沉默SCAP基因对奶牛乳腺上皮细胞脂滴的影响
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摘要
旨在探究沉默固醇调节元件结合蛋白裂解激活蛋白(SREBP cleavage-activating protein,SCAP)对奶牛乳腺上皮细胞脂滴的影响,本研究构建了SCAP短发夹RNA慢病毒载体,包装感染奶牛乳腺上皮细胞,通过在培养基中添加嘌呤霉素筛选获得SCAP基因沉默稳转细胞株,采用Real-time PCR和Western blot观察基因沉默细胞株中SCAP基因和蛋白的表达,尼罗河红和油红O染色脂滴检测SCAP基因沉默及SCAP质粒瞬时转染对细胞脂滴形成的影响。结果表明,本研究成功构建了奶牛SCAP基因沉默慢病毒重组载体,感染细胞后经过5 mg·L~(-1)嘌呤霉素筛选得到SCAP基因沉默稳转细胞株。基因检测发现,各个沉默细胞株的SCAP基因表达显著降低(P<0.05或P<0.01)。与对照组相比,RNAi-SCAP3细胞中的SCAP蛋白表达降低为对照组的0.49倍(P<0.05),脂代谢基因SCD和FAS表达也显著下降(P<0.05)。脂滴染色发现,RNAi-SCAP3沉默细胞株的脂滴含量显著低于对照组细胞(P<0.01),脂滴直径≤2.0μm的小脂滴个数增多,而直径≥2.5μm的大脂滴个数减少;将SCAP真核表达载体瞬时转染RNAi-SCAP3细胞株后发现,SCAP基因的过表达显著减少了细胞中小脂滴比例而增加了大脂滴比例(P<0.05)。本研究成功构建了SCAP基因沉默稳转细胞株,发现SCAP的表达影响了细胞脂滴直径的大小。
The aim of this study was to investigate the effect of silencing SCAP gene on formation of lipid droplet in dairy mammary epithelial cells. SCAP short hairpin RNA lentivirus vector was constructed, the bovine mammary epithelial cells were packed and infected. SCAP gene silencing cell lines were selected by applying puromycin in culture medium. Effects of SCAP gene silencing on the expression of SCAP gene and protein were detected by Real-time PCR and Western blot, moreover, the effects of SCAP gene silencing and transient transfection of SCAP plasmid on lipid droplet formation were detected using Nile red and Oil red O staining. The results showed that lentivirus recombinant vector with SCAP gene silencing was constructed successfully. SCAP gene silencing stable cell line was obtained via screening of 5 mg·L~(-1) puromycin. The expression of SCAP gene in all silent cell lines were significantly reduced(P<0.05 or P<0.01). Compared with control group, the expression of SCAP protein in RNAi-SCAP3 cell lines were decreased by 0.49 fold(P<0.05), the expression of SCD and FAS genes were significantly decreased(P<0.05) in RNAi-SCAP3 cells. Lipid droplet content in RNAi-SCAP3 cell lines was significantly lower than that in control cells(P<0.01). The number of small lipid droplets with size≤ 2.0 μm were increased, while the number of large lipid droplets with size≥2.5 μm were decreased. After transient transfection of SCAP eukaryotic expression vector into RNAi-SCAP3 gene silencing cell lines, overexpression of SCAP significantly decreased the percentage of small lipid droplets and increased the percentage of large lipid droplets(P<0.05). In this study, SCAP gene silencing cell lines were successfully constructed, and the size of lipid droplets were affected by the expression of SCAP.
The aim of this study was to investigate the effect of silencing SCAP gene on formation of lipid droplet in dairy mammary epithelial cells. SCAP short hairpin RNA lentivirus vector was constructed, the bovine mammary epithelial cells were packed and infected. SCAP gene silencing cell lines were selected by applying puromycin in culture medium. Effects of SCAP gene silencing on the expression of SCAP gene and protein were detected by Real-time PCR and Western blot, moreover, the effects of SCAP gene silencing and transient transfection of SCAP plasmid on lipid droplet formation were detected using Nile red and Oil red O staining. The results showed that lentivirus recombinant vector with SCAP gene silencing was constructed successfully. SCAP gene silencing stable cell line was obtained via screening of 5 mg·L~(-1) puromycin. The expression of SCAP gene in all silent cell lines were significantly reduced(P<0.05 or P<0.01). Compared with control group, the expression of SCAP protein in RNAi-SCAP3 cell lines were decreased by 0.49 fold(P<0.05), the expression of SCD and FAS genes were significantly decreased(P<0.05) in RNAi-SCAP3 cells. Lipid droplet content in RNAi-SCAP3 cell lines was significantly lower than that in control cells(P<0.01). The number of small lipid droplets with size≤ 2.0 μm were increased, while the number of large lipid droplets with size≥2.5 μm were decreased. After transient transfection of SCAP eukaryotic expression vector into RNAi-SCAP3 gene silencing cell lines, overexpression of SCAP significantly decreased the percentage of small lipid droplets and increased the percentage of large lipid droplets(P<0.05). In this study, SCAP gene silencing cell lines were successfully constructed, and the size of lipid droplets were affected by the expression of SCAP.
