| MicroRNAs(miRNAs)are important short gene fragments in various tissues,about 22 nucleotides in length.miRNAs are also important regulatory factors in lipid metabolism.Lipid is the main component of milk,which is important for the growth and development of infants.Therefore,it has become an important research in screening for miRNAs that affects the metabolism of lipid and affects the viability of mammary epithelial cells.MiR-29 b is a key miRNA,involved in many important life processes,including cancer cells proliferation and migration and intestinal epithelial cells proliferation;in addition,there are significant differences in the expression of miR-29 b in the high-fat and low-fat dairy cow mammary tissue,suggesting that miR-29 b may be a key factor in the regulation of lipid metabolism.MiR-29 b participates in the process of milk fat metabolism and apoptosis by regulating the target genes.Therefore,we first predicted the possible target genes of mi R-29 b by bioinformatic analysis.The results showed that miR-29 b could identify 5730 binding sites in 4353 transcripts.We initially locked the LPL and TDG genes as the target genes for this study through functional analysis.There are specific loci for identification with the miR-29 b seed sequence(AGCACCA)in LPL 3'-UTR(1802-1809 bp)and TDG3'-UTR(1057-1064 bp).According to the binding site,vectors were designed,including LPL-3'-UTR-WT(TGGTGCTA),LPL-3'-UTR-mut(CAAGTACG),TDG-3'-UTR-WT(TGGTGCTA),TDG-3'-UTR-mut(CAAGTACG).The dual luciferase reporter system detection showed that luciferase activity was significantly lower in group of miR-29 b mimics co-transfected with TDG-3'-UTR-WT than the control group(p<0.05),no significant difference between miR-29 b mimics co-transfected TDG-3'-UTR-mut and and the control group(p>0.05);luciferase activity was significantly lower in group of mi R-29 b mimics co-transfected with LPL-3'-UTR-WT than the control group(p<0.05),there was no significant difference between miR-29 b mimics co-transfected with LPL-3'-UTR-mut group and the control group(p>0.05),TDG and LPL are further verified the target genes of miR-29 b.To understand the effects of miR-29 b on MECs,vectors were transfected into MECs separately.The expression of green fluorescent was obvious under fluorescence microscopy,suggesting that transfection was successful,and then real-time fluorescence quantitative analysis showed that mi R-29 b was successfully expressed or interfered.Then the real-time fluorescent quantitative assay showed that miR-29 b mimics significantly decreased TDG and LPL mRNA(p<0.01);proteins of TDG and LPL genes were significantly higher expression in miR-29 b inhibitor group than miR-29 b mimics group by western blotting;the above results shown that mi R-29 b could play a role in degradation of target genes mRNA and inhibit protein translation.To investigate the effects of miR-29 b on fat metabolism and apoptosis,miR-29 b was overexpressed or disrupted in mammary epithelial cells(MECs).Then the apoptosis rate was analyzed by flow cytometry,mi R-29 b mimics(8.78%),mi R-29 b inhibitor group(21.86%)and mi R-shNC group(17.1%).Higher expression of mi R-29 b could inhibit the apoptosis of mammary epithelial cells,however the silence of miR-29 b could promote the apoptosis of mammary epithelial cells.Then,we transfected mi R-29 b mimics,miR-29 b inhibitor and miR-shNC into mammary epithelial cells,and triglycerides were detected by kit;the results showed that mi R-29 b mimics significantly increased the triglyceride formation,but the effect of mi R-29 b inhibitor on triglyceride was unobvious.According to the above results,we speculated that miR-29 b might inhibit the apoptosis of mammary epithelial cells and promote the formation of triglyceride by down-regulating target genes TDG and LPL,which may provide the theoretical basis and analysis of the underlying molecular mechanisms for fat metabolism. |
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