The Function Of KLF3 And KLF15 In Bovine Preadipocyte Differentiation And Lipid Metabolism

Beef as a high protein and low cholesterol meat food has been favored by many people.Its tenderness and flavor can be improved with the increase of intramuscular fat content.The formation and deposition of animal fat are complex physiological processes regulated by a variety of transcription factors,which are closely related to the differentiation of fat cells and lipid metabolism.The KLF family of transcription factors(Krupple-like factors,KLFs)is a very important class of zinc finger transcription factor,plays an important role in the regulation of animal growth,cell proliferation and differentiation and apoptosis,especially in differentiation and lipid metabolism of fat cells.The mammalian KLF gene family consists of 17 members namelyKLF1 to KLF17;The members KLF3 and KLF15 of this family,plays an important role in the adipocyte differentiation and lipid metabolism,but the effects on the bovine preadipocyte differentiation and lipid metabolism and its regulation mechanism is still unclear.In order to explore the effects of bovine KLF3 and KLF15 on adipocyte differentiation and lipid metabolism,bovine preadipocytes were studied by adenovirus mediated over expression and siRNA technology.At the same time,we preliminarily explored the interaction between bovine KLF3 and KLF15 at the transcriptional level,and their effects on some important genes related to adipocyte differentiation and lipid metabolism.In this experiment,the regulation mechanism of KLF3 and KLF15 genes were studied in adipocyte differentiation and lipid metabolism at cellular and molecular levels.The main results were as following:1? We found that KLF3 gene was expressed highly in rumen and adipose tissues in both adult and newborn Qinchuan cattle,while low expression was found in the skeletal muscle.This result revealed that bovine KLF3 gene might play an important role in the regulation of bovine adipocyte differentiation and lipid metabolism.KLF15 gene was highly expressed in liver,kidney,and fat in both newborn and adult Qinchuan cattle,but low expression was found in lung.It revealed that KLF15 gene might plays an important role in adipocyte differentiation and lipid metabolism.2? Adinovirus over-expression vector of KLF15 gene was constructed,high titer recombinant adenovirus over-expression system was optimized in bovine preadipocyte.KLF15 gene was silenced through siRNA interference system suitable in bovine preadipocyte.Overexpression of KLF15 gene showed that the expression of PPAR? and C/EBP? were up-regulated,while the expression levels of key genes of lipid metabolism,FAS,ACC and FABP4 were also significantly upregulated(P<0.05).However,the expression of these key genes was significantly(p<0.05)down regulated through siRNA silenced KLF15 gene.Oil red O staining and triglyceride content were detected.It was found that over expression of KLF15 gene promoted triglyceride accumulation in bovine adipocytes,similarly KLF15 silenced gene inhibited the accumulation of triglyceride in bovine adipocytes.3?The siRNA of KLF3 with high interference efficiency was screened out.Interference of KLF3 on second day of transfection showed 73% interference,and protein level was also significantly down regulated,so it can be used for subsequent interference experiments.After transfection of Si-KLF3 to bovine preadipocytes,the expression of PPAR? and C/EBP? were down regulated significantly(P<0.05),and the expression of key genes of lipid metabolism FAS,ACC? and FABP4 also decreased significantly(P<0.05).And the determination of oil red O and triglyceride content was inhibited by interference KLF3 gene in bovine adipocytes4?The TSS(Transcription start site)of KLF3 was found at 39 bp upstream of translation initiation site(ATG).We defined it as +1,and compared with the KLF3 mRNA sequence(NM_)transcript initiation site reported in the NCBI database.Thus the validated transcription start site was found 6 bp downstream of the reported mRNA sequence of the KLF3 gene.The 5'flanking sequence 1594 bp of the bovine KLF3 gene was cloned.In-594,-376 and-114 bp were found with three single nucleotide polymorphisms.We found 8 haplotypes.the level of intramuscular fat content(IFC)of individuals with the Hap7/7 diplotype was much higher(P<0.05)than that of individuals with other diplotypes.The promoter activity with the H7 haplotype was higher(P<0.05)than the H8 haplotype,consistent with the association analysis.Polymorphisms in transcription factor binding sites of the KLF3 promoter region also affected transcriptional activity of KLF3,which subsequently might influence intramuscular fat content in Qinchuan cattle.5? the analysis of luciferase activity of the deletion fragment shows that the core promoter region of KLF3 gene is located at-264 /-76 bp,and it is found that there are three KLF15 binding sites in KLF3 core promoter.Overexpression vector of KLF15 and control(Ad-CMV-NC)was transfected into bovine adipocytes found that overexpression of KLF15 gene enhanced KLF3 segment deletion promoter activity,also by RNA interference(siRNA)and the KLF15 binding site directed mutagenesis,were co-transfected with plasmid pGL-264/-76 and KLF15 siRNA,as well as control(si-NC)and point mutation of pGL3-264 was transfected into bovine primary preadipocytes.It was found that the activity of pGL--264 promoter was decreased.This shows that KLF15 promotes the transcriptional activity of KLF3 promoter in bovine adipocytes.Interference of KLF3 gene KLF 15 gene was significantly downregulated at the transcriptional level,and it may be due to the micRNA co-regulating the KLF 3 and KLF 15 gene.