Regulation Of KLF4 On Intramuscular Preadipocyte Differentiation In Goat

Krüppel-like factors(KLFs),as transcriptional regulatory proteins,involved in the regulation of a wide range of physiological processes,such as cell proliferation,differentiation,apoptosis,and embryonic development.Recent studies have found that many KLFs involved in the regulation of adipocyte differentiation.KLF4,as a member of the KLF family,plays an important biological role in the regulation of adipocyte adipogenic differentiation about this,however,previous studies mainly focused on human and mice cell lines,and only predicted sequences of goat KLF4 could be found in NCBI,it is not clear that the regulation and possible mechanism of KLF4 in the differentiation of goat intramuscular preadipocytes.In this study,RT-PCR,real-time quantitative PCR,cell culture,overexpression,interference,RNA-Seq technology and bioinformatics analysis were used to demonstrate the effect of KLF4 on intramuscular preadipocyte differentiation in goat.The main results of this study are as follows:(1)The sequence of goat KLF4 gene was 1536 bp,encoding a protein of 486 amino acids,possessesing three typical highly conserved Cys2/His2 zinc finger structures of KLF family.(2)The relative expression level of KLF4 was highest in spleen,which significantly higher than other tissues(P<0.01),KLF4 gene also kept a high expression spleen and subcutaneous adipose tissue,and relatively lower in heart and muscle tissues.(3)The recombinant adenovirus vector Ad-KLF4 was successfully constructed and packaged in 293 cells.The efficiency of siRNA sequences targeting goat KLF4 was 71.62%,85.11% and 61.50% respectively.(4)The mRNA expression level of KLF4 decreased firstly and then increased in goat intramuscular precursor adipocyte differentiation process,reached the lowest level on the 1 d after differentiation,and not significant after 2 d.Lipid accumulation decreased after KLF4 overexpression,while increased after KLF4 RNA interference.The mRNA expression of C/EBPβ was extremely significantly upregulated(P<0.01)and PPARγ was significantly upregulated(P<0.05)after KLF4 overexpression,while PPARγ was extremely significantly downregulated(P<0.01)and C/EBPβ was significantly downregulated(P<0.01).The mRNA expression level of KLF2,5,6,7 were extremely significantly downregulated after KLF4 overexpression(P<0.01),and extremely significantly upregulated after KLF4 interference(P<0.01).(5)A total of 286 differential genes were screened by RNA-Seq,of which 52 genes were up-regulated and 234 genes were down-regulated,which were mainly enriched in metabolic processes,catalytic activities,and biological regulation.The downstream target genes and signaling pathway of KLF4 related to the differentiation of adipocytes,SLC2A3,S1PR1,NR4A1,SOCS2,Wnt5 b et.al and Hippo signaling pathway,Jak-STAT signaling pathway,TNF signaling pathway et.al were by RNA-Seq data analysis.The above results indicate KLF4 may regulate the expression of CEBPβ and PPARγ through antagonism with KLF2,5,6,and 7,and play an inhibitory role in the early stage of goat intramuscular preadipocyte differentiation.The results of this study provide important data to further elucidate the molecular mechanism of KLF4 gene regulating goat intramuscular precursor adipocytes adipogenic differentiation.