硒结合蛋白1生物功能及抗肿瘤作用机理的研究
发布时间：2018-09-06 10:50
【摘要】：硒做为一种人体必须的微量元素对于人体各种机能的提高和多种疾病的预防和治疗都具有潜在的重要价值,长期较低的硒摄入量对人体健康具有多种不利的影响。由于我国约有70%的土地处在全球的缺硒带上,其上产出的各种动、植物和微生物产品都属于缺硒产品,而无机码的摄入又会对人体产生多种毒副作用,因此硒蛋白做为一种有机硒的代表成为各种富硒食品和硒相关药品研究的热点。然而硒蛋白的生物功能多样,作用靶点广泛,具体抗病机制尚不清楚,阻碍了其在医药和食品中的进一步应用,因此对硒蛋白结构和功能以及抗病分子机制的研究具有重要意义。硒结合蛋白1(SBP1)做为一种新型的含硒蛋白可能参与了人体细胞高尔基体蛋白运输等多个重要的生化进程,并通过与多种其它蛋白的相互作用影响细胞内的信号传导发挥其潜在的抑癌功能。本研究旨在通过蛋白组学的方法对SBP1的生物功能进行研究揭示其参与癌症发生的具体信号通路和分子机制,并通过SBP1与谷胱甘肽过氧化物酶(GPx1)的相互作用研究以及两者与硒的协同作用揭示新的肿瘤发病机制,进一步了解SBP1的结构,并为硒所介导的个性化肿瘤预防提供重要的理论基础。主要研究结果分述如下:1.研究了SBP1在体内和体外对肿瘤细胞生物学行为的影响利用诱导型病毒载体pRetroX-Tight-Pur构建了SBP1哺乳动物细胞诱导质粒pRetroX-Tight-Pur-SBP1,并通过单克隆筛选得到了SBP1稳转诱导结肠癌细胞HCT116-TetSBP1。使用该细胞在体外培养条件下大量诱导SBP1蛋白的表达,并测定肿瘤细胞各种生物学行为的变化。通过对细胞增殖,细胞迁移和细胞凋亡的检测发现SBP1的诱导表达对体外培养肿瘤细胞的增殖和迁移影响不大,能够诱导部分细胞发生凋亡但效果不显著。将诱导SBP1表达的HCT116-TetSBP1细胞注射到裸鼠体内,并在体内维持SBP1的诱导表达,检测其对肿瘤细胞生长和转移的影响,发现体内诱导SBP1表达不仅显著抑制了肿瘤细胞在裸鼠皮下的生长而且显著减少了肿瘤细胞向肺部的转移。2.利用iTRAQ蛋白组学的方法研究了SBP1的体内抑癌功能从裸鼠皮下种植瘤形成的肿瘤组织中提取蛋白,使用iTRAQ蛋白组学的方法鉴定差异表达的蛋白,结果共鉴定到1975个蛋白,其中差异显著蛋白132个(p0.05)。通过生物信息学GO生物功能分析和KEGG信号通路分析,筛选到了13个细胞脂代谢相关蛋白,其中5个为表达上调蛋白包括DKK1,HSP60,DHCR7,ANXA4和AGPAT5;8个为表达下调蛋白包括LPCAT2,GPx5,GAPDH,NME2,ALDH2,BPIFA3,PPIA和FABP4。另外筛选到7个细胞糖代谢相关蛋白包括GAA,GAPDH,ALDH2,TXN,EN03,UGDH和LUM,全部为表达下调蛋白。对iTRAQ实验结果进行了Western blotting验证,显示iTRAQ分析结果准确可靠。通过检测多种信号通路蛋白表达的变化,表明SBP1对细胞脂代谢和糖代谢以及癌症相关的多重信号通路都有影响。由此推测SBP1通过影响细胞糖代谢和脂代谢相关蛋白的表达参与了细胞内多个信号通路的信号传导,包括EMT通路、WNT信号通路、JNK信号通路、NOTCH信号通路和NFkB信号通路,并通过这些癌症相关信号通路引起一系列下游蛋白的激活或抑制,从而发挥其抑癌功能。3.研究了SBP1硒结合位点对其功能的影响对SBP1硒结合位点第57位氨基酸进行了单核苷酸突变,使其密码子由TGC突变为GGC,编码的氨基酸由半胱氨酸(Cys,C)突变为甘氨酸(Gly,G),去除了原有氨基酸上的一个巯基,使得SBP1失去了结合硒的能力,而对其蛋白质结构不会产生太大的影响。构建了突变SBP1哺乳动物细胞表达质粒pIRES2-SBP1C57G,将其转入人结肠癌细胞HCT116检测对SBP1功能的影响。结果显示:(1)硒造成的细胞毒性在有SBP1表达的细胞中导致的死亡率为38.2%,而在SBP1C57G表达的细胞中导致的死亡率为64.4%,因此SBP1C57G的点突变显著降低了SBP1对细胞的保护作用;(2)通过不同MCF7细胞中SBP1半衰期的检测,表明两种不同多态性的GPx1蛋白(GPx1A5P和GPx1A7L)的存在都会将SBP1蛋白的半衰期从45h延长到60h左右,而SBP1C57G的半衰期在GPx1存在时依然会从60h减少到45h,表明SBP1硒结合位点的突变破坏了这两种蛋白之间的结合;(3)通过多个信号通路蛋白和细胞内ATP含量的检测,表明SBP1C57G引起了细胞ATP总含量的降低,进而导致不依赖于信号通路的多个蛋白磷酸化的减少。4.研究了SBP1和GPx1两种蛋白在体外的相互作用构建了大肠杆菌重组表达载体PQE80L-SBP1和pQE80L-GPx1,并利用大肠杆菌表达宿主SoluBL21实现了SBP1的可溶性表达。通过镍柱纯化和分子筛纯化方法分别得到了电泳纯的SBP1蛋白和GPx1蛋白。利用两种纯化蛋白在体外进行相互作用后,发现经过Superdex 200凝胶层析的洗脱时间从原有的34 min(SBP1)和30 min(GPx1)分别延长至42.5 min和37 min,可能是由于两种蛋白之间形成了多蛋白复合体所造成的。从人结肠癌细胞HCT116中获取与SBP1和GPx1相互作用蛋白的研究表明,两种蛋白具有相同的作用蛋白硒代半胱氨酸特异延长因子(EEFSEC)。
