MiR-32-5p Regulates Differential Accumulation Of Intramuscular And Subcutaneous Fat Via Suppressing KLF3 In Pigs

Both intramuscular fat(IMF)and subcutaneous fat(SCF)contents are important factors that affect the economic benefits of the pig industry.In which less subcutaneous fat and more intramuscular fat is favourite.However,the mechanism that affects the differential fat deposition between IMF and SCF remains unclear.Therefore,clarifying the molecular mechanism that regulates digfferential deposition of intramuscular fat and subcutaneous fat is of high economic value and production significance.We picked the KLF3 gene as a candidate gene to study the mechanism that regulates differential deposition of intramuscular fat and subcutaneous fat in Erhualian pigs according to the earlier transcriptome data,and we further investigated the mechanism that regulates KLF3.This study first selected 6 Erhualian individuals from high IMF content group and 6 Erhualian individuals from low IMF content group,and we detected the expression of KLF3 gene in the two groups by real-time-quantitative PCR(RT-qPCR)technology,results showed that KLF3 was significantly negative correlated with IMF content(r=-0.83,P<0.001).Subsequently,we identified the expression of KLF3 in IMF and SCF by RT-qPCR.The results showed that the expression of KLF3 was significantly higher in intramuscular fat than that of subcutaneous fat(P<0.01,n=4).These indicate that KLF3 has an inhibitory effect on fat deposition,and its expression is higher in intramuscular fat,which means the inhibition in intramuscular fat is greater than that in subcutaneous fat.Then,overexpression vectors and siRNA were used to transfect preadipocyte cells to study the function of KLF3.RT-qPCR results showed that the overexpression of KLF3 can significantly inhibit the expression of PPARγ(P<0.05),C/EBPα(P<0.01)and FABP4(P<0.01).Oil red O staining and triglyceride content showed that the triglyceride was significantly down regulated(P<0.01);and the interference of siRNA can significantly upregulate the expression of PPARγ(P<0.01)、C/EBPα(P<0.05)and FABP4(P<0.05),Oil red O staining and the triglyceride content showed that the triglyceride content was significantly upregulated(P<0.001).In order to study the regulation mechanism for differential expression of KLF3,we predicted miRNAs that might target KLF3 through the miRNA prediction website and screened 5 of them as candidates according to our earlier transcriptome data.RT-qPCR results showed that only miR-32-5p expression and KLF3 expression were both significantly negative correlated in different intramuscular fat content group(r=-0.79,P<0.01)and different fat tissues of intramuscular fat and subcutaneous fat(r=-0.90,P<0.01)among the 5 candidates.In order to further determine the targeting relationship between miR-32-5p and KLF3,a fluorescent vector of the wild-type 3’UTR of the KLF3 gene was constructed,and a mutant vector with a mutation in the binding region was constructed by point mutation.Dual luciferase reporter gene experiment showed that miR-32-5p can target the wild-type S’UTRvector of the KLF3 gene and inhibit its luciferase activity(P<0.05),but has no significant effect on the mutant vector’s fluorescence activity(P>0.05).Experiments in preadipocytes further confirmed that the transfection of miR-32-5p mimics could significantly down-regulate the expression of KLF3 gene(P<0.05),and the expression of PPARy(P<0.001)and FABP4(P<0.05)were upregulated in different degrees.Oil red O staining and the triglyceride content showed that the transfection of miR-32-5p mimics could significantly promote the deposition of triglyceride(P<0.001),and this process could be abolished by the overexpression of KLF3.This study finds that the expression of KLF3 gene could negatively regulate fat deposition in pigs,and miR-32-5p could regulate the differential fat deposition of intramuscular fat and subcutaneous fat in pigs via targeting KLF3.