The Function Of SIRT2 Gene In Bovine Preadipocyte Differentiation And Its Transcriptional Regulation Analysis

Sirtuins are NAD⁺-dependent deacetylases and play critical role in growth,patterning,aging,metabolism and so on.Sirtuins have received growing attention,as their implications in the most important human diseases such as longevity and aging,cancer,energy and metabolic disorders.Mammals harbor seven different sirtuins named SIRT1-SIRT7,which differ in the celluar location and functions.Among them,SIRT2 is the predominantly cytoplasmic protein,and through its downstream targets it participates in various physiological processes,including adipogenesis,fatty acid oxidation,insulin sensitivity,gluconeogenesis and so on.To date,little is known about the function of the bovine SIRT2gene in adipogenesis,and also its transcriptional regulatory mechanism is not clear.The aim of this research was to study the function of the bovine SIRT2 gene in adipogenesis at both the celluar and molecular level,using the adenovirus-mediated gene over-expression and down-regulation biotechnology.Furthermore,the characterization and activity of the bovine SIRT2 were analyzed using bioinformatics and dual-luciferase reporter assay.Finally,the analysis of the transcriptional regulation mechanism of SIRT2 gene was performed using site-specific mutagenesis,electrophoretic mobility shift assay（EMSA）and chromatin immunoprecipitation（ChIP）technologies.The main results were as follows:（1）The complete CDS region of the bovine SIRT2 gene was obtained.And the CDS region was 1173 bp in length,encoding 390 amino acids.Recombinant adenoviral expression vector carrying bovine SIRT2 CDS was successfully constructed,and after being packaged and amplificated in HEK 293 A cell lines,the high titer adenoviruses Ad-SIRT2 were acquired.（2）By screening in HEK 293 A cell lines,one shRNA targeting the bovine SIRT2 gene was obtained with the interference efficiency of 74.7%,named shRNA-1002.After the recombination of shRNA-1002 with adenovirus-backbone-vector,and the subsequent packaging and amplification in HEK 293 A cell lines,the high titer adenviruses Ad-shRNA-1002 were obtained.（3）The bovine preadipocytes were obtained using enzyme digestion method.The detection of the effectiveness of recombinant adenovirus using real-time PCR was performed after transfecting the bovine preadipocytes with adenovirus for 72 h.Results showed that compared with the Ad-CMV-NC,the mRNA content of SIRT2 in the Ad-SIRT2 group increased by 316.3（P<0.01）.Moreover,compared with the Ad-shRNA-NC,transfection of Ad-shRNA-1002 resulted in a decrease of 68.3%in SIRT2 mRNA content（P<0.01）.These results suggested that Ad-SIRT2 and Ad-shRNA-1002 could be used for over-expression and inhibiton of the bovine SIRT2,respectively.（4）Oil O staining results showed that over-expression of SIRT2 in bovine preadipocytes could decrease the accumulation of lipid droplets,significantly inhibiting the differentiation of preadipocytes.On the contrary,the inhibition of SIRT2 could improve the accumulation of lipid droplets,significantly promoting the differentiation of preadipocytes.（5）After induced differentiation of the bovine preadipocytes for 6 d,the detection of the key transcription factors related to adipogenesis and important functional genes related to metabolism of lipid droplets was performed by real-time PCR in the SIRT2 gene over-expression and inhibition groups,respectively.Results indicated over-expression of SIRT2 downregulated C/EBPδand PPARγmRNA expression level,and also the mRNA level of FABP4（fatty acid binding protein 4）,FAS（fatty acid synthase）and LPL（lipoprotein lipase）（P<0.05）.Whereas down-regulation of SIRT2 could significantly improve the mRNA expression level of C/EBPδ,PPARγ,FABP4,FAS and LPL（P<0.05）.However,both the over-expression and down-regulation of SIRT2 had no significant influence on C/EBPβ,C/EBPδand SREBP1（P>0.05）.（6）Using 5’-rapid amplification of cDNA ends,several transcriptional start sites（TSS）were identified in the bovine SIRT2 gene.The guanine residue（G）,which was located 85 bp upstream of the translation initiation site,was designated as+1,and it came to an agreement with the 5’end position of the SIRT2 mRNA（NM₀01113531）provided in the GenBank.（7）A 2017 bp fragment（-1956/+61）of the bovine SIRT2 5’flanking region from the bovine genome was cloned.Stepwise 5’end deletion analysis indicated the putative SIRT2core promoter region was located in-178/+4.Bioinformatics analysis predicted that the binding sites of SP1,E2F1,YY1,XBP1 and so on were in bovine SIRT2 core promoter.However,the classical eukaryotic promoter elements TATA box and CAAT box were not found,suggesting that SIRT2 promoter was a TATA-less promoter.（8）After site-specific mutation of YY1 binding site,the SIRT2 promoter transcriptional activity decreased by about 60%（P<0.01）.Further,results of analyses by EMSA and ChIP respectively confirmed the combination of YY1 with the bovine SIRT2 promoter in vitro and in vivo.Collectively,YY1 positively regulated the transcriptional activity of SIRT2.In conclusion,the bovine SIRT2 gene suppressed the adipocyte differentiation;YY1positively regulated the transcriptional activity of SIRT2.Also,a simple sketch map of the interaction among YY1,SIRT2 pathway and PPARγwas obtained based on both the prior findings and current findings.Our results provided the evidence to comprehend the function of bovine SIRT2 in adipocyte differentiation.Furthermore,our findings were helpful for understanding the characterization of SIRT2 gene,and also broadened the gene regulatory network related to adipogenesis.Consequently,our study not only provided theory evidence for further studying and better understanding the complicated molecular mechanism of fat formation and deposition,but also laid theoretical basis for enhancing the meat quality of beef cattle.