| Liver X receptorα(LXRα) gene is a member of the nuclear receptor supper family, which can be highly expressed in macrophages, adipocyte, skeletal muscle cell and intestine cell which have an active effect on lipid metabolism. LXRαgene is a tissue-specific expression gene and has a key regulation role in cholesterol homeostasis, lipogenesis, inflammation and glucose homeostatsis. In this study, we cloned the cDNA sequence of LXRαgene from the mammary gland of Xinong Saanen dairy goat and constructed the overexpression of LXRαgene and its shRNA adenovirus. Then we treated the goat mammary gland epithelial cell with its shRNA adenovirus, LXRαagonist TO901317 and its over- expression adenovirus and we detected the mRNA expression level by RT-qPCR of six different genes, including LXRα, Fatty acid synthase (FASN), Heart-Fatty acid binding proteins (H-FABP), Acety1-CoA carboxy1ase (ACACA),Sterol regulatory element binding protein-1 (SREBP-1), Peroxisome proliferator activated receptorγ(PPARγ), which provided an experimental basis for the study of LXRαgene on fatty acid metabolism. The main results were as follows:(1) Through the RT-PCR & RACE technique, we cloned LXRαgene cDNA sequence in Xinong Saanen dairy goat mammary gland and got the GenBank number (GU332719). The whole cDNA sequence of LXRαgene was 1654bp, including 1344bp CDS, 150bp 5′untranslated region (5′UTR) and 160bp 3′UTR. The homology of goat LXRαgene CDS region with Bos taurus, Sus scrofa, Mus musculus, Rattus norvegicus, Homo sapiens were 98%, 93%, 87%, 87%, 90% respectively; The homology of 5′UTR and 3′UTR with Bos taurus, Sus scrofa, Mus musculus, Homo sapiens were 94%, 89%, 84%, 80% and 98%, 87%, 81%, 84% respectively.(2) We detected that the most effective concentration for TO901317 treated mammary epithelial cell is 10-6mol/L. RT-qPCR results showed that the expression of FASN, ACACA, SREBP-1 gene was up-regulated, H-FABP & PPARγgenes were down-regulated after 10-6mol/L TO901317 treated mammary epithelial cell.(3) By cotransfecting HEK 293 Cell, we obtained two shRNA sequences (pENTR/ CMV-GFP/U6- Lsh-490 & pENTR/CMV-GFP/ U6- Lsh-923) which could cause obvious interference effect. Then, we generated the two recombinant adenovirus vectors by LR recombination and the adenovirus were successfully packed in HEK 293 Cell. The titers of the two adenoviruses were both 5×108 PFU/mL. After that, we detected the MOI (Multiplicity Of Infection) of these two adenoviruses, the results showed that the MOI of pENTR/ CMV-GFP/ U6-Lsh-490 & pENTR/CMV-GFP/ U6- Lsh-923 were 30 and 75 in goat mammary epithelial cell respectively. The RNA interference effect of pENTR/ CMV-GFP/U6- Lsh-490 adenovirus was up to 70% in mammary epithelial cell. After pENTR/ CMV- GFP/U6- Lsh-490 adenovirus infected into mammary epithelial cell, FASN & ACACA genes were down-regulated, SREBP-1 & PPARγgenes were up-regulated.(4) We constructed the shuttle vector of LXRαgene. After homologous recombination of this vector, we transfected the vecotor into HEK 293 cell. Then we generated the overexpression recombinant adenovirus of LXRαgene (Ad-LXRα) and its titer was 5×107 PFU/mL. The MOI of Ad-LXRαwas 250 in mammary epithelial cell. At last, we infected the Ad-LXRαvector into mammary epithelial cell, the RT-qPCR showed that the mRNA expression of LXRα, FASN, ACACA, SREBP-1 genes were up-regulated. H-FABP & PPARγgenes were down-regulated.In this study, we cloned the cDNA sequence of LXRαgene and constructed the RNA interference adenovirus targeted to LXRαgene & LXRαoverexpression adenovirus vectors. The expression of FASN & ACACA genes were down-regulated, SREBP-1 & PPARγgenes were up-regulated after RNA interference experiment. Meanwhile, the expression of FASN, ACACA and SREBP-1 genes were up-regulated, PPARγgene was down-regulated after overexpression of LXRαgene. In a word, the results provided an experiment basis for the further function study of LXRαgene. |
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