Abstract:Abstract Tail-interacting protein 47 gene (TIP47), known as mannose 6-phosphate receptors binding protein 1 (M6PRBP1), is a member of the (perilipin/ADRP/TIP47, PAT) family, which plays a crucial regulatory role during lipid droplets (LD) biogenesis and secretion process. Milk fat, one of the important nutrients in goat (Capra hircus) milk, is especially rich in short and medium chain fatty acids, which can be absorbed by human (Homo sapiens) body easily. At the same time, the polyunsaturated fatty acids in goat milk have a good effect on anti-oxidation. However, the role of TIP47 on fatty acid metabolism in goat mammary gland is poorly understood. Thus, studying the function of the TIP47 on formation of the milk LD has a great significance to regulate the nutritional value of goat milk. RT- PCR was used to clone TIP47 CDS. The Block-iT short hairpin RNA (shRNA) interference system was used to design specific shRNAs according to the CDS of TIP47 mRNA and 293A cell line was used to package and amplify the adenovirus in this experiment. Then using qRT-PCR to detect the relative expression level of genes and oil red O was used to identify the changes of lipid droplets in intracellular. The full-length 1 314 bp CDS of TIP47 (GenBank No. HQ846827) was cloned onto pDsRed1-C1vector which could express the red fluoresence protein. Three pairs of pENTR/CMV-GFP/U6-494/828/888 and pDsRed1-C1-TIP47 were co-transfected into 293A cells to filter out the most interference efficient shRNA. The results showed that shRNA-888 could markedly reduce the red fluorescence, so shRNA-888 was considered to inhibit the expression of TIP47 significantly. Then shRNA-888 was packaged and amplified high-titer virus Ad-shRNA-888 into 293A cells. The titer of the Ad-shRNA-888 was 108 U/mL and the optimal multiplicity of infection (MOI) was 200 in goat mammary epithelial cells. Compared with the control, the expression of TIP47 and adipose triglyceride lipase (ATGL) mRNA were downregulated by 61% (P<0.01) and 15% respectively, and expression of fatty acid synthase (FASN) and hormone-sensitive lipase (HSL) mRNA were upregulated to 1.77 (P<0.05) and 2.8 (P<0.01) folds but no noticeable changes for the mRNA expression of stearoyl-CoA desaturase (SCD) when treated mammary epithelial cells with the Ad-shRNA-888 after 72 h. And the result of oil red O staining showed that 100 μmol/L oleic acid could significantly increase the accumulation of lipid droplets in mammary epithelial cells, however the intracellular accumulation of lipid droplets had a downward trend after treating with the Ad-shRNA-888 but did not fully suppress the formation of intracellular lipid droplets. Taken together, the qRT-PCR results indicated that Ad-shRNA-888 could effectively downregulate the expression of TIP47, which contributed to changing fatty acid metabolism related genes significantly in goat mammary gland epithelial cells, including FASN and HSL. Moreover, the result of oil red O staining suggested that there might be other genes involved in the process of the formation of lipid droplets in a way of coordinated control. Here, our study demonstrated that TIP47 played a vital role in fatty acid metabolism during the dairy goat lactation process, and provided the basic data for further research in regulating fatty acid metabolism.
[1]郝娟, 罗军, 王紫骞等.西农萨能羊 尾部相互作用蛋白基因的 克隆、序列结构分析与组织表达[J].农业生物技术学报, 2011, 19(5):864-872[2]刘梅芳, 徐国恒.家族蛋白在细胞内脂滴代谢过程中的作用[J].科学进展, 2006, 37(2):103-107[3]Barbero P, Buell E, Zulley S, Pfeffer S R.TIP47 is not a component of lipid droplets[J].Biological Chemistry, 2001, 276(26):24348-24351[4]Bickel P E, Tansey J T, Welte M A.PAT proteins,an ancient family of lipid droplet proteins that regulate cellular lipid stores[J].Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2009, 1791(6):419-440[5]Bulankina AV, Deggerich A, Wenzel D, et al.TIP47 functions in the biogenesis of lipid droplets,[J].Journal of Cell Biology, 2009, 185(4):641-655[6]Cebo, C, H.Caillat,FBouvier and PMartin.. Major proteins of the goat milk fat globule membrane.[J].Journal?of Dairy?Science, 2010, 93(3):868-876[7]Carr RM, Patel RT, Rao V, et al.Reduction of TIP47 improves hepatic steatosis and glucose homeostasis in mice[J].Am J Physiol Regul Integr Comp Physiol, 2012, 302(8):996-1003[8]Hengbo Shi, Jiangjiang Zhu, Jun Luo, et al.Genes regulating lipid and protein metabolism are highly expressed in mammary gland of lactating dairy goats[J].Functional & Integrative Genomics, 2015, 15(3):309-321[9]Kang Y, Hengbo S, Jun L, et al.PPARG modulated lipid accumulation in dairy GMEC via regulation of ADRP gene[J].J Cell Biochem, 2015, 116(1):192-201[10]Londos C, Brasaemle D L, Gruia-Gray J, et al.Perilipin: unique proteins associated with intracellular neutral lipiddroplets in adipocytes and steroidogenic cells[J].Biochem SocTrans, 1995, 23(3):611-615[11]Li J, Luo J, Xu H, et al.Fatty acid synthase promoter: characterization,and transcriptional regulation by sterol regulatory element binding protein-1 in goat mammary epithelial cells[J].Gene, 2015, 561(1):157-164[12]Lampidonis A D, Argyrokastritis A, Stravopodis D J, et al.Cloning and functional characterization of the ovine Hormone Sensitive Lipase (HSL) full-length cDNAs: an integrated approach[J].Gene, 2008, 416(1-2):30-43[13]McManaman JL, Russell TD, Schaack J, et al.Molecular determinants of milk lipid secretion[J].Journal of mammary gland biology and neoplasia, 2007, 12(4):259-268[14]Pawella LM, Hashani M, Eiteneuer E, et al.Perilipin discerns chronic from acute hepatocellular steatosis[J].J Hepatol, 2014, 60(3):33-42[15]Shi H, Yu K, et al.Adipocyte differentiation-related protein promotes lipid accumulation in goat mammary epithelial cells[J].J Dairy Sci, 2015, 98(10):6954-64[16]Sztalryd C, XuG, Dorward H, et al.Perilipin A is essential for the translocation of hormone-sensitive lipase during lipolytic activation[J].J Cell Biol, 2003, 161(6):1093-1103[17]Than N G,Sumegi B,Than G N.Cloning and sequence analysis of cDNAs encoding human placental tissue Protein 17 (PPl7) variants[J].Eur J Biochem, 1998, 258(2):752-757[18]Tansey JT, Sztalryd C, G ruia-Gray J, et al.Perilipin ablation results in a leanmouse with aberrant adipocyte lipolysis,enhanced leptin production,and resistance to diet-induced obesity[J].Proc Natl Acad Sci USA, 2001, 98(11):6494-6499[19]Vollmert C, Schoenborn V, Heid I M, et al.The ATGL gene is associated with free fatty acids,triglycerides,and type 2 diabetes[J].Diabetes, 2006, 55(5):1270-1275[20]Wolins NE, Quaynor BK, Skinner JR, et a.S3-12, Adipophilin, and TIP47 package lipid in adipocytes,[J].journal of biological chemistry, 2005, 280(19):19146-19155