MicroRNA-141 Regulates Milk Fat Metabolism In Bovine Mammary Gland Epithelial Cells By Targeting The SIRT1 Gene

Milk fat metabolism is a complex procedure which is regulated by many factors.SIRT1 is a member of the Sirtuin family and a kind of deacetylase.Besides,it is highly conserved in mammals.SIRT1 is also one of the silent message regulators in the mammalian.SIRT1 is highly expressed in the adipocyte and it can inhibit the formation of fat.However the study of the effects of SIRT1 on the milk fat metabolism has seldom been reported in the bovine mammary epithelial cells.MicroRNAs(miRNAs)act as post-transcription regulators.They can indirectly influence the fat metabolism process by regulating the expression of the target gene.In our previous studies,we used high thoughput sequencing technique to identify the expression patterns of microRNAs in mammary glands between peak lactation and early lactation.The result showed that the expression of bta-miR-141 were significantly different(P<0.05).Bioinformatics analysis showed that bta-miR-141 targets SIRT1 gene.In this study,the mammary epithelial cell and human renal epithelial cell lines(293T)were used to investigate the function and regulatory mechanism of miR-141 in bovine mammary gland epithelial cells by Oil Red O stain?Triglyceride quantification?Dual-Glo luciferase assay?qRT-PCR and Western Blot.The results were as follow:1.Functional analysis of bta-miR-141:miR-141 mimic and mimic-NC were transfected into mammary gland epithelial cells respectively.The oil red O staining experiment was carried out after 48 hours.The results were as follows:Compared to mimic-NC group,the fat content of miR-141 mimic group was significantly increased(P<0.05).The effects of miR-141 on milk fat metabolism in mammary epithelial cells was further verified by triglyceride content assay.The results showed that the triglyceride content of miRNA-141 mimic group was significantly higher than mimic-NC group(P<0.05).The above results indicate that miR-141 had a function of promoting milk fat metabolism in mammary gland epithelial cells of dairy cows.2.Validation of the targeting relationship between bta-miR-141 and SIRT1 gene:The 3'UTR of SIRT1 gene was inserted into the pmirGLO vector to construct the dual luciferase wild-type vector and mutant vector.They were co-transfected with miR-141 mimic and mimic-NC into human renal epithelial cell line(293T),and the fluorescence activity was measured.The results were as follows:After overexpressed of miR-141,the ratio of firefly enzyme/sea-renal enzyme activity(F/R)in wild-type vector group was significantly lower than mimic-NC group(P<0.05);the ratio of firefly enzyme/sea-renal enzyme activity in mutant vector group had no significant difference(P>0.05).Further,miR-141 mimic,mimic-NC,miR-141 inhibitor,and inhibitor-NC were transfected into mammary gland epithelial cells.The results of qRT-PCR showed that overexpression of miR-141 was able to significantly inhibit the expression of SIRT1(P<0.05).The results of Western Blot verification were consistent with the results of qRT-PCR.The above results indicated that miR-141 could inhibit the expression of SIRT1 gene at the mRNA and protein levels,and the SIRT1 gene is the target gene of miR-141.3.Whether bta-miR-141 affect other genes related to milk fat metabolism by regulating the SIRT1 gene:miR-141 mimic and mimic-NC were transfected into mammary gland epithelial cells respectively.The expression of SREBF1,FASN and PPARy were detected by qRT-PCR.Compared to mimic-NC group,the expression of SREBF1,FASN and PPARy were increased.Interfering RNA(siRNA)knockdowned the expression of SIRT1 gene expression.SiSIRTl,miR-141 mimic,mimic-NC and siSIRTl-NC were co-transfected into mammary gland epithelial cells respectively.The expression of SREBFJ,FASN and PPARy were detected by qRT-PCR.The results showed that they were significantly increased after co-transfection of siSIRTl with mimic-NC group,siSIRTl-NC with mimic group(P<0.05).After inhibiting the expression,of SIRT1 by siSIRT1,miR-141 mimic was transfected into the cells.The expression of SREBF1,FASN and PPARy were not significantly changed(P>0.05).These results indicate that miR-141 affected the genes involved in milk fat metabolism by regulating the SIRT1 gene.In summary,the bta-miR-141 affects the process of milk fat metabolism by targeting SIRT1 gene in mammary gland epithelial cells.And miR-141 promoted the expression of genes related to milk fat metabolism by inhibiting the expression of SIRT1 gene.This study would provide a basic theory to reveal the function and regulation mechanism of microRNA on the milk fat level of dairy cows.