Effects Of Overexpression And RNAi Of LXR? Gene On Lipid Metabolism In Dairy Goat Mammary Epithelial Cells

Liver X receptor ?,one of the superfamily of nuclear receptors,is involved in the regulation of the body's metabolism of fatty acids and cholesterol.LXR? forms heterodimer with the retinoid X receptor?RXR?binding to the promoters of target genes that paly important roles in the process of fatty acid and cholesterol metabolism to regulate their activity and expression,such as sterol regulatory element-binding protein-1c?SREBP-1c?,and fatty acid synthase?FASN?.Further affecting the the metabolism of fatty acids and cholesterol in the body.In this experiment,we extracted total RNA from Xinong saanen dairy goat mammary gland,and then designed primers for cloning the sequence of LXR? using RT-PCR.A recombinant adenovirus vector was constructed into 293 A cells after cloning the right sequence for infecting goat mammary epithelial cells hoping to overexpression the gene,and we designed siRNA according to the sequence of LXR? in order to interfere the gene expression.To further clarify the function of this gene in the regulation of lipid metabolism in mammary gland.Following are the main results obtained in this study:?1?The total RNA was extracted from Xinong saanen dairy goat mammary gland,and then designed primers based on the Capra hircus' s LXR? genesequence in GenBank?XM₀18062857?for cloning the sequence of LXR? using RT-PCR.The whole CDS of LXR? is 1368 bp in length,encoding 455 amino acids.The similarity of CDS region of goat LXR? gene with Bos taurus,Ovis aries,Homo sapiens and Mus musculus were 98%,98%,87% and 85%,amino acid sequence were 99%,99%,90% and 91%.?2?We successfully constructed the adenovirus shuttle vector pAdTrack-CMV-LXR? containing LXR? gene,which was transformed into BJ5183 competent cell of Escherichia coli to obtain the recombinant adenovirus vector pAdeasy-LXR? by using homologous recombination after digesting by restriction endonuclease Pme ?.And then pAdeasy-LXR? was transfected into 293 A cells to package and amplify the recombinant adenovirus.The titer of virus was 109 U/mL.We determined its best multiplicity of infection?MOI?in goat mammary epithelial cells was 200.Then we infected goat mammary epithelial cells with virus and T0901317 to detect the expression of LXR? and some lipid metabolism related genes using RT-qPCR after 48 h,the result showed that: the mRNA level of LXR? increased by about 100 times;SREBP-1,FASN,stearoyl-CoA desaturase?SCD-1?,acyl-CoA synthetases?ACSL1?,ACSL3,acetyl-coenzyme A-carboxylase?ACC??and elongase of verylong chain fatty acids?ELVOL6?genes were all significantly increased,the expression of FADS1 gene was no obviously change;compared with the control group,the virus and T0901317 could increase the number of intracellular lipid droplets,the intracellular triglyceride content and monounsaturated fatty acids C18:1 content?P<0.05?.?3?Based on the CDS sequence of LXR? gene,we designed and synthesis the siRNA to transfect goat mammary epithelial cells for 48 h.Then using RT-qPCR to detect the effects of RNA interference.The result showed that the mRNA expression of LXR? gene was decreased by about 88%,and level of diacylgycerol acyltransferase 2?DGAT2?was reduced 30%,while SREBP1 was increased 50%.After treating cells siRNA and T0901317,the mRNA level of SREBP1 and FSAN genes were decreased by about 15%and 35% respectively;the expression of ASCL1 was significantly increased about 35%;the expression of fatty acid desaturase 1?FADS1?,ELOVL6,SCD and liver x receptor ??LXR??genes had no significant change;compared with the control group,the siRNA could decrease the intracellular triglyceride content.All above,we successfully cloned the whole CDS of LXR ? gene and obtained the high titer recombinant adenovirus.Overexpression of LXR? gene in Xinong Saanen goat mammary epithelial cell could increase the mRNA levels ofgenes related to milk fat and increase the number of intracellular lipid droplets.And interference of the gene had definite inhibitory effect on the expression of genes related to milk fat and intracellular triglyceridesynthesis.These results provide basis for further clarify the function of this gene in the regulation of lipid metabolism in mammary gland.