蛋白磷酸化相关遗传变异与结直肠癌的易感性研究

发布时间：2018-04-20 07:00

本文选题：结直肠癌 + 磷酸化　；参考：《华中科技大学》2015年博士论文

【摘要】：结直肠癌是全球的第四大恶性肿瘤。我国每年结直肠癌的新发病例数估计高达25万,且近年来发病率一直呈现上升趋势,其防治已刻不容缓。大量流行病学研究和分子功能学实验表明,结直肠癌是一种由遗传和环境长期共同作用引起的复杂性疾病。目前已揭示的环境危险因素包括烟草、酒精、高脂饮食和缺乏锻炼等。但暴露于相同或相似环境的人群中,仅有少部分个体最终进展为结直肠癌,提示机体遗传因素在结直肠癌的易感性中也占据了重要地位。随着基因组学的迅猛发展,目前结直肠癌的全基因组关联研究和候选基因研究已鉴定出不少易感区域和位点,但仍不足以填补结直肠癌遗传机制上的空白,亟需我们从更多角度深入挖掘潜在的易感位点,帮助构建该病的遗传易感图谱。蛋白磷酸化是一种非常重要的真核细胞翻译后修饰,几乎参与了细胞生命活动的各个环节,该修饰调节系统主要由蛋白激酶(催化磷酸化反应)、蛋白磷酸酶(催化去磷酸化反应)和底物蛋白组成。异常的磷酸化调节可导致蛋白稳定性下降,功能活性异常,进而影响细胞增殖、分化和凋亡等,最终参与疾病发生。通过整合基因组学和磷酸化蛋白组学的数据,人们已发现越来越多的磷酸化相关基因变异与肿瘤有关,并且发生在激酶、磷酸酶和底物分子上的遗传变异可显著影响人群的患癌易感性。目前激酶与肿瘤的研究已近成熟,而磷酸酶／底物与肿瘤的相关研究虽然取得一定成果,但我们对其的认识大多停留在探索阶段,亟需更多的人群研究和功能实验加以阐明。因此,本研究从底物和磷酸酶的角度出发,采用病例-对照人群研究和生物信息学分析,佐以功能学实验的方法,探索蛋白磷酸化相关遗传变异与中国人群结直肠癌发病风险之间的关联。第一部分磷酸化相关遗传变异ADD1-rs4963与结直肠癌的易感性研究研究背景：ADD1(α-adducin)蛋白是细胞骨架的重要组成部分,在细胞增殖、运动和通讯中发挥着重要作用。之前的研究表明,ADD1基因可能对肿瘤发生有潜在作用。最近的研究显示,位于该基因上的磷酸化相关错义多态rs4963(Ser586Cys)与非贲门胃癌的发病风险相关,提示该位点可能对消化系统肿瘤的遗传易感性有潜在影响。研究目的：探索ADD1-rs4963与中国人群结直肠癌发病风险之间的关联；分析基因-环境交互作用对结直肠癌易感性的潜在影响。研究方法：采用以医院为基础的病例-对照研究策略,通过主流的TaqMan分型技术对纳入的1054名原发性结直肠癌病例和1128名健康对照进行ADD1-rs4963的基因型别检测。采用Pearson's χ2检验和独立样本t检验评价病例组和对照间的基本特征分布(包括年龄、性别、吸烟和饮酒)。使用疾病比值比(OR)和95%置信区间(95%CI)作为效应指标,运用非条件logistic回归分析ADD1-rs4963与结直肠癌发病风险之间的关联,以及ADD1基因和吸烟(或饮酒)暴露的交互作用。研究结果：ADD1-rs4963的等位基因频率和基因型频率在结直肠癌病例组和健康对照组中均存在统计学差异(等位基因频率比较：P=0.011；基因型频率比较：P=0.028)。