Regulation Of KLF11 On Intramuscular Preadipocytes Differentiation In Goat

Krüppel-like factors(KLFs)family is a type of transcriptional regulatory protein,and multiple family members have been shown to be key participants in the transcriptional network of mammalian preadipocyte formation,adipogenesis,adipogenesis,and obesity.As a member of KLFs,KLF11 plays an important regulatory role in the adipogenic differentiation of adipocytes.However,the research of KLF11 in adipocytes mostly focuses on human and mouse cell lines.Whether KLF11 plays a regulatory role in the differentiation of intramuscular preadipocytes in goats is still unclear.Therefore,this study used RT-PCR,real-time fluorescent quantitative PCR(q PCR),vector construction,cell culture,RNA interference,overexpression,triglyceride determination and dual luciferase reporter detection system and other test methods.Using the above-mentioned methods to reveal the regulatory effect of KLF11 on the differentiation of goat intramuscular preadipocytes and its possible mechanism.(1)Cloning,bioinformatics and tissue expression analysis of goat KLF11Successfully cloned goat KLF11 gene sequence 1752 bp,of which CDS region is 1518 bp,encoding 505 amino acid residues.KLF11 gene was widely expressed in various goat tissues,and high expressed in heart,longissimus dorsi and abdominal fat(P<0.01),while low expressed in other tissues.(2)The effect of KLF11 on goat intramuscular preadipocyte differentiationThe relative expression trend of KLF11 in goat intramuscular preadipocytes was significant increased and then downward and the expression level of KLF11 peaked at 48 h after induction.Designed and synthesized the siRNA sequence for KLF11,and successfully constructed the KLF11 overexpression vector(pc DNA-KLF11)based on the obtained goat KLF11 sequence.The results showed that after interference with KLF11 in goat intramuscular adipocytes,the accumulation of intracellular lipid droplets increased,and the expression levels of GPAM,DGAT2,TIP47,FASN,ACC,PPARγ and C/EBPβ increased significantly(P<0.05),while the expression levels of DLK1 and ATGL were significantly reduced(P<0.05).The relative expression of KLF1,KLF2,KLF4,KLF5,KLF6,KLF8,KLF10,KLF14 and KLF16 were significantly down-regulated(P<0.05),while the expression of KLF7 and KLF15 were significantly up-regulated(P<0.05).After overexpression of KLF11,the accumulation of lipid droplets in goat intramuscular adipocytes decreased,and the expression levels of GPAM,DGAT2,TIP47,FASN,ACC,PPARγ,C/EBPα and C/EBPβ were significantly decreased(P<0.05).The relative expression of KLF2,KLF3,KLF4,KLF5,KLF6 and KLF15 were significantly down-regulated(P<0.05),while the expression of KLF10 and KLF17 were significantly up-regulated(P<0.05).(3)The possible mechanism of KLF11 regulating the goat intramuscular adipocytes differentiationSuccessfully cloned goat PPARγ gene promoter sequence 1993 bp,and the promoter luciferase reporter gene vector of goat PPARγ was constructed.The goat PPARγ promoter luciferase reporter gene vector and the C/EBPβ promoter luciferase reporter gene vector stored in the laboratory and the goat KLF11 overexpression vector were co-transfected into goat intramuscular adipocytes.The results showed that goat the promoter activity of PPARγand C/EBPβ was significantly enhanced(P<0.01).In conclusion,we successfully cloned and obtained the sequence of KLF11 gene in goats,and clarified its tissue and cellular timing expression characteristics.Overexpression and interference showed that KLF11 promoted the differentiation of goat intramuscular precursor adipocytes,and this regulation may be achieved through PPARγ and C/EBPβ in cooperation with some members of the family.The results of this study provide important data support for further revealing the molecular mechanism of KLF11 gene in regulating goat fat deposition.