Post-transcriptional Regulation Of FASN By RNA Binding Protein HuR In Human Adipocyte Differentiation

Objective: To investigate the posttranscriptional regulation of RNA binding protein HuR on FASN in human adipocyte differentiation.Methods:(1)adipogenesis was induced by h ADSC,and adipogenesis was detected by oil red O staining.The m RNA and protein levels of HuR and FASN were detected by RT-q PCR and western blotting.(2)After overexpression and knockdown of HuR,the m RNA and protein expression levels of HuR and FASN were detected by RT-q PCR and western blotting,respectively.The binding level of HuR and FASN was detected by RNA-IP.(3)The eukaryotic expression vectors of FASN m RNA 3’utr truncated and point mutant reporter gene were constructed,and the activities of reporter genes were detected by transfection of 293 T cells.(4)The stability of FASN m RNA was detected by actinomycin D assay.Changes in adipogenesis in cells were detected by oil red O staining.Results:(1)During the induction of h ADSC into adipocytes,the expression levels of HuR mrna and protein and FASNm RNA and protein were increased gradually and the differences were statistically significant(all P<0.05).(2)RIP results show that HuR and FASN have higher binding abundance;Reporter gene detection after truncated and point mutant FASN 3 ’UTR: Compared with the control group,the activity of reporter genes containing No.1 ARE truncated body had little change,while the activity of reporter genes containing No.2 ARE truncated body significantly increased.Compared with the control group,there was little change in the reporter gene activity after the 1 ARE mutation,and significantly decreased the reporter gene activity after the 2 ARE mutation(all P < 0.01).(3)After HuR overexpression,FASN m RNA expression level was not significantly changed,protein expression level was significantly up-regulated,and the number of adipocyte generation was significantly increased with statistical significance(all P<0.05).After HuR knockdown,FASN m RNA expression level had no significant changes,m RNA stability and protein expression levels were significantly down-regulated,and the number of adipocyte generation was significantly reduced with statistical significance(all P<0.05).Conclusion:(1)HuR can specifically bind to FASN m RNA 3’utr and participate in regulating the function of FASN m RNA 3’utr;(2)HuR mainly enhances the translation level of FASN protein by improving the stability of FASN m RNA,and promotes the synthesis of triglycerides and the differentiation of human adipocytes.