[Abstract]:Selenium, as a necessary trace element for human body, has potential important value for the improvement of human function and the prevention and treatment of various diseases. Long-term low selenium intake has many adverse effects on human health. Selenoprotein, as a representative of organic selenium, has become a research hotspot in various selenium-rich foods and selenium-related drugs. However, selenoprotein has a variety of biological functions, a wide range of targets, and the specific mechanism of disease resistance is still unclear. Selenium binding protein 1 (SBP1), as a new selenium-containing protein, may be involved in many important biochemical processes such as Golgi protein transport in human cells and may be involved in many other proteins. The purpose of this study is to reveal the specific signaling pathways and molecular mechanisms involved in the development of cancer by proteomics methods, and to study the interaction between SBP1 and glutathione peroxidase (GPx1) and the biological functions of SBP1. The synergistic effects of SBP1 and selenium reveal new mechanisms of tumor pathogenesis, further understand the structure of SBP1, and provide an important theoretical basis for selenium-mediated personalized tumor prevention. The main results are summarized as follows: 1. The effects of SBP1 on the biological behavior of tumor cells in vitro and in vivo were studied using inducible virus vector pRetroX-Tight-Pur. SBP1 mammalian cell-induced plasmid pRetroX-Tight-Pur-SBP1 was constructed, and the stable transfection of SBP1 into colon cancer cell HCT116-TetSBP1 was obtained by monoclonal screening. The expression of SBP1 protein was induced by SBP1 cells in vitro and the changes of biological behavior of tumor cells were measured. Migration and apoptosis detection showed that the induction of SBP1 had little effect on the proliferation and migration of tumor cells in vitro, and could induce apoptosis of some cells, but the effect was not significant. In vivo, the expression of SBP1 not only significantly inhibited the growth of tumor cells subcutaneously in nude mice, but also significantly reduced the metastasis of tumor cells to the lungs. 2. Using iTRAQ proteomics method, the antitumor function of SBP1 in vivo was studied. Proteins were extracted from tumor tissues formed by subcutaneous implantation of tumor in nude mice and iTRAQ eggs were used. The results showed that 132 of the 1975 proteins were significantly different (p0.05). Through bioinformatics GO functional analysis and KEGG signaling pathway analysis, 13 proteins related to lipid metabolism were screened. Five of them were up-regulated proteins, including DKK1, HSP60, DHCR7, ANXA4 and AGPAT5. Seven proteins related to glucose metabolism, including GAA, GAPDH, ALDH2, BPIFA3, PPIA and FABP4, were screened out, including GAA, GAPDH, ALDH2, TXN, EN03, UGDH and LUM. All of them were down-regulated proteins. Western blotting was used to verify the results of iTRAQ assay. The results showed that iTRAQ analysis was accurate and