| The mammary gland is the main organ of mammalian lactation,where fatty acid metabolism is quite active.Micro RNAs(miRNAs)are endogenous non-coding RNAs with a length of 18-24 nucleotides,which can participate in the regulation of cell life activities through target genes,including lipid metabolism.The expression level of miR-424-5p varies significantly in different lactation stages of dairy goats.However,there is no research on the regulation of miR-424-5p on the lipid metabolism in mammary glands of dairy goats.Therefore,the study on the transcriptional regulation of miR-424-5p in dairy goats is of great significance to the mechanism of lipid synthesis in ruminants.In this study,dairy goat mammary epithelial cells were used as research materials to explore the regulation of miR-424-5p on cellular fatty acid metabolism.The study found that miR-424-5p directly targets 3`UTR of INSR gene to play a regulatory role by bioinformatics and dual luciferase reporter system.The overexpression plasmid and si RNA interference method are used to explore the influence of INSR on the lipid metabolism of mammary epithelial cells,so as to reveal the molecular mechanism of miR-424-5p regulating fatty acid metabolism in dairy goats through INSR.The main findings are as follows:1.The effect of miR-424-5p on fatty acid metabolism of breast epithelial cells.Overexpression of miR-424-5p significantly up-regulated the accumulation of triglycerides and lipid droplets in breast epithelial cells.The m RNA expression level of fatty acid metabolism-related genes like FASN was significantly down-regulated(P<0.05).The m RNA expression levels of DGAT1 and SCD1 were significantly up-regulated(P<0.05).After inhibiting the expression of miR-424-5p,the triglyceride content was significantly down-regulated(P<0.05).The lipid droplet content had a downward trend but not significant(P>0.05).And the expression of the fatty acid extension-related gene ELOVL6 m RNA was significantly up-regulated;Thus,miR-424-5p participates in the regulation of lipid metabolism in breast epithelial cells.2.Bioinformatics analysis showed that INSR gene is one of the target genes of miR-424-5p.And overexpression of miR-424-5p causes the m RNA level of INSR to decrease significantly(P<0.01),meanwhile inhibition of miR-424-5p causes the m RNA level of INSR to increase significantly(P<0.05).Dual luciferase experiments have further proved that miR-424-5p targets the INSR and inhibits its expression.3.We designed PCR primers according to the predicted sequence of INSR in Gen Bank,and the CDS sequence of goat INSR was cloned.The sequence length is 4110 bp,encoding1369 amino acids.The protein molecular weight is 154.75 KDa,which is mainly located on the cell membrane.The results of homology analysis showed that except for goats,the INSR of dairy goats have the closest relationship with goats,followed by sheep,and the farthest relationship with mice.4.Construction of pcDNA3.1-INSR recombinant overexpression plasmid,after transfection into goat mammary epithelial cells,INSR gene m RNA and protein expression levels were significantly up-regulated(P<0.01),lipid metabolism-related genes DGAT1 and SCD1 m RNA expression levels were significantly down-regulated.The internal triglyceride content and lipid droplet accumulation were significantly reduced(P<0.01).C18:0 was significantly up-regulated in the composition of fatty acids(P<0.01).In addition,overexpression of INSR significantly increased the phosphorylation levels of Akt and m TOR proteins(P<0.01).It indicates that overexpression of INSR gene can inhibit the lipid synthesis of breast epithelial cells through Akt/m TOR signaling pathway.5.The expression of INSR interfered by si RNA technology.After si RNA-INSR is transfected into breast epithelial cells,the m RNA and protein expression levels of INSR gene were significantly reduced(P<0.01).And the m RNA expression levels of lipid metabolism-related genes like FASN,ACACA,ELOVL6 were significantly down-regulated(P<0.05),while DGAT1 and DGAT2 m RNA levels were significantly up-regulated(P<0.01).After interfering with INSR,the phosphate of Akt and m TOR proteins were significantly down-regulated(P<0.05),which indicates that INSR participates in the lipid metabolism process of breast epithelial cells by affecting the Akt/m TOR signaling pathway.The above results show that miR-424-5p can regulate the milk fat metabolism process of goat mammary epithelial cells through INSR.This study provides the theoretical and experimental basis for the regulatory network and mechanism of miRNA on fatty acid metabolism in mammary epithelial cells of dairy goats. |
