Transcriptional Regulation Study Of Bovine Smad2 And Smad3 Gene

TGF-beta signaling pathway play an important role in the regulation of muscle and fat growth and development,which is a strong inhibitor during myoblasts and preadipocytes differentiation.The Smads protein play a crucial role in the TGF-?signaling pathway,which can transfer signals from the cell membrane to the nucleus and thereby regulate the transcription of the target genes.Smad2/Smad3 is R-Smads,which are activated by Activin/TGF-?.Studies have demonstrated that Smad3,rather than Smad2 plays a key role in inhibiting myoblasts and preadipocytes differentiation regulated by TGF-?1.In view of the potential role of Smad3 in inhibiting the differentiation of myoblasts and precursor adipocytes,this study investigated the role of Smad3 play in the regulation of bovine myoblasts and precursor adipocytes differentiation through adenovirus-mediated the Smad3 genes overexpression and silencing.At the same time,although the amino acid similarity between Smad2 and Smad3 is high,there are significant differences in the structure,function and transcriptional regulation.In this experiment,a preliminary study about the transcriptional regulation mechanism of bovine Smad2 and Smad3 genes and the regulatory relationship between them at transcriptional level was conducted.The main results are shown as follows:?1?The full-length CDS region of Qinchuan cattle Smad3 gene was successfully cloned.The length of the nucleic acid sequence was 1278 bp and encoded 425 amino acid residues.A shRNA sequence with an interference efficiency of 83.3%targeting the Smad3 gene was screened.We constructed the adenoviral shuttle vectors pDC316-mCMV-EGFP-bSMAD3 and pDC316-EGFP-ShRNA-bSMAD3-1405 of Qinchuan cattle Smad3 gene.The two recombinant plasmids which carried the target gene Smad3 were co-transfected into HEK293A cells,respectively,the virus was packaged and expanded to obtain high-titer.LaSRT method was used to measure virus titer.The results showed that the titer of both AD-bSmad3 and AD-NC?vacant virus?were 1×10¹⁰ PFU/mL.Moreover,AD-ShRNA-bSMAD3 and AD-ShRNA-NC?empty virus?adenovirus titers were also 1 x 10¹⁰PFU/mL?2?qRT-PCR results showed that overexpression Smad3 in bovine preadipocytes can significantly inhibit the expression of adipogenic marker genes PPAR?,FABP4,C/EBP?and C/EBP?,and interference Smad3 in bovine preadipocytes can promote the expression of PPAR?,FABP4,C/EBP?and C/EBP?.Oil red O staining results also showed that overexpression Smad3 in preadipocytes inhibited the accumulation of lipid droplets,and silence Smad3 in preadipocytes can promote the lipid droplet content in preadipocytes increased.In conclusion,Smad3 gene can significantly inhibit the differentiation of Qinchuan cattle preadipocytes.Similarly,overexpression Smad3 gene in myoblasts significantly inhibited the expression of MyoD and MyoG,which are the differentiation markers of myoblasts,and interference Smad3 can promote the expression of the MyoD and MyoG gene.The observation of cell morphology also showed that the length of the myotubes became longer after interfering with the Smad3.?3?The 5'flanking sequence of Smad3 was cloned,the leghth is 1143 bp.The double luciferase assay system showed that the core promoter region of the Smad3 gene was between-337 and-41 bp.Through the site-directed mutation of the transcription factor binding sites,the role of KLF6,KLF15,KLF7,and MZF1 in maintaining the promoter activity of the Smad3 gene was determined;in vitro EMSA experiments further confirmed that KLF6,KLF15,KLF7,and MZF1 are the key transcription factors of the Smad3 gene.KLF6-siRNA,KLF15-siRNA,KLF7-siRNA,MZF1-siRNA and control siRNA were transfected into bovine preadipocytes and myoblasts,qRT-PCR results showed that KLF6,KLF15,KLF7 and MZF1are necessary for maintaining the basic transcriptional activity of the bovine Smad3 promoter.?4?Real-time PCR assay showed that Smad2 and Smad3 genes were widely expressed in various tissues of Qinchuan cattle and the amino acid similarity between them was as high as89.2%.During the differentiation of myoblasts,the expression of both Smad2 and Smad3showed the same trend.The expression of Smad2 and Smad3 increased significantly along with the formation of myotubes.qRT-PCR and Western-blot results showed that Smad3inhibited the expression of Smad2 whether in the bovine myoblasts which were in high serum medium,proliferation culture medium?GM:F12/DMEM+20%FBS?or low serum medium,differentiation culture medium?DM:F12/DMEM+2%HS?.?5?The 5'-terminal cDNA amplification method?SMART RACE cDNA Amplification?was used to determine the transcription initiation site of bovine Smad2 gene.Compared with the transcription start site of the Smad2 gene mRNA sequence?NM₀01046218?was reported in NCBI,we advanced the 5'UTR of the Smad2 gene by 5 bp.The 5'flanking sequence of bovine Smad2 gene was obtained by 1243 bp.The double luciferase assay system showed that the core promoter region of Smad2 gene was located at-158/-35bp.By site-directed mutation of transcription factor binding sites,it was determined that C/EBP?and C/EBP?bind to the core promoter region of Smad2 gene.In vitro EMSA experiments,it has been further confirmed that C/EBP?and C/EBP?transcription factors can bind to the core promoter region of the bovine Smad2 gene and regulate its promoter activities.?6?Both the dual-luciferase assay system and qRT-PCR results showed that the regulation of Smad2 gene by C/EBP?and C/EBP?varied according to the serum concentration.Under the culture condition of low serum medium,C/EBP?and C/EBP?transcription factors inhibit the expression of Smad2 gene.Under high serum medium conditions,C/EBP?and C/EBP?transcription factors promote the expression of Smad2 gene.Similarly,we found that serum concentrations also influence the regulation of C/EBP?and C/EBP?by the Smad3 gene.Smad3 gene promotes the expression of C/EBP?and C/EBP?gene in bovine myoblasts under DM culture conditions,while Smad3 gene inhibits C/EBP?and C/EBP?gene expression in bovine myoblasts under GM culture conditions..The double luciferase assay system has further confirmed that Smad3 gene may regulate the expression of Smad2 gene through regulating the expression of transcription factors C/EBP?and C/EBP?.In conclusion,this article used the adenovirus to mediate gene overexpression and interference techniques to explore the role of Smad3 gene in regulation of bovine preadipocytes and myoblasts differentiation and some functional genes which play an important role in the muscle and fat growth and differentiation;the regulatory mechanisms at the transcriptional level between the Smad2 and Smad3 genes have also been explored,in order to further investigate the role of TGF-?signaling pathways play in the formation of bovine muscle and fat.It can also provide a certain theoretical basis and lay the foundation for the next step in the breeding of Qinchuan cattle.