reliable. The changes of protein expression of SBP1 signaling pathway indicate that SBP1 has effects on cell lipid metabolism, glucose metabolism and cancer-related multiple signaling pathways. The pathway, NOTCH signaling pathway and NFkB signaling pathway can activate or inhibit a series of downstream proteins through these cancer-related signaling pathways, thus exerting their anticancer function. 3. The effect of SBP1 selenium binding site on its function was studied. The single nucleotide mutation of amino acid at position 57 of SBP1 selenium binding site was carried out to make its codon protrude from TGC. The mutant SBP1 mammalian cell expression plasmid pIRES2-SBP1C57G was constructed and transformed into human colon cancer. The results showed that: (1) Selenium-induced cytotoxicity resulted in a mortality rate of 38.2% in SBP1-expressing cells and 64.4% in SBP1C57G-expressing cells, so point mutation of SBP1C57G significantly reduced the protective effect of SBP1 on SBP1 cells; (2) SBP1 in different MCF7 cells was induced by point mutation of SBP1C57G. Half-life test showed that the existence of two different polymorphisms of GPx1 protein (GPx1A5P and GPx1A7L) would prolong the half-life of SBP1 protein from 45 hours to about 60 hours, while the half-life of SBP1C57G would still be reduced from 60 hours to 45 hours in the presence of GPx1, suggesting that the mutation of SBP1 selenium binding site destroyed the binding between the two proteins; (3) through multiple Detection of signal pathway proteins and intracellular ATP levels showed that SBP1C57G caused a decrease in total ATP content in cells, which led to a decrease in phosphorylation of multiple proteins independent of signal pathways. 4. The interaction between SBP1 and GPx1 proteins in vitro was studied to construct recombinant expression vectors PQE80L-SBP1 and pQE80L-GPx1, and the recombinant expression vectors PQE80L-GPx1 were constructed. Soluble expression of SBP1 was achieved by E.coli expression host SoluBL21. Electrophoretic purified SBP1 and GPx1 proteins were obtained by nickel column purification and molecular sieve purification, respectively. After interaction between the two purified proteins in vitro, it was found that the elution time was 34 min (SBP1) and 30 min (SBP1) after Superdex 200 gel chromatography. (GPx1) was prolonged to 42.5 min and 37 min respectively, possibly due to the formation of multiple protein complexes between the two proteins. Studies on the protein interacting with SBP1 and GPx1 obtained from human colon cancer cell HCT116 showed that the two proteins had the same function protein, selenocysteine specific elongation factor (EEFSEC).
