Txnip基因siRNA慢病毒表达质粒构建及促进猪前体脂肪细胞分化
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摘要
硫氧还蛋白互作蛋白(thioredoxin interacting protein, Txnip)是一种氧化还原调节蛋白质,与硫氧还蛋白结合并抑制其活性,调节细胞氧化还原状态,影响细胞多种生理过程,然而其在猪脂肪细胞分化中的作用尚不明确。本文设计合成3对靶向猪Txnip基因的shRNA寡核苷酸,分别连接于重组慢病毒载体pGLV_3/H_1/GFP+Puro构建siRNA表达质粒。测序验证后,与包装质粒共转染293T细胞,获得滴度1×10~8 pfu/mL的慢病毒干扰质粒。以MOI值100转染原代培养猪前体脂肪细胞,转染率均达80%以上,其中Txnip-shRNA-2转染细胞Txnip基因沉默率达75%。转染Txnip-shRNA-2的猪前体脂肪细胞用成脂分化培养液诱导后,每隔1 d检测细胞成脂分化及相关基因表达。结果发现,其分化比阴性对照质粒转染或未转染细胞显著增强(P<0.05),PPARγ和FAS mRNA表达水平显著提高(P<0.05)。本文构建siRNA慢病毒表达质粒能有效干扰猪Txnip基因表达,Txnip表达沉默可通过上调PPARγ表达促进猪前体脂肪细胞分化。本研究提示,Txnip可能是猪脂肪细胞分化的抑制因子。
Thioredoxin interacting protein(Txnip), a redox regulatory protein, binds to thioredoxin(Trx) and inhibits its activity. Txnip regulates the redox state in various physiological processes of cells, but its role in differentiation of porcine preadipocyte is not clear. In this study, 3 pairs of shRNA oligonucleotides targeting porcine Txnip gene were designed, synthesized and inserted to recombinant lentivirus vector pGLV_3/H_1/GFP+ Puro to construct siRNA expression plasmids. After sequencing verification, the recombinant shRNA expression lentiviruses were co-transfected with packaging plasmids into 293 T cells, and the packaged lentiviral interference plasmids with a titer of 1×10~8 pfu/mL were obtained. When the primary cultured porcine preadipocytes were transfected with these packaged plasmids, the transfection efficiencies were over 80%, with 75% silencing efficiency for Txnip gene in the Txnip-shRNA-2 transfected cells(P<0.01). After porcine preadipocytes transfected with Txnip-shRNA-2 were induced by adipogenic differentiation medium, the adipogenic differentiation of the cells was detected every other day. The results showed that the differentiation of the cells transfected with Txnip-shRNA-2 was significantly promoted(P<0.05) and expression level of PPARγ and FAS mRNA increased significantly(P<0.05) as compared with that of the cells transfected with/without negative control virus. The constructed siRNA lentiviral expression plasmid could effectively interfere with the Txnip expression of porcine preadipocytes, and Txnip expression-silencing stimulated differentiation of the cells through up-regulating PPARγ expression. It was speculated that Txnip could be an inhibitor of porcine adipocyte differentiation.
Thioredoxin interacting protein(Txnip), a redox regulatory protein, binds to thioredoxin(Trx) and inhibits its activity. Txnip regulates the redox state in various physiological processes of cells, but its role in differentiation of porcine preadipocyte is not clear. In this study, 3 pairs of shRNA oligonucleotides targeting porcine Txnip gene were designed, synthesized and inserted to recombinant lentivirus vector pGLV_3/H_1/GFP+ Puro to construct siRNA expression plasmids. After sequencing verification, the recombinant shRNA expression lentiviruses were co-transfected with packaging plasmids into 293 T cells, and the packaged lentiviral interference plasmids with a titer of 1×10~8 pfu/mL were obtained. When the primary cultured porcine preadipocytes were transfected with these packaged plasmids, the transfection efficiencies were over 80%, with 75% silencing efficiency for Txnip gene in the Txnip-shRNA-2 transfected cells(P<0.01). After porcine preadipocytes transfected with Txnip-shRNA-2 were induced by adipogenic differentiation medium, the adipogenic differentiation of the cells was detected every other day. The results showed that the differentiation of the cells transfected with Txnip-shRNA-2 was significantly promoted(P<0.05) and expression level of PPARγ and FAS mRNA increased significantly(P<0.05) as compared with that of the cells transfected with/without negative control virus. The constructed siRNA lentiviral expression plasmid could effectively interfere with the Txnip expression of porcine preadipocytes, and Txnip expression-silencing stimulated differentiation of the cells through up-regulating PPARγ expression. It was speculated that Txnip could be an inhibitor of porcine adipocyte differentiation.
