MicroRNA-125a-5p Affects Adipocytes Proliferation,differentiation And Fatty Acid Composition Of Porcine Intramuscular Fat

The intramuscular fat(IMF)content and fatty acid composition are two important factors which decide the quality of the meat products,and they are directly related to consumer’s health.The lipid deposition of intramuscular preadipocytes is influenced by various factors such as inheritance,nutririon and environment.Among these factors,regulatory molecules play an important role in the post-transcriptional regulation of the adipogenesis related genes.Micro RNAs(miRNAs,miRs)are ubiquitous regulatory molecules in eukaryotic cells,and they involve in the development of preadipocytes by repressing the expression of the target genes.Recently,micro RNA-125a-5p(miR-125a-5p)was identified as an important regulator of adipogenesis.However,the molecular mechanism of how miRNA affecting porcine intramuscular adipocytes development is still mostly unclear.In this study,porcine intramuscular preadipocytes were used as experimental material to verify the protential function of miR-125a-5p during the proliferation and differentiation through overexpressing or repressing the expression of miR-125a-5p.Furthermore,the target genes prediction and a luciferase reporter assay demonstrated that KLF13 and ELOVL6 are target genes of miR-125a-5p.Then,we detected the fatty acid composition of porcine intramuscular preadipocytes which were transfected with miR-125a-5p mimics.The main results of this study are as follows:(1)The expression of miR-125a-5p was negatively correlated with the content of intramuscular fat,and the expression of miR-125a-5p was significantly regulated during the differentiation of porcine intramuscular preadipocytes.(2)During the proliferation,porcine intramuscular preadipocytes were transfected with miR-125a-5p mimics,inhibitors,or a negative control(NC),respectively.We found that overexpressing miR-125a-5p enhanced the expression level of proliferation related genes significantly(p<0.05)and reduced the expression level of apoptosis pathway related genes(p<0.05)while transefecting miR-125a-5p inhibitors was opposite to the overexpression of miR-125a-5p;Similarly,the CCK8 and Edu proliferation analysis showed that miR-125a-5p could promote the proliferation of porcine intramuscular preadipocytes.(3)During the proliferation,we also overexpressed or inhibited the expression level of miR-125a-5p by treating porcine intramuscular preadipocytes with mimics or inhibitors,respectively.The Oil Red O staining and triglyceride analysis demonstrated that adipogenesis was blunted in mimics group(p<0.05).By contrast,in the inhibitors group the adipogenesis was strengthened(p<0.05).In addition,quantitative analysis of lipid related genes showed that overexpression of miR-125a-5p decreased the expression level of C/EBPα,PPAR,FABP4,FATP4 and FASN(p<0.05),while the inhibition of miR-125a-5p was contrary to the overexpression of miR-125a-5p.These results demonstrated that miR-125a-5p could reduce the differentiation of porcine intramuscular preadipocytes.(4)miR-125a-5p significantly increaseed the mitochondrial content of the porcine intramuscular preadipocytes.The expression level of(Tfam,Mterf1)and mitochondrial energy metabolism related genes(Cox5b,TMEM70,Cox8 b,Uqcr10,ATP6)increased significantly(p<0.05)when overexpressed miR-125a-5p.These genes were repressed when miR-125a-5p was inhibited.(5)We proved the relationship between miR-125a-5p and the target genes of KLF13 and ELOVL6 by the dual luciferase reporter system which suggested that miR-125a-5p played its role in the regulation of KLF13 and ELOVL6 genes.(6)Porcine intramuscular preadipocytes were transfected with miR-125a-5p mimics then collected and determined the fatty acid composition.Results showed that the content of saturated fatty acid(SFA)decreased significantly(p<0.05)and monounsaturated fatty acids(MUFA)/SFA ratios increased significantly,while there were no significant impact on polyunsaturated fatty acids(PUFA)/SFA and n-6/n-3(omega-6/omega-3)ratios(p>0.05)).This suggested that miR-125a-5p participated in the regulation of fatty acid composition in porcine intramuscular fat precursor cells.