【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R73-3
本文编号：2226126
[Abstract]:Selenium, as a necessary trace element for human body, has potential important value for the improvement of human function and the prevention and treatment of various diseases. Long-term low selenium intake has many adverse effects on human health. Selenoprotein, as a representative of organic selenium, has become a research hotspot in various selenium-rich foods and selenium-related drugs. However, selenoprotein has a variety of biological functions, a wide range of targets, and the specific mechanism of disease resistance is still unclear. Selenium binding protein 1 (SBP1), as a new selenium-containing protein, may be involved in many important biochemical processes such as Golgi protein transport in human cells and may be involved in many other proteins. The purpose of this study is to reveal the specific signaling pathways and molecular mechanisms involved in the development of cancer by proteomics methods, and to study the interaction between SBP1 and glutathione peroxidase (GPx1) and the biological functions of SBP1. The synergistic effects of SBP1 and selenium reveal new mechanisms of tumor pathogenesis, further understand the structure of SBP1, and provide an important theoretical basis for selenium-mediated personalized tumor prevention. The main results are summarized as follows: 1. The effects of SBP1 on the biological behavior of tumor cells in vitro and in vivo were studied using inducible virus vector pRetroX-Tight-Pur. SBP1 mammalian cell-induced plasmid pRetroX-Tight-Pur-SBP1 was constructed, and the stable transfection of SBP1 into colon cancer cell HCT116-TetSBP1 was obtained by monoclonal screening. The expression of SBP1 protein was induced by SBP1 cells in vitro and the changes of biological behavior of tumor cells were measured. Migration and apoptosis detection showed that the induction of SBP1 had little effect on the proliferation and migration of tumor cells in vitro, and could induce apoptosis of some cells, but the effect was not significant. In vivo, the expression of SBP1 not only significantly inhibited the growth of tumor cells subcutaneously in nude mice, but also significantly reduced the metastasis of tumor cells to the lungs. 2. Using iTRAQ proteomics method, the antitumor function of SBP1 in vivo was studied. Proteins were extracted from tumor tissues formed by subcutaneous implantation of tumor in nude mice and iTRAQ eggs were used. The results showed that 132 of the 1975 proteins were significantly different (p0.05). Through bioinformatics GO functional analysis and KEGG signaling pathway analysis, 13 proteins related to lipid metabolism were screened. Five of them were up-regulated proteins, including DKK1, HSP60, DHCR7, ANXA4 and AGPAT5. Seven proteins related to glucose metabolism, including GAA, GAPDH, ALDH2, BPIFA3, PPIA and FABP4, were screened out, including GAA, GAPDH, ALDH2, TXN, EN03, UGDH and LUM. All of them were down-regulated proteins. Western blotting was used to verify the results of iTRAQ assay. The results showed that iTRAQ analysis was accurate and reliable. The changes of protein expression of SBP1 signaling pathway indicate that SBP1 has effects on cell lipid metabolism, glucose metabolism and cancer-related multiple signaling pathways. The pathway, NOTCH signaling pathway and NFkB signaling pathway can activate or inhibit a series of downstream proteins through these cancer-related signaling pathways, thus exerting their anticancer function. 3. The effect of SBP1 selenium binding site on its function was studied. The single nucleotide mutation of amino acid at position 57 of SBP1 selenium binding site was carried out to make its codon protrude from TGC. The mutant SBP1 mammalian cell expression plasmid pIRES2-SBP1C57G was constructed and transformed into human colon cancer. The results showed that: (1) Selenium-induced cytotoxicity resulted in a mortality rate of 38.2% in SBP1-expressing cells and 64.4% in SBP1C57G-expressing cells, so point mutation of SBP1C57G significantly reduced the protective effect of SBP1 on SBP1 cells; (2) SBP1 in different MCF7 cells was induced by point mutation of SBP1C57G. Half-life test showed that the existence of two different polymorphisms of GPx1 protein (GPx1A5P and GPx1A7L) would prolong the half-life of SBP1 protein from 45 hours to about 60 hours, while the half-life of SBP1C57G would still be reduced from 60 hours to 45 hours in the presence of GPx1, suggesting that the mutation of SBP1 selenium binding site destroyed the binding between the two proteins; (3) through multiple Detection of signal pathway proteins and intracellular ATP levels showed that SBP1C57G caused a decrease in total ATP content in cells, which led to a decrease in phosphorylation of multiple proteins independent of signal pathways. 4. The interaction between SBP1 and GPx1 proteins in vitro was studied to construct recombinant expression vectors PQE80L-SBP1 and pQE80L-GPx1, and the recombinant expression vectors PQE80L-GPx1 were constructed. Soluble expression of SBP1 was achieved by E.coli expression host SoluBL21. Electrophoretic purified SBP1 and GPx1 proteins were obtained by nickel column purification and molecular sieve purification, respectively. After interaction between the two purified proteins in vitro, it was found that the elution time was 34 min (SBP1) and 30 min (SBP1) after Superdex 200 gel chromatography. (GPx1) was prolonged to 42.5 min and 37 min respectively, possibly due to the formation of multiple protein complexes between the two proteins. Studies on the protein interacting with SBP1 and GPx1 obtained from human colon cancer cell HCT116 showed that the two proteins had the same function protein, selenocysteine specific elongation factor (EEFSEC).
【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R73-3
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