引文
[1] Patwari P,Higgins LJ,Chutkow WA,et al.The interaction of thioredoxin with Txnip.Evidence for formation of a mixed disulfide by disulfide exchange [J].J Biol Chem,2006,281(31):21884-21891
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[3] Gasiorek JJ,Mikhael M,Garcia-Santos D,et al.Thioredoxin-interacting protein regulates the differentiation of murine erythroid precursors [J].Exp Hematol,2015,43(5):393-403.e2
[4] Schuster S,Johnson CD,Hennebelle M,et al.Oxidized linoleic acid metabolites induce liver mitochondrial dysfunction,apoptosis,and NLRP3 activation in mice [J].J Lipid Res,2018,59(9):1597-1609
[5] 邓文珍,李杨,贾彦军,等.过表达硫氧还蛋白相互作用蛋白(TXNIP)通过激活p38MAPK通路促进MIN6细胞凋亡 [J].细胞与分子免疫学杂志(Deng WZ,Li Y,Jia YJ,et al.Over-expression of thioredoxin-interacting protein promotes apoptosis of MIN6 cells via activating p38MAPK pathway [J].Chin J Cell Mol Immunol),2017,33(10):1323-1327
[6] Kim SY,Suh HW,Chung JW,et al.Diverse functions of VDUP1 in cell proliferation,differentiation,and diseases [J].Cell Mol Immunol,2007,4(5):345-351
[7] Schulze PC,De Keulenaer GW,Yoshioka J,et al.Vitamin D3-upregulated protein-1 (VDUP-1) regulates redox-dependent vascular smooth muscle cell proliferation through interaction with thioredoxin [J].Circ Res,2002,91(8):689-695
[8] Yoshioka J,Schulze PC,Cupesi M,et al.Thioredoxin-interacting protein controls cardiac hypertrophy through regulation of thioredoxin activity [J].Circulation,2004,109(21):2581-2586
[9] Park JW,Lee SH,Woo GH,et al.Downregulation of TXNIP leads to high proliferative activity and estrogen-dependent cell growth in breast cancer [J].Biochem Biophys Res Commun,2018,498(3):566-572
[10] Rajalin AM,Micoogullari M,Sies H,et al.Upregulation of the thioredoxin-dependent redox system during differentiation of 3T3-L1 cells to adipocytes [J].Biol Chem,2014,395(6):667-677
[11] Chutkow WA,Lee RT.Thioredoxin regulates adipogenesis through thioredoxin-interacting protein (Txnip) protein stability [J].J Biol Chem,2011,286(33):29139-29145
[12] Robinson KA,Brock JW,Buse MG.Posttranslational regulation of thioredoxin-interacting protein [J].J Mol Endocrinol,2012,50(1):59-71
[13] Bharti V,Tan H,Chow D,et al.Glucocorticoid upregulates thioredoxin-interacting protein in cultured neuronal cells [J].Neuroscience,2018,384:375-383
[14] Wu N,Zheng B,Shaywitz A,et al.AMPK-dependent degradation of TXNIP upon energy stress leads to enhanced glucose uptake via GLUT1 [J].Mol Cell,2013,49(6):1167-1175
[15] Parikh H,Carlsson E,Chutkow WA,et al.TXNIP regulates peripheral glucose metabolism in humans [J].PLoS Med,2007,4(5):e158
[16] Jung H,Kim DO,Byun JE,et al.Thioredoxin-interacting protein regulates haematopoietic stem cell ageing and rejuvenation by inhibiting p38 kinase activity [J].Nat Commun,2016,7:13674
[17] Mortuza R,Chen S,Feng B,et al.High glucose induced alteration of SIRTs in endothelial cells causes rapid aging in a p300 and FOXO regulated pathway [J].PLoS One,2013,8(1):e54514
[18] 张国华,卢建雄,杨具田,等.葡萄糖和含腺苷分子通过碳水化合物反应元件结合蛋白(ChREBP)诱导猪脂肪细胞硫氧还蛋白互作蛋白(Txnip)的表达 [J].农业生物技术学报(Zhang GH,Lu JX,Yang JT,et al.Glucose and adenosine-containing molecule stimulate expression of thioredoxin interacting protein(Txnip) mRNA through carbohydrate response element binding protein(ChREBP) in primary cultured porcine adipocytes [J].J Agric Biotechnol),2014,22(3):280-288
