MiR-9-5p Promotes Rabbit Preadipocyte Differentiation By Targeting Leptin

MicroRNAs are endogenous short-chain(22-24 nt)non-coding small RNAs.Studies have shown that microRNA plays an important role in adipocyte differentiation and adipose metabolism.miR-9-5p regulates the differentiation and proliferation of various cancer cells,hepatocytes and fibroblasts,but the regulation of adipocyte differentiation by miR-9-5p has not been reported.LEPTIN regulates lipid metabolism by mediating AMPK and JAK/STAT signaling pathways,and LEPTIN is a potential target gene of miR-9-5p.Therefore,it is speculated that miR-9-5p may affect the differentiation of preadipocytes.In this experiment,the pre-adipocytes of newly born New Zealand white rabbits were isolated and cultured.The preadipocytes were transfected by synthetic mir-9-5P analogues and induced to differentiate.Then the levels of related gene mRNA were detected.Subsequently,pis-CHECH-LEPTIN-3'U TR and pis-CHECH-Mut-LEPTIN-3'UTR recombinant plasmids were constructed to detect luciferase activity.In order to further explore the relationship between LEPTIN gene and the differentiation of rabbit preadipocytes,cells were transfected with siRNA,and the levels of related gene mRNA were detected.Finally,Western-blotting was used to detect the protein levels of PPAR gamma and LEPTIN after overexpression and inhibition of expression.The main results are as follows:(1)The expression of microRNA-9-5P was continuous up-regulated in the process of inducing the differentiation of preadipocytes,and the expression of microRNA-9-5P at 0 d was very significantly lower than that at 9 d(P < 0.01).(2)The expression of LEPTIN gene in perirenal adipose tissue of rabbits aged from 1 to 3 months has been continuous up-regulated,and the expression level at 3 months of age was significantly higher than that at 1 month of age(P < 0.01).(3)After transfection of miR-9-5p mimic,the expression of miR-9-5p was significantly up-regulated(P<0.01).The results showed that mimic could promote the differentiation of preadipocytes.The levels of PPAR?,C/EBP? and FABP4 genes were significantly increased(P < 0.01),and the contents of fatty acids and triglycerides were significantly increased(P < 0.01).Transfection of miR-9-5p inhibitor significantly reduced the expression of miR-9-5p in cells(P < 0.01).The results showed that inhibitor could inhibit lipid differentiation of the preadipocytes,and the content of fatty acid and triglyceride decreased significantly(P < 0.01),and the mRNA levels of PPAR?,C/EBP? and FABP4 genes of differentiation related genes were significantly decreased(P < 0.01).The results showed that inhibitor could inhibit the differentiation of preadipocytes.The levels of PPAR?,C/EBP? and FABP4 genes were significantly decreased(P < 0.01),and the contents of fatty acids and triglycerides were significantly decreased(P < 0.01).Overexpression of microRNA-9-5p could up-regulate the expression of PPAR? gene and protein(P < 0.01).After inhibiting the expression of microRNA-9-5p,the expression level of PPAR? gene and protein was down-regulated(P < 0.01).(4)Target gene prediction results showed that LEPTIN gene was a potential target gene of microRNA-9-5p,and the binding site was conservative.Double luciferase assay showed that LEPTIN gene is a target gene.(5)Discovered by interfering with the expression of endogenous leptin gene in cells,the expression of LEPTIN decreased significantly,while the expression of differentiation-related genes C/EBP?,PPAR? gamma and FABP4 increased significantly(P < 0.01),which was consistent with the results of over-expression of microRNA-9-5p.In conclusion,microRNA-9-5p promotes the differentiation of rabbit preadipocytes by targeting the LEPTIN gene.