Lnc-TRTMFS Regulates Milk Fat Synthesis In Dairy Cow Mammary Epithelial Cells By Adsorbing MiR-132x And Targeting RAI14

Milk fat is an important index to evaluate the quality of milk.The level of milk fat directly determines the quality and taste of milk and dairy products.Lnc RNA(longnoncoding RNA)is a kind of RNA longer than 200 nt,which participates in the regulation of life process in the form of RNA.It can participate in downstream signal pathways to regulate biological processes by competitively combining miRNA to regulate the expression of m RNA.In this study,based on the sequencing data of mammary gland tissue during lactation and dry period,lnc RNA(named lnc-TRTMFS,Transcripts related to milk fat synthesis),which may be related to the regulation of milk fat synthesis,was selected to verify its role in milk fat synthesis in dairy cow mammary epithelial cells.Double luciferase activity analysis and other techniques were used to analyze the interaction between lnc-TRTMFS and miRNA in the regulation of milk fat synthesis and competitive regulatory genes.The results are as follows:1.Functional verification of Lnc-TRTMFS: when BMECs was transfected with lnc-TRTMFS overexpression vector and small interference RNA(siRNA),it was found that lnc-TRTMFS overexpression significantly promoted triglyceride accumulation and lipid droplet formation in BMECs,up-regulated the expression and phosphorylation of genes related to milk fat synthesis and key molecules of mTOR signal pathway 4EBP1 and P70S6K,while interfered with lnc-TRTMFS,suggesting that lnc-TRTMFS may regulate milk fat synthesis in BMECs through mTOR signal pathway.2.Lnc-TRTMFS targeted miRNA screening and validation: using prediction software Bibiserv2 and miRBase,lnc-TRTMFS was predicted to have targeted binding relationships with miR-1468-5p,miR-1468,miR-1260 b,miR-21-5p,and miR-132x;Transfection of siRNA lnc-TRTMFS in BMECs resulted in a significant upregulation of miR-132 x,while transfection with OP-lnc-TRTMFS resulted in a significant downregulation of miR-132 x.Through dual fluorescence reporting experiments,it was found that the lnc-TRTMFS-WT-miR-132 x group showed a significant downregulation of fluorescence compared to the lnc-TRTMFS-MT-miR-132 x group,lnc-TRTMFSWT-NC group,and lnc-TRTMFS-MT-NC group,indicating a targeted binding relationship between lnc-TRTMFS and miR-132 x.3.Functional verification of miR-132 x and screening of target genes: transfection of miR-132x-mimics and miR-132x-inhibitor in BMECs showed that miR-132 xmimics significantly inhibited triglyceride accumulation,lipid droplet formation and lipid related gene expression in BMECs,while the result of transfection of miR-132 xinhibitor was the opposite.Through the prediction of Bibiserv2 and miRBase software and double fluorescein report experiment,it was found that RAI14 was the target gene of miR-132x;Meanwhile,miR-132 x inhibitor treatment promoted the expression of mTOR,4EBP1,P70S6 K m RNA,protein,and phosphorylated protein,while miR-132 x mimics showed the opposite results.It is suggested that Lnc-TRTMF and miR-132 x may play a role in milk fat synthesis through RAI14 regulating mTOR signal pathway.4.The role of the lnc-TRTMF-miR-132 x RAI14 relationship in the regulation of milk fat synthesis: through cotransfection experiments,we found that OP-lnc-TRTMFS can alleviate the inhibitory effects of miR-132 x mimics on triglyceride accumulation,lipid droplet formation,and RAI14 expression,while miR-132 x inhibitor can salvage the inhibitory effects of siRNA-lnc-TRTMFS on triglyceride accumulation,lipid droplet formation,and RAI14 expression.Indicating that the lnc-TRTMF miR-132 x RAI14 relationship plays an important regulatory role in milk fat synthesis.To sum up,lnc-TRTMFS regulates the expression of RAI14 through competitive binding to miR-132 x,and then regulates milk fat synthesis through mTOR pathway,which lays a theoretical foundation for the analysis of the molecular regulation mechanism of milk fat synthesis in breast epithelial cells.