[19] Kong J,Li YC.Molecular mechanism of 1,25-dihydroxyvitamin D3 inhibition of adipogenesis in 3T3-L1 cells [J].Am J Physiol Endocrinol Metab,2006,290(5):E916-924
[20] Rayalam S,Della-Fera MA,Ambati S,et al.Enhanced effects of 1,25(OH)(2)D(3) plus genistein on adipogenesis and apoptosis in 3T3-L1 adipocytes [J].Obesity (Silver Spring),2008,16(3):539-546
[21] Blumberg JM,Tzameli I,Astapova I,et al.Complex role of the vitamin D receptor and its ligand in adipogenesis in 3T3-L1 cells [J].J Biol Chem,2006,281(16):11205-11213
[22] Nimitphong H,Holick MF,Fried SK,et al.25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3promote the differentiation of human subcutaneous preadipocytes [J].PLoS One,2012,7(12):e52171
[23] Narvaez CJ,Simmons KM,Brunton J,et al.Induction of STEAP4 correlates with 1,25-dihydroxyvitamin D3 stimulation of adipogenesis in mesenchymal progenitor cells derived from human adipose tissue [J].J Cell Physiol,2013,228(10):2024-2036
[24] Mahajan A,Stahl CH.Dihydroxy-cholecalciferol stimulates adipocytic differentiation of porcine mesenchymal stem cells [J].J Nutr Biochem,2009,20(7):512-520
[25] Zhuang H,Lin Y,Yang G.Effects of 1,25-dihydroxyvitamin D3 on proliferation and differentiation of porcine preadipocyte in vitro [J].Chem Biol Interact,2007,170(2):114-123
[26] 卢建雄,张国华,李昌辉,等.ChREBP基因siRNA表达质粒构建及对原代培养猪脂肪细胞生脂的影响 [J].中国农业科学(Lu JX,Zhang GH,Li CH,et al.Construction of siRNA expression plasmids targeting ChREBP gene and its effect on lipogenesis in primary cultured porcine adipocytes [J].Scientia Agric Sinica),2013,46(24):5196-5204
[27] Ramirez-Zacarias JL,Castro-Munozledo F,Kuri-Harcuch W.Quantitation of adipose conversion and triglycerides by staining intracytoplasmic lipids with Oil red O [J].Histochemistry,1992,97(6):493-497
[28] 王斌,刘昆梅,郭乐,等.基于CRISPR/Cas9技术AEG-1基因敲除神经细胞系的构建及AEG-1基因敲除介导的神经细胞周期阻滞和细胞凋亡抑制 [J].中国生物化学与分子生物学报(Wang B,Liu KM,Guo L,et al.Construction of the AEG-1 knockout neural cell line based on the CRISPR /Cas9 technique and inhibition of cell cycle and cell apoptosis mediated by AEG-1 knockout [J].Chin J Biochem Mol Biol),2018,34(11):1201-1210
[29] Lu J,Holmgren A.The thioredoxin antioxidant system [J].Free Radic Biol Med,2014,66:75-87
[30] Yoshihara E,Masaki S,Matsuo Y,et al.Thioredoxin/Txnip:redoxisome,as a redox switch for the pathogenesis of diseases [J].Front Immunol,2014,4:514
[31] Chutkow WA,Birkenfeld AL,Brown JD,et al.Deletion of the alpha-arrestin protein Txnip in mice promotes adiposity and adipogenesis while preserving insulin sensitivity [J].Diabetes,2010,59(6):1424-1434
[32] Aprile M,Cataldi S,Ambrosio MR,et al.PPARgammaDelta5,a naturally occurring dominant-negative splice isoform,impairs PPARgamma function and adipocyte differentiation [J].Cell Rep,2018,25(6):1577-1592
[33] Ji S,Doumit ME,Hill RA.Regulation of adipogenesis and key adipogenic gene expression by 1,25-dihydroxyvitamin D in 3T3-L1 cells [J].PLoS One,2015,10(6):e0126142
[34] Morrison JA,Pike LA,Sams SB,et al.Thioredoxin interacting protein (TXNIP) is a novel tumor suppressor in thyroid cancer [J].Mol Cancer,2014,13:62