引文
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[2] AVRAMIS C A,WANG H,MCBRIDE B W,et al.Physical and processing properties of milk,butter,and cheddar cheese from cows fed supplemental fish meal[J].J Dairy Sci,2003,86(8):2568-2576.
[3] LOPEZ C,BRIARD-BION V,MENARD O,et al.Phospholipid,sphingolipid,and fatty acid compositions of the milk fat globule membrane are modified by diet[J].J Agric Food Chem,2008,56(13):5226-5236.
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[5] CHONG B M,REIGAN P,MAYLE-COMBS K D,et al. Determinants of adipophilin function in milk lipid formation and secretion[J]. Trends Endocrinol Metab, 2011,22(6):211-217.
[6] 王苗,罗军,许会芬,等.山羊INSIG1基因超表达对乳腺上皮细胞中脂质合成的影响[J].畜牧兽医学报,2016,47(9):1806-1816.WANG M,LUO J,XU H F,et al.Effect of INSIG1 overexpression on the lipid synthesis in goat mammary gland epithelial cells[J].Acta Veterinaria et Zootechnica Sinica,2016,47(9):1806-1816.(in Chinese)
[7] BROWN M S,GOLDSTEIN J L.A proteolytic pathway that controls the cholesterol content of membranes,cells,and blood[J].Proc Natl Acad Sci U S A,1999,96(20):11041-11048.
[8] 韩立强,孙宇,付彤,等.奶牛乳腺上皮细胞中SCAP和SREBP1蛋白调控SCD基因表达的作用研究[J].畜牧兽医学报,2018,49(3):652-658.HAN L Q,SUN Y,FU T,et al.Effect of SCAP and SREBP1 proteins on regulation of SCD gene expression in dairy mammary epithelial cells[J].Acta Veterinaria et Zootechnica Sinica,2018,49(3):652-658.(in Chinese)
[9] CHENG X,LI J Y,GUO D L.SCAP/SREBPs are central players in lipid metabolism and novel metabolic targets in cancer therapy[J].Curr Top Med Chem,2018,18(6):484-493.
[10] JENSEN K K,TADIN-STRAPPS M,WANG S P,et al.Dose-dependent effects of siRNA-mediated inhibition of SCAP on PCSK9,LDLR,and plasma lipids in mouse and rhesus monkey[J].J Lipid Res,2016,57(12):2150-2162.
[11] MOON Y A.The SCAP/SREBP pathway:a mediator of hepatic steatosis[J].Endocrinol Metab,2017,32(1):6-10.
[12] XU H F,LUO J,MA G Z,et al.Acyl-CoA synthetase short-chain family member 2 (ACSS2) is regulated by SREBP-1 and plays a role in fatty acid synthesis in caprine mammary epithelial cells[J].J Cell Physiol,2018,233(2):1005-1016.
[13] XU H F,LUO J,TIAN H B,et al.Rapid communication:lipid metabolic gene expression and triacylglycerol accumulation in goat mammary epithelial cells are decreased by inhibition of SREBP-1[J].J Anim Sci,2018,96(6):2399-2407.
[14] BICHI E,FRUTOS P,TORAL P G,et al.Dietary marine algae and its influence on tissue gene network expression during milk fat depression in dairy ewes[J].Anim Feed Sci Technol,2013,186(1-2):36-44.
[15] BIONAZ M,LOOR J J.Gene networks driving bovine milk fat synthesis during the lactation cycle[J].BMC Genomics, 2008,9(1):366.
