The Effect Of MicroRNA-331-3p On Preadipocytes Proliferation And Differentiation And Fatty Acid Accumulation In Laiwu Pigs

MicroRNAs are a class of endogenous single-stranded non-coding small RNAs found in eukaryotic organisms.The length of microRNAs is about 18-25 nt.MicroRNAs are strictly conservative among different species.They have the function of regulating post-transcriptional translation of genes.They can participate in various life activities,including the development of organisms,the immune process of organisms,and even participate in the occurrence of cancer and the formation of mature adipocytes.The formation of mature adipocytes is a complex biological process involving many factors,which includes three main stages,including the formation of precursor adipocytes by mesenchymal stem cells,the growth and proliferation of precursor adipocytes and the differentiation of precursor adipocytes into mature adipocytes.The latter two stages are important parts of mature adipocyte formation.It is of great significance to reveal the molecular mechanism of adipocyte formation and apply it to animal breeding.In this study,we selected differentially expressed miR-331-3p for further study through the results of transcriptome sequencing and reading related literature.miR-331-3p has been proved to be involved in the proliferation,differentiation and fatty acid accumulation of porcine adipocytes.The main results are as follows:1.After over-expression of miR-331-3p,CCK8 reagent was used to detect the growth status of cells.It was found that the growth rate of cells slowed down.The results of flow cytometry analysis showed that the proportion of cells in G0/G1 phase increased,and the proportion of cells in S phase decreased,which was consistent with the results of CCK8 experiment.After over-expression of miR-331-3p,cell cycle-related genes were detected by qRT-PCR.It was found that the levels of genes related to promoting cell proliferation were significantly decreased,while those related to inhibiting cell proliferation were significantly increased.The results showed that miR-331-3p could inhibit the proliferation of porcine precursor adipocytes.2.After overexpression of miR-331-3p,the preadipocytes were induced to differentiate and the expression trend of miR-331-3p during the differentiation of preadipocytes was detected by qRT-PCR.The expression change of PPARgamma,a marker gene of preadipocyte differentiation,was detected during the differentiation.It was found that miR-331-3p could promote the differentiation of preadipocytes.3.In this study,the genes DLST and SL25A1 related to fatty acid metabolism were predicted by bioinformatics.The changes of luciferase expression in double luciferase reporting system confirmed that DLST was the target gene of miR-331-3p.Enzymes encoded by the DLST gene are present in the citrate pyruvate cycle pathway,which transports acetyl-COA,essential for fatty acid synthesis,from mitochondria to cytoplasm.According to the fluorescence quantitative results of DLST,SLC25A1 genes and upstream and downstream genes of their pathways,especially ACLY and FAS genes,fatty acid synthesis increased after overexpression of miR-331-3p.Oil red O staining and lipid droplet quantification showed that fatty acid synthesis increased after over-expression of miR-331-3p,while inhibitor transfection had the opposite effect.In turn,miR-331-3p could promote fatty acid synthesis through citratepyruvate cycling pathway.In conclusion,this study systematically analyzed the regulation of miR-331-3p on the proliferation,differentiation and fatty acid accumulation of preadipocytes,and finally proved that miR-331-3p can inhibit the proliferation of preadipocytes,promote their differentiation into mature adipocytes and promote fatty acid accumulation.