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| Effects of RNAi Expression of TIP47 on Lipid Droplet Formation and the Expression of Related Genes in Goat (Capra hircus) Mammary Epithelial Cells |
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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.
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Received: 09 August 2016
Published: 13 January 2017
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Corresponding Authors:
Jun LUO
E-mail: luojun@nwsuaf.edu.cn
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