[16] 唐彬秩,屈艺,赵凤艳,等.以慢病毒为载体的整合素β8 RNAi在新生鼠脑内干扰效率的研究[J].四川大学学报:医学版,2018,49(3):325-330.TANG B Z,QU Y,ZHAO F Y,et al.Investigation of lentiviral vectors based integrin β8 RNAi system in neonatal rats’ brain[J].Journal of Sichuan University:Medical Science Edition,2018,49(3):325-330.(in Chinese)
[17] 孙丽,宗秋芳,吴森,等.猪FUT2基因沉默对其所在通路基因表达及小肠上皮细胞E.coli F18黏附能力的影响[J].畜牧兽医学报,2017,48(11):2034-2045.SUN L,ZONG Q F,WU S,et al.The effects of FUT2 gene silencing on the expression of genes involved in glycosphingolipid biosynthesis-globo series pathway and the adhesion of E.coli F18 to small intestinal epithelial cells[J].Acta Veterinaria et Zootechnica Sinica, 2017,48(11):2034-2045.(in Chinese)
[18] 曾阳阳,朱弘炎,田玉民,等.猪skMLCK基因RNAi慢病毒载体的构建及干扰效率测定[J].畜牧与兽医,2016,48(2):70-73.ZENG Y Y,ZHU H Y,TIAN Y M,et al.Construction of RNAi lentiviral vector targeting porcine skMLCK gene and determination of interference efficiency[J].Animal Husbandry and Veterinary Medicine,2016,48(2):70-73.(in Chinese)
[19] 蓝林祥,杜艳涛,赵威,等.Myosin Va RNAi慢病毒载体的构建及对肺癌PG细胞运动和迁移能力的影响[J].解剖学报,2009,40(6):891-896.LAN L X,DU Y T,ZHAO W,et al.Construction of a lentivirus vector for RNA interference of myosin Va and its effect on motility and migration of P G cells[J].Acta Anatomica Sinica,2009,40(6):891-896.(in Chinese)
[20] COUVREUR S,HURTAUD C,MARNET P G,et al.Composition of milk fat from cows selected for milk fat globule size and offered either fresh pasture or a corn silage-based diet[J].J Dairy Sci,2007,90(1):392-403.
[21] MESILATI-STAHY R,MIDA K,ARGOV-ARGAMAN N. Size-dependent lipid content of bovine milk fat globule and membrane phospholipids[J]. J Agric Food Chem,2011, 59(13): 7427-7435.
[22] WALTHER T C,FARESE R V.Lipid droplets and cellular lipid metabolism[J].Annu Rev Biochem,2012,81(1):687-714.
[23] JEONG J,LISINSKI I,KADEGOWDA A K G,et al.A test of current models for the mechanism of milk-lipid droplet secretion[J]. Traffic,2013,14(9):974-986.
[24] RAMBOLD A S,COHEN S,LIPPINCOTT-SCHWARTZ J.Fatty acid trafficking in starved cells:regulation by lipid droplet lipolysis,autophagy,and mitochondrial fusion dynamics[J].Dev Cell,2015,32(6):678-692.
[25] BICKEL P E,TANSEY J T,WELTE M A.PAT proteins,an ancient family of lipid droplet proteins that regulate cellular lipid stores[J].Biochim Biophys Acta, 2009,1791(6):419-440.
[26] COHEN B C,SHAMAY A,ARGOV-ARGAMAN N.Regulation of lipid droplet size in mammary epithelial cells by remodeling of membrane lipid composition-a potential mechanism[J].PLoS One,2015,10(3):e0121645.
[27] COHEN B C,RAZ C,SHAMAY A,et al.Lipid droplet fusion in mammary epithelial cells is regulated by phosphatidylethanolamine metabolism[J].J Mammary Gland Biol Neoplasia,2017,22(4):235-249.
[28] ZHANG R,MA H M,GAO Y,et al.Th-POK regulates mammary gland lactation through mTOR-SREBP pathway[J].PLoS Genet,2018,14(2):e1007211.
[29] GONG J Y,SUN Z Q,WU L Z,et al.Fsp27 promotes lipid droplet growth by lipid exchange and transfer at lipid droplet contact sites[J].J Cell Biol,2011,195(6):953-963.
[30] 张菡,朱河水,樊晓娜,等.奶牛SREBP1蛋白对FASN基因启动子的转录调控分析[J].农业生物技术学报,2018,26(4):652-659.ZHANG H,ZHU H S,FAN X N,et al.Transcriptional regulation analysis of FASN gene promoter by SREBP1 protein in bovine (Bos taurus)[J].Journal of Agricultural Biotechnology,2018,26(4):652-659.(in Chinese)
[31] RAWSON R B,DEBOSE-BOYD R,GOLDSTEIN J L,et al.Failure to cleave sterol regulatory element-binding proteins (SREBPs) causes cholesterol auxotrophy in Chinese hamster ovary cells with genetic absence of SREBP cleavage-activating protein[J].J Biol Chem,1999,274(40):28549-28556.
