IRS-1Regulates The Differentiation Of Preadipocytes Mediated By MiR-143

ObjecitveTo figure out the mechanism of differentiation of mesenchymal stem cell(MSC) differentiating to adipocytes, which was one of the key breakthrough to the prevention and treatment of metabolic diseases, our previous study showed that insulin receptor substrate1(IRS-1) could regulate MSC differentiate to osteoblasts and adipocytes. With the establishment of IRS-1knock-out mice, we found out the differences of miRNA and gene expressing profiling in bone mesenchymal stem cells(BMSC). On this basis, this study would further explore the specific mechanism and application of IRS-1regulating preadipocytes.MethodWith extracting adipose tissue, liver and skeletal muscle from IRS-1homozygous, heterozygous and wild-type mouse, we detected the expression level of miR-143by RT-qPCR and cloned the binding sequences between IGF1R3’-UTR and miR-143as well as its mutated sequences. These sequences inserted into the luciferase reporter vector and transfected miRNA mimics, miRNA inhibitor with reporter gene vector into cells, which were analysed its activity by using the dual luciferase assay. During the process of3T3-L1differentiation, we used Western-blot and RT-qPCR to detect the level of IGFIR mRNA and miR-143to analyze the expression and relationship between IGFIR and miR-143. Then we observed the changes of adipocytes after transfecting miR-143mimics and miR-143inhibitor into cells and collected the serum from normal, metabolically healthy obesity, metabolically abnormal obesity. Finally we extracted RNA and took reverse transcription and RT-qPCR technique to detect the expression level of miR-143to compare the expression among groups.ResultOur research group found that the target genes of miR-143contain IGFIR by seperating the BMSC of IRS-l-/-mice, access to extract its protein, RNA and analyse the miRNA chip, PCR array and the result of target gene predict software. We used RT-qPCR to detect the expression levels in adipose tissue, liver, skeletal muscle of IRS-1homozygous, heterozygous and wild type mice. The result showed that the expression of miR-143in the IRS-l-/-mice was down-regulated, which was consistent with the miRNA chip’s analysis. Thus we took the dual luciferase assay to find that miR-143mimics could inhibit luciferase activity and miR-143inhibitor could enhance the luciferase activity. The miR-143inhibition was weaked after the mutation. Therefore we confirmed that IGF-1R was the target gene of miR-143, which may be involved in the complex regulation of lipid metabolism of IRS-1. In order to further validation, we used Western-blot and RT-qPCR technology to detect the expression levels of miR-143and IGF1R during differention process to adipocyte. The results showed that in the prosess of3T3-L1adipogenic differentiation, the protein level of IGF1R stayed rising until the sixth day, then decreased at the ninth day. MiR-143was up-regulated, while IGF1R mRNA was down-regulated. Next we transfected miR-143mimics and miR-143inhibitor into the cells. The results showed that the number of the lipid droplets in the miR-143mimics group were more than the inhibitor group. In addition, we collected the serum samples of normal people, metabolically healthy obesity, metabolically abnormal obesity. Through the RT-qPCR technology, we found that miR-143was expressed in metabolically healthy obesity more than in metabolically abnormal obesity.Conclusion1. IRS-1gene knocked out could result in the down-regulation of miR-143, which further up-regulated its target gene—IGF1R participating in the lipid metabolism.2. MiR-143was up-regulated in the process of preadipocytes differentiating to adipocytes and was abundant in the serum of metabolically healthy obesity population.