Study On The Regulation Of MiR-128-1-5p In Proliferation And Differentiation Of Sheep Preadipocyte

Sheep is an important economic livestock,which can produce meat,wool,skin,milk and other products.The content of adipose tissue is critical to meat quality in terms of meat performance.The proliferation and differentiation of preadipocytes are two important biological processes for studying adipogenesis,which are affected by a variety of regulatory factors.As an important regulatory factor,miRNA(micro RNA)can inhibite the mRNA transcription process or directly degrade the mRNA by binding to the target gene,and plays a regulatory role in the post-transcription of mRNA.Therefore,it is particularly significant to study the regulation of miRNAs during the proliferation and differentiation of sheep preadipocytes,which will help to further understand lipid mechanisms and provide theoretical basis for improving carcass quality.In this study,1-month-old Dorper lamb were used as test objects.Full-length CDS of KLF11 gene were cloned by reverse transcription,PCR amplification and sequencing.The nucleic acid and protein structures of KLF11 were analyzed by bioinformatics.The target genes of miR-128-1-5p was predicted by using miRcode and miRWalk softwares,and the target relationship between them were verified by double fluorescence report test.Through lentivirus infection,KLF11 was overexpressed or inhibited during the differentiation of sheep preadipocyte,and the effects of KLF11 on adipocyte differentiation were studied by q PCR,Western blotting and oil red O staining.After miR-128-1-5p was overexpressed or inhibited by transfection of miR-128-1-5p mimic or inhibitor,q PCR,Western blotting and oil red O staining were used to investigate the mechanism of miR-128-1-5p targeting KLF11 and KLF2 in the process of sheep preadipocyte differentiation.By overexpression of miR-128-1-5p,the expression of proliferation marker genes were detected by q PCR,the cell proliferation was detected by CCK-8 and Ed U,and the effect of miR-128-1-5p on the proliferation of sheep preadipocytes was studied.The main results of this study are as follows:(1)The results of sheep KLF11 gene cloning and bioinformatics analysis showed that the complete sequence of sheep KLF11 CDS(coding sequence)was 1518 bp,which consisted of three exons,GC content was higher than AT content,and it encoded 505 amino acids.KLF11 protein molecular weight was 53783.58 u,isoelectric point was 8.55,there was no transmembrane region and signal peptide structure,and KLF11 protein belonged to unstable hydrophobic protein.KLF11 protein contained fifty-five phosphorylation sites,two O-glycosylation sites,and three tandem C2H2 zinc finger structures at the C end.The zinc finger structures acted as transcription factors by recognizing specific DNA sequences.(2)There was a binding site between miR-128-1-5p and KLF11-5’-UTR and KLF2-3’-UTR,respectively.(3)In infected sheep preadipocytes with KLF11 lentivirus expression system,it was found that the expression of adipogenic differentiation marker genes were significantly reduced(**P<0.01)after overexpression of KLF11,but the results were opposite after KLF11 was inhibited.After overexpression of miR-128-1-5p,the expression of KLF11 was extremely significantly decreased(**p<0.01).The mRNA expression of adipogenic differentiation marker genes were extremely significantly increased(**P<0.01)in the miR-128-1-5p overexpressing cells,and more lipid droplets were produced therefrom.The results were opposite after miR-128-1-5p was inhibited.The results showed that KLF11 had an inhibitory effect on fat differentiation.miR-128-1-5p could promote the differentiation of sheep preadipocytes by down-regulating the expression of KLF11.(4)After miR-128-1-5p was overexpressed during the proliferation of sheep preadipocyte,the expression of proliferation marker genes,cell activity and number of new cells were significantly reduced(*p<0.05).The results showed that miR-128-1-5p inhibited the proliferation of sheep preadipocyte.(5)The expression of miR-128-1-5p generally increased,while the expression of KLF2 decreased after transfection with miR-128-1-5p mimic into sheep preadipocyte.The expression of KLF2 was extremely significantly decreased(**p<0.01)after miR-128-1-5p was overexpressed.The mRNA expression of the adipogenic differentiation marker genes were all increased in different degrees(*P<0.05).A large number of lipid droplets were produced therefrom.The results were opposite after miR-128-1-5p was inhibited.The results showed that there was a negative correlation between the expressions of miR-128-1-5p and KLF2,and miR-128-1-5p may promote the differentiation of sheep preadipocytes by down-regulating the expression of KLF2.