Overexpression And RNA Interference Of TRIB3 Gene On Lipid Metabolism In Dairy Goat Mammary Epithelial Cells

Tribbles pseudokinase 3(TRIB3)is a class of serine/threonine kinase-like proteins with conserved domains of serine/threonine protein kinases.However,it lacks the key amino acids and ATP binding regions for enzyme-triggering reactions,therefore,TRIB3 has no catalytic activity and can be used as regulatory proteins involved in cell proliferation,differentiation,apoptosis,glycolipid metabolism and tumorigenesis.TRIB3 not only regulates lipid metabolism through key genes affecting fat synthesis metabolism,but also can be induced by external stimuli to affect cell lipid metabolism.The effect of TRIB3 on lipid metabolism in goat mammary epithelial cells(GMECs)was investigated by constructing interfering RNA and overexpression vector.The changes of lipid expression and intracellular lipid content were detected by q RT-PCR and Western Blot,and the mechanism of TRIB3 gene affecting lipid metabolism in GMECs was studied.The main findings of this study are as follows.1.TRIB3 gene cloning,sequence analysis and tissue differential expression.The specific primers were designed by Primer Premier 5.0 according to the TRIB3 gene sequence(XM＿018057667.1)of Capra hircus in the Gen Bank.The results showed that the complete CDS region of TRIB3 gene was 1,074 bp,encoding 357 amino acids;The molecular weight of the TRIB3 protein is 39.6 ku,isoelectric point is 7.8,and subcellular localization shows that TRIB3 is mainly presented in the nucleus.That sequence homologies of Xinong Saanen dairy goat with sheep(Ovis aries),cattle(Bos taurus)and mouse(Mus musculus)are 99%,96% and 71%,respectively,and sheep are the most closest species with dairy goat;the TRIB3 gene had the highest expressed in breast tissue and the lowest in kidney tissue.2.Effects of overexpression TRIB3 on lipid metabolism in GMECs.We successfully constructed the pc DNA3.1(+)vector containing TRIB3 gene,which was transformed into GMECs.The m RNA expression of TRIB3 gene increased by 40-fold,which significantly changed the m RNA expression level of lipid metabolism related genes.The result showed that the m RNA expression of Glycerol-3-phosphate acyltransferase 1(GPAM),Diacylglycerolacyl transferase 1(DGAT1),Perilipin 1(PLIN1),Peroxisome proliferator-activated receptor(PPARg)was significantly down-regulated(P<0.05),overexpression TRIB3 significantly decreased the total triglyceride content(TAG)and intracellular lipid droplet accumulation(P<0.05).Besides,for the intracellular fatty acid C16:0 was significantly down-regulated while the C18:0 was significantly up-regulated(P<0.05).Moreover,p-Akt/PPARg protein levels were significantly downregulated and PPARg promoter activity significantly downregulated(P<0.05).The results suggest that overexpression TRIB3 gene inhibited lipid metabolism in GMECs through Akt signaling pathway.3.Effects of interference TRIB3 on lipid metabolism in GMECs.The si RNA of TRIB3 gene were designed based on CDS sequence of TRIB3 gene and transferred into GMECs,the results showed that si RNA-588 leaded to the significantly decreased m RNA expression of TRIB3 gene by about 60% and the m RNA expression of Stearoyl-Co A desaturase1(SCD1),Lipoprotein lipase(LPL),DGAT1,PLIN1,Liver X receptors alpha(LXRα),PPARg,Sterol regulatory element binding protein 1(SREBP1)were significantly up-regulated compared with control group(P<0.05).The TAG and intracellular lipid droplet accumulation were increased.The cellular fatty acid composition C16:0 was significantly up-regulated and C18:0 significantly down-regulated(P<0.05).The expression of p-Akt,p-m TOR,PPARg protein levels were significantly upregulated through interference of TRIB3,which indicates that TRIB3 is involved in the Akt signaling pathway and affects the lipid metabolism process by inhibiting the phosphorylation of Akt.Through interference and overexpression to explore the function of TRIB3 gene,the lipid metabolism in GMECs can be made further studys,and the mechanism of milk lipid metabolism network could be improved,consequently,we can lay a theoretical basis for improving the quality of goat milk.