Mechanism Of LncRNA MPFAST Regulating Proliferation And Fatty Acid Synthesis Of Bovine Mammary Epithelial Cells

Mammary gland epithelial cells are the functional unit of mammary gland,which can synthesize milk fat,protein and carbohydrate.The number and lactation capacity of mammary gland epithelial cells are the key factors of lactation.lnc RNAs and miRNAs participate in mammary gland development and lactation by regulating gene expression.According to the RNA-Seq results of mammary gland tissues during the lactation and dry period of dairy cows,lnc RNA TCONS-00067521(named lnc RNA MPFAST,mammary proliferation and fatty acids synthesis-associated transcript)expressed significantly higher during lactation period than that during dry period,suggesting that MPFAST may be involved in lactation regulation of bovine,and milk fat is the main energy component of milk.Therefore,this study first investigated the effects of MPFAST on the proliferation and fatty acid synthesis of bovine mammary epithelial cell lines(BMECs)by CCK8,Ed U and oil red O staining tests.Further RT-q PCR,Western blot and other methods were performed to explore the regulation molecular mechanism of BMECs proliferation and fatty acid synthesis,and the obtained results were as follows:1.MPFAST overexpression vector and interference vector were transfected into BMECs,respectively,CCK8 result showed that compared with the control group,the overexpression of MPFAST significantly increased the cell viability of BMECs(P<0.01),while interference with MPFAST significantly decreased the cell viability of BMECs(P<0.05);Ed U result showed that compared with the control group,the overexpression of MPFAST promoted the proliferation of BMECs,while the interference of MPFAST inhibited the proliferation of BMECs.In addition,the overexpression of MPFAST significantly promoted the expression of proliferation-related gene CCND1 at m RNA and protein levels(P<0.01),while the interference of MPFAST opposite compared with the control group(P<0.01).2.The effects of MPFAST on fatty acid synthesis of BMECs were detected by oil red O staining and RT-q PCR.Oil red O staining showed that compared with the control group,the overexpression of MPFAST promoted the formation of lipid droplets in BMECs,while the interference with MPFAST inhibited the formation of lipid droplets in BMECs.RT-q PCR results showed that compared with the control group,the overexpression of MPFAST significantly promoted the expression of fatty acid synthesis genes FASN and ACACA in BMECs(P<0.01,P<0.05),the results of interfering with MPFAST were opposite,indicating that MPFAST promoted the synthesis of fatty acids in BMECs.3.Subcellular localization studies found that MPFAST was expressed in both the nucleus and cytoplasm.Dual luciferase assay showed that MPFAST could bind to miR-103.RT-q PCR result further showed that the overexpression of MPFAST significantly inhibited the expression of miR-103(P<0.01),and the result was opposite after MPFAST inhibition.CCK8 and Ed U assays showed that the inhibition of miR-103 promoted the proliferation of BMECs compared with the control group.Dual luciferase assay verified that miR-103 could bind to PIK3R1,RT-q PCR as well as Western blot results further indicated that PIK3R1 was the target gene of miR-103.In addition,it was found that compared with the control group,the overexpression of MPFAST significantly increased the expression of target gene PIK3R1 at m RNA and protein levels(P<0.01),while interference with MPFAST expression resulted in the opposite.4.In order to explore the effect of MPFAST on PI3K/AKT signaling pathway,RT-q PCR and Western blot results showed that compared with the control group,the overexpression of MPFAST significantly increased the relative expression levels of AKT m RNA,AKT protein and p-AKT protein in BMECs(P<0.01,P<0.05),and the overexpression of MPFAST significantly increased the relative expression levels of mTOR and SREBP1 mRNA in BMECs(P<0.001,P<0.05),and the result of interfering MPFAST expression was opposite.In conclusion,MPFAST in BMECs regulated the expression of downstream target gene PIK3R1 by inhibiting miR-103 and affected the expression of key genes of PI3K/AKT signaling pathway and the phosphorylation of AKT protein,ultimately promoting the proliferation and the synthesis of fatty acids of BMECs,which would provide a theoretical basis for revealing the regulatory mechanism of lncRNA in lactation.