Function Research Of Alternative Polyadenylation Of HO1 Gene During 3T3-L1 Adipogenesis

Post-transcriptional regulation that depends on the function of 3' regulatory regions(3'UTR)is an important control mechanism governing gene expression in eukaryotes.Alternative polyadenylation(APA)makes genes regulated differently after transcription by producing 3'UTR variants with different lengths.Sequence and structure of different3'UTR produced by APA determine the stability,localization,and m RNA expression.Heme oxygenase 1(HO1)is a 32-k Da protein ubiquitously expressed and inducible in many tissues.In recent years,the functions of HO1 in oxidative stress,energy metabolism,cell proliferation,and adipocyte differentiation have been gradually revealed,and the complex relationship between the regulatory network of HO1 needs to be improved.However,the research on the regulatory network of HO1 in adipocyte differentiation still needs to be improved.Therefore,this study explored the function of HO1 gene selective polyadenylation in the differentiation of 3T3-L1 cells.The main research results are as follows:(1)Knockdown of HO1 expression in 3T3-L1 preadipocytes can significantly promote 3T3-L1 preadipocyte differentiation and increase lipid droplet accumulation.(2)The 3'RACE results showed that both bovine and murine HO1 genes can produce two different variants through APA.(3)Luciferase experiments show that mouse and cow HO1 long and short 3'UTR have different regulatory abilities,and the short 3'UTR has higher luciferase activity.The3'UTR overexpression vector with different lengths of mouse HO1 was constructed and transfected into HEK293 T.the results of q RT-PCR and Western blot showed that the m RNA and protein expression levels of short variants were higher than long.(4)The miRNA155-5P and miRNA377-3P can directly bind to the 3'UTR region of mouse HO1 long variant and inhibit its expression,while the short variant is not affected.(5)Both the long and short variants of HO1 can inhibit the differentiation of 3T3-L1 preadipocytes and reduce lipid droplets' accumulation.However,the effect of short variants is more evident than long.(6)In 3T3-L1 cells,miRNA155-5P and miRNA377-3P can reduce inhibiting cell fat differentiation caused by overexpression of HO1 long variant.(7)In 3T3-L1 cells,interfering miR155-5P and miR377-3P can rescue the HO1 long variant and enhance the inhibitory effect of HO1 on 3T3-L1 preadipocyte differentiation.In a word,this study clarified a new post-transcriptional regulation mechanism that regulates HO1.HO1 can escape the inhibition of miR155-5P and miR377-3P by producing shorter variants from APA,and different inhibit the differentiation of 3T3-L1 preadipocytes.It provides a theoretical basis for further enriching the molecular mechanism of HO1 in adipogenesis.