T3-E1 into osteoblasts;and promoted the expression of ALP,BGLAP,Osterix,OPN and BSP that genes related to osteogenic differentiation,and up-regulated the protein levels of ALP,Runx2,Osterix;On the other hand,inhibited the differentiation of ST2 cells and MC3T3-E1 cells into osteoblasts by inhibiting the level of miR-142a-5p.3?miR-142a-5p LV successfully infected BMSCs and up-regulated the expression of miR-142a-5p,and found that it promoted the differentiation of BMSCs into osteoblasts and promoted the expression of ALP,BGLAP,BSP and Osterix that genes related to osteogenic differentiation,and up-regulated protein levels of ALP,Runx2,and Osterix.4?Dual luciferase reporter gene technology showed that miR-142a-5p agomir can reduce Nfia3'UTR luciferase activity;Transfection of miR-142a-5p agomir in ST2 cells can down-regulated the expression of Nfia protein,while transfection of miR-142a-5p antagomir up-regulated Nfia protein expression;cell rescue experiments showed that Nfia attenuated the promoting effect of miR-142a-5p agomir on osteogenic differentiation of ST2 cells.5 ? Overexpression of ?-catenin can increases miR-142a-5p promoter luciferase activity,and the site-directed mutagenesis with the expected binding site of TCF7L2 can alleviate the increase in miR-142a-5p promoter luciferase activity caused by ?-catenin overexpression;ST2 cells were transfected with ?-catenin and the expression of miR-142a-5p was up-regulated;the expression of miR-142a-5p was down-regulated after DKK1 treatment;Chromatin immunoprecipitation(CHIP)detected the physical binding of ?-catenin to themiR-142a-5p promoter.Conclusion:1?miR-142a-5p was highly expressed in the bone tissues suggesting that miR-142a-5p may be involved in the differentiation of osteoblasts.2?miR-142a-5p promoted osteogenic differentiation of BMSCs,ST2,and MC3T3-E1 cells.3 ?miR-142a-5p may promote osteoblast differentiation by targeting inhibition of Nfia expression.4??-catenin can bind to the promoter of miR-142a-5p and regulate the expression of miR-142a-5p in precursor cells.