在校正了年龄、性别、吸烟和饮酒因素后,与CC基因型相比,CG基因型和GG基因型的结直肠癌发病风险分别上升25%(OR=1.25,95%CI=1.02-1.55,P=0.036)和35%(OR=1.35,95%CI=1.06.1.72,P=0.015)；显性模型显示,CG和GG基因型个体发生结直肠癌的风险是携带CC基因型个体的1.28倍(OR=1.28,95%CI=1.05-1.57,P=0.014)；加性模型分析发现,每增加一个G等位基因,结直肠癌发病风险增加0.16倍(OR=1.16,95%CI=1.03-1.31,P=0.016)。以上分析提示该位点与结直肠癌的易感性显著相关。我们进一步探索了ADD1基因与吸烟/饮酒的交互作用,但未发现阳性结果。研究结论：位于ADD1基因上的磷酸化相关遗传变异rs4963显著影响了中国人群结直肠癌的发病风险。本研究首次报道了ADD1基因与结直肠癌的易感性相关,进一步促进了我们对结直肠癌遗传机制的理解。研究创新：首次探索了ADD1基因与中国人群结直肠癌发病之间的关联,一定程度弥补了国内在蛋白磷酸化相关遗传变异与肿瘤关联研究的空白。第二部分经典蛋白酪氨酸磷酸酶家族的遗传错义多态与结直肠癌的易感性研究研究背景：蛋白酪氨酸磷酸酶可拮抗酪氨酸激酶的效应,维持细胞内酪氨酸蛋白的磷酸化平衡,在调控细胞生长、增殖、凋亡和运动等方面发挥了十分重要的作用。经典蛋白酪氨酸磷酸酶是该大家族中发现最早、研究最为广泛的一个亚类,目前已被报道与多种人类肿瘤密切相关。研究目的：系统评价经典蛋白酪氨酸磷酸酶基因家族上的遗传错义多态与中国人群结直肠癌发生风险之间的关联；深入分析候选位点与吸烟／饮酒之间的潜在交互效应;进一步探索阳性位点的生物学功能,分析其在结直肠癌发病中的潜在作用。研究方法：首先通过生物信息学系统挖掘经典蛋白酪氨酸磷酸酶基因家族上的遗传错义多态,然后采用两阶段病例-对照研究分析候选位点与结直肠癌发病之间的关联,运用非条件logistic回归评估候选位点在调整了常见混杂因素(年龄、性别、吸烟和饮酒)后的疾病效应值,以及候选基因与环境暴露之间的交互作用,最后通过蛋白功能实验进一步探索阳性关联位点的潜在生物学效应。研究结果：前期筛选一共得到25个候选位点。第一阶段的人群研究共纳入1096例结直肠癌和1816例匹配对照,其中我们鉴定出2个阳性位点,分别是PTPN12基因上的rs3750050(P-adjusted=2.220×10-4,FDR-adjusted=0.004)和PTPN23基因上的rs6780013(P-adjusted=3.645×10-4,FDR-usted=0.003).我们接着在另一地区纳入的767例结直肠癌和1215例匹配对照的人群中对这2个位点进行了关联研究,发现PTPNl2-rs750050得到成功验证。合并分析显示,rs3750050-G是结直肠癌发病的风险等位基因,每增加一个G风险等位基因,结直肠癌的罹患风险将上升25％(OR=1.25,95％CI=1.14.1.36,P=8.158×10-7).基因-环境交互分析表明,PTPN12-rs3750050还与吸烟存在显著交互(P吸烟=0.013),同时具备GG危险基因型和吸烟暴露时,其结直肠癌的发病风险明显增高(OR=1.50,95％CI=1.24.1.83).进一步的Western Blot结果揭示,该位点的G风险等位基因可明显削弱PTPN12蛋白对下游Src因子的去磷酸化作用,可能在结直肠癌致病过程中发挥一定作用。研究结论：1.PTPN12基因与中国人群的结直肠癌易感性相关,rs3750050-G可显著增加结直肠癌的发生风险。 2.PTPN12-rs3750050与吸烟存在显著的交互作用,吸烟暴露进一步增加了危险基因型携带者罹患结直肠癌的风险。 3.PTPN12-rs3750050影响了PTPN12蛋白的磷酸酶