[2] Hwang J,Suh HW,Jeon YH,et al.The structural basis for the negative regulation of thioredoxin by thioredoxin-interacting protein [J].Nat Commun,2014,5:2958
[3] Gasiorek JJ,Mikhael M,Garcia-Santos D,et al.Thioredoxin-interacting protein regulates the differentiation of murine erythroid precursors [J].Exp Hematol,2015,43(5):393-403.e2
[4] Schuster S,Johnson CD,Hennebelle M,et al.Oxidized linoleic acid metabolites induce liver mitochondrial dysfunction,apoptosis,and NLRP3 activation in mice [J].J Lipid Res,2018,59(9):1597-1609
[5] 邓文珍,李杨,贾彦军,等.过表达硫氧还蛋白相互作用蛋白(TXNIP)通过激活p38MAPK通路促进MIN6细胞凋亡 [J].细胞与分子免疫学杂志(Deng WZ,Li Y,Jia YJ,et al.Over-expression of thioredoxin-interacting protein promotes apoptosis of MIN6 cells via activating p38MAPK pathway [J].Chin J Cell Mol Immunol),2017,33(10):1323-1327
[6] Kim SY,Suh HW,Chung JW,et al.Diverse functions of VDUP1 in cell proliferation,differentiation,and diseases [J].Cell Mol Immunol,2007,4(5):345-351
[7] Schulze PC,De Keulenaer GW,Yoshioka J,et al.Vitamin D3-upregulated protein-1 (VDUP-1) regulates redox-dependent vascular smooth muscle cell proliferation through interaction with thioredoxin [J].Circ Res,2002,91(8):689-695
[8] Yoshioka J,Schulze PC,Cupesi M,et al.Thioredoxin-interacting protein controls cardiac hypertrophy through regulation of thioredoxin activity [J].Circulation,2004,109(21):2581-2586
[9] Park JW,Lee SH,Woo GH,et al.Downregulation of TXNIP leads to high proliferative activity and estrogen-dependent cell growth in breast cancer [J].Biochem Biophys Res Commun,2018,498(3):566-572
[10] Rajalin AM,Micoogullari M,Sies H,et al.Upregulation of the thioredoxin-dependent redox system during differentiation of 3T3-L1 cells to adipocytes [J].Biol Chem,2014,395(6):667-677
[11] Chutkow WA,Lee RT.Thioredoxin regulates adipogenesis through thioredoxin-interacting protein (Txnip) protein stability [J].J Biol Chem,2011,286(33):29139-29145
[12] Robinson KA,Brock JW,Buse MG.Posttranslational regulation of thioredoxin-interacting protein [J].J Mol Endocrinol,2012,50(1):59-71
[13] Bharti V,Tan H,Chow D,et al.Glucocorticoid upregulates thioredoxin-interacting protein in cultured neuronal cells [J].Neuroscience,2018,384:375-383
[14] Wu N,Zheng B,Shaywitz A,et al.AMPK-dependent degradation of TXNIP upon energy stress leads to enhanced glucose uptake via GLUT1 [J].Mol Cell,2013,49(6):1167-1175
[15] Parikh H,Carlsson E,Chutkow WA,et al.TXNIP regulates peripheral glucose metabolism in humans [J].PLoS Med,2007,4(5):e158
[16] Jung H,Kim DO,Byun JE,et al.Thioredoxin-interacting protein regulates haematopoietic stem cell ageing and rejuvenation by inhibiting p38 kinase activity [J].Nat Commun,2016,7:13674
[17] Mortuza R,Chen S,Feng B,et al.High glucose induced alteration of SIRTs in endothelial cells causes rapid aging in a p300 and FOXO regulated pathway [J].PLoS One,2013,8(1):e54514
[18] 张国华,卢建雄,杨具田,等.葡萄糖和含腺苷分子通过碳水化合物反应元件结合蛋白(ChREBP)诱导猪脂肪细胞硫氧还蛋白互作蛋白(Txnip)的表达 [J].农业生物技术学报(Zhang GH,Lu JX,Yang JT,et al.Glucose and adenosine-containing molecule stimulate expression of thioredoxin interacting protein(Txnip) mRNA through carbohydrate response element binding protein(ChREBP) in primary cultured porcine adipocytes [J].J Agric Biotechnol),2014,22(3):280-288