[2] AVRAMIS C A,WANG H,MCBRIDE B W,et al.Physical and processing properties of milk,butter,and cheddar cheese from cows fed supplemental fish meal[J].J Dairy Sci,2003,86(8):2568-2576.
[3] LOPEZ C,BRIARD-BION V,MENARD O,et al.Phospholipid,sphingolipid,and fatty acid compositions of the milk fat globule membrane are modified by diet[J].J Agric Food Chem,2008,56(13):5226-5236.
[4] MCMANAMAN J L,REYLAND M E,THROWER E C.Secretion and fluid transport mechanisms in the mammary gland:comparisons with the exocrine pancreas and the salivary gland[J].J Mammary Gland Biol Neoplasia,2006,11(3-4):249-268.
[5] CHONG B M,REIGAN P,MAYLE-COMBS K D,et al. Determinants of adipophilin function in milk lipid formation and secretion[J]. Trends Endocrinol Metab, 2011,22(6):211-217.
[6] 王苗,罗军,许会芬,等.山羊INSIG1基因超表达对乳腺上皮细胞中脂质合成的影响[J].畜牧兽医学报,2016,47(9):1806-1816.WANG M,LUO J,XU H F,et al.Effect of INSIG1 overexpression on the lipid synthesis in goat mammary gland epithelial cells[J].Acta Veterinaria et Zootechnica Sinica,2016,47(9):1806-1816.(in Chinese)
[7] BROWN M S,GOLDSTEIN J L.A proteolytic pathway that controls the cholesterol content of membranes,cells,and blood[J].Proc Natl Acad Sci U S A,1999,96(20):11041-11048.
[8] 韩立强,孙宇,付彤,等.奶牛乳腺上皮细胞中SCAP和SREBP1蛋白调控SCD基因表达的作用研究[J].畜牧兽医学报,2018,49(3):652-658.HAN L Q,SUN Y,FU T,et al.Effect of SCAP and SREBP1 proteins on regulation of SCD gene expression in dairy mammary epithelial cells[J].Acta Veterinaria et Zootechnica Sinica,2018,49(3):652-658.(in Chinese)
[9] CHENG X,LI J Y,GUO D L.SCAP/SREBPs are central players in lipid metabolism and novel metabolic targets in cancer therapy[J].Curr Top Med Chem,2018,18(6):484-493.
[10] JENSEN K K,TADIN-STRAPPS M,WANG S P,et al.Dose-dependent effects of siRNA-mediated inhibition of SCAP on PCSK9,LDLR,and plasma lipids in mouse and rhesus monkey[J].J Lipid Res,2016,57(12):2150-2162.
[11] MOON Y A.The SCAP/SREBP pathway:a mediator of hepatic steatosis[J].Endocrinol Metab,2017,32(1):6-10.
[12] XU H F,LUO J,MA G Z,et al.Acyl-CoA synthetase short-chain family member 2 (ACSS2) is regulated by SREBP-1 and plays a role in fatty acid synthesis in caprine mammary epithelial cells[J].J Cell Physiol,2018,233(2):1005-1016.
[13] XU H F,LUO J,TIAN H B,et al.Rapid communication:lipid metabolic gene expression and triacylglycerol accumulation in goat mammary epithelial cells are decreased by inhibition of SREBP-1[J].J Anim Sci,2018,96(6):2399-2407.
[14] BICHI E,FRUTOS P,TORAL P G,et al.Dietary marine algae and its influence on tissue gene network expression during milk fat depression in dairy ewes[J].Anim Feed Sci Technol,2013,186(1-2):36-44.
[15] BIONAZ M,LOOR J J.Gene networks driving bovine milk fat synthesis during the lactation cycle[J].BMC Genomics, 2008,9(1):366.