[19] Kong J,Li YC.Molecular mechanism of 1,25-dihydroxyvitamin D3 inhibition of adipogenesis in 3T3-L1 cells [J].Am J Physiol Endocrinol Metab,2006,290(5):E916-924
[20] Rayalam S,Della-Fera MA,Ambati S,et al.Enhanced effects of 1,25(OH)(2)D(3) plus genistein on adipogenesis and apoptosis in 3T3-L1 adipocytes [J].Obesity (Silver Spring),2008,16(3):539-546
[21] Blumberg JM,Tzameli I,Astapova I,et al.Complex role of the vitamin D receptor and its ligand in adipogenesis in 3T3-L1 cells [J].J Biol Chem,2006,281(16):11205-11213
[22] Nimitphong H,Holick MF,Fried SK,et al.25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3promote the differentiation of human subcutaneous preadipocytes [J].PLoS One,2012,7(12):e52171
[23] Narvaez CJ,Simmons KM,Brunton J,et al.Induction of STEAP4 correlates with 1,25-dihydroxyvitamin D3 stimulation of adipogenesis in mesenchymal progenitor cells derived from human adipose tissue [J].J Cell Physiol,2013,228(10):2024-2036
[24] Mahajan A,Stahl CH.Dihydroxy-cholecalciferol stimulates adipocytic differentiation of porcine mesenchymal stem cells [J].J Nutr Biochem,2009,20(7):512-520
[25] Zhuang H,Lin Y,Yang G.Effects of 1,25-dihydroxyvitamin D3 on proliferation and differentiation of porcine preadipocyte in vitro [J].Chem Biol Interact,2007,170(2):114-123
[26] 卢建雄,张国华,李昌辉,等.ChREBP基因siRNA表达质粒构建及对原代培养猪脂肪细胞生脂的影响 [J].中国农业科学(Lu JX,Zhang GH,Li CH,et al.Construction of siRNA expression plasmids targeting ChREBP gene and its effect on lipogenesis in primary cultured porcine adipocytes [J].Scientia Agric Sinica),2013,46(24):5196-5204
[27] Ramirez-Zacarias JL,Castro-Munozledo F,Kuri-Harcuch W.Quantitation of adipose conversion and triglycerides by staining intracytoplasmic lipids with Oil red O [J].Histochemistry,1992,97(6):493-497
[28] 王斌,刘昆梅,郭乐,等.基于CRISPR/Cas9技术AEG-1基因敲除神经细胞系的构建及AEG-1基因敲除介导的神经细胞周期阻滞和细胞凋亡抑制 [J].中国生物化学与分子生物学报(Wang B,Liu KM,Guo L,et al.Construction of the AEG-1 knockout neural cell line based on the CRISPR /Cas9 technique and inhibition of cell cycle and cell apoptosis mediated by AEG-1 knockout [J].Chin J Biochem Mol Biol),2018,34(11):1201-1210
[29] Lu J,Holmgren A.The thioredoxin antioxidant system [J].Free Radic Biol Med,2014,66:75-87
[30] Yoshihara E,Masaki S,Matsuo Y,et al.Thioredoxin/Txnip:redoxisome,as a redox switch for the pathogenesis of diseases [J].Front Immunol,2014,4:514
[31] Chutkow WA,Birkenfeld AL,Brown JD,et al.Deletion of the alpha-arrestin protein Txnip in mice promotes adiposity and adipogenesis while preserving insulin sensitivity [J].Diabetes,2010,59(6):1424-1434
[32] Aprile M,Cataldi S,Ambrosio MR,et al.PPARgammaDelta5,a naturally occurring dominant-negative splice isoform,impairs PPARgamma function and adipocyte differentiation [J].Cell Rep,2018,25(6):1577-1592
[33] Ji S,Doumit ME,Hill RA.Regulation of adipogenesis and key adipogenic gene expression by 1,25-dihydroxyvitamin D in 3T3-L1 cells [J].PLoS One,2015,10(6):e0126142
[34] Morrison JA,Pike LA,Sams SB,et al.Thioredoxin interacting protein (TXNIP) is a novel tumor suppressor in thyroid cancer [J].Mol Cancer,2014,13:62
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