[16] 唐彬秩,屈艺,赵凤艳,等.以慢病毒为载体的整合素β8 RNAi在新生鼠脑内干扰效率的研究[J].四川大学学报:医学版,2018,49(3):325-330.TANG B Z,QU Y,ZHAO F Y,et al.Investigation of lentiviral vectors based integrin β8 RNAi system in neonatal rats’ brain[J].Journal of Sichuan University:Medical Science Edition,2018,49(3):325-330.(in Chinese)
[17] 孙丽,宗秋芳,吴森,等.猪FUT2基因沉默对其所在通路基因表达及小肠上皮细胞E.coli F18黏附能力的影响[J].畜牧兽医学报,2017,48(11):2034-2045.SUN L,ZONG Q F,WU S,et al.The effects of FUT2 gene silencing on the expression of genes involved in glycosphingolipid biosynthesis-globo series pathway and the adhesion of E.coli F18 to small intestinal epithelial cells[J].Acta Veterinaria et Zootechnica Sinica, 2017,48(11):2034-2045.(in Chinese)
[18] 曾阳阳,朱弘炎,田玉民,等.猪skMLCK基因RNAi慢病毒载体的构建及干扰效率测定[J].畜牧与兽医,2016,48(2):70-73.ZENG Y Y,ZHU H Y,TIAN Y M,et al.Construction of RNAi lentiviral vector targeting porcine skMLCK gene and determination of interference efficiency[J].Animal Husbandry and Veterinary Medicine,2016,48(2):70-73.(in Chinese)
[19] 蓝林祥,杜艳涛,赵威,等.Myosin Va RNAi慢病毒载体的构建及对肺癌PG细胞运动和迁移能力的影响[J].解剖学报,2009,40(6):891-896.LAN L X,DU Y T,ZHAO W,et al.Construction of a lentivirus vector for RNA interference of myosin Va and its effect on motility and migration of P G cells[J].Acta Anatomica Sinica,2009,40(6):891-896.(in Chinese)
[20] COUVREUR S,HURTAUD C,MARNET P G,et al.Composition of milk fat from cows selected for milk fat globule size and offered either fresh pasture or a corn silage-based diet[J].J Dairy Sci,2007,90(1):392-403.
[21] MESILATI-STAHY R,MIDA K,ARGOV-ARGAMAN N. Size-dependent lipid content of bovine milk fat globule and membrane phospholipids[J]. J Agric Food Chem,2011, 59(13): 7427-7435.
[22] WALTHER T C,FARESE R V.Lipid droplets and cellular lipid metabolism[J].Annu Rev Biochem,2012,81(1):687-714.
[23] JEONG J,LISINSKI I,KADEGOWDA A K G,et al.A test of current models for the mechanism of milk-lipid droplet secretion[J]. Traffic,2013,14(9):974-986.
[24] RAMBOLD A S,COHEN S,LIPPINCOTT-SCHWARTZ J.Fatty acid trafficking in starved cells:regulation by lipid droplet lipolysis,autophagy,and mitochondrial fusion dynamics[J].Dev Cell,2015,32(6):678-692.
[25] BICKEL P E,TANSEY J T,WELTE M A.PAT proteins,an ancient family of lipid droplet proteins that regulate cellular lipid stores[J].Biochim Biophys Acta, 2009,1791(6):419-440.
[26] COHEN B C,SHAMAY A,ARGOV-ARGAMAN N.Regulation of lipid droplet size in mammary epithelial cells by remodeling of membrane lipid composition-a potential mechanism[J].PLoS One,2015,10(3):e0121645.
[27] COHEN B C,RAZ C,SHAMAY A,et al.Lipid droplet fusion in mammary epithelial cells is regulated by phosphatidylethanolamine metabolism[J].J Mammary Gland Biol Neoplasia,2017,22(4):235-249.
[28] ZHANG R,MA H M,GAO Y,et al.Th-POK regulates mammary gland lactation through mTOR-SREBP pathway[J].PLoS Genet,2018,14(2):e1007211.
[29] GONG J Y,SUN Z Q,WU L Z,et al.Fsp27 promotes lipid droplet growth by lipid exchange and transfer at lipid droplet contact sites[J].J Cell Biol,2011,195(6):953-963.
[30] 张菡,朱河水,樊晓娜,等.奶牛SREBP1蛋白对FASN基因启动子的转录调控分析[J].农业生物技术学报,2018,26(4):652-659.ZHANG H,ZHU H S,FAN X N,et al.Transcriptional regulation analysis of FASN gene promoter by SREBP1 protein in bovine (Bos taurus)[J].Journal of Agricultural Biotechnology,2018,26(4):652-659.(in Chinese)
[31] RAWSON R B,DEBOSE-BOYD R,GOLDSTEIN J L,et al.Failure to cleave sterol regulatory element-binding proteins (SREBPs) causes cholesterol auxotrophy in Chinese hamster ovary cells with genetic absence of SREBP cleavage-activating protein[J].J Biol Chem,1999,274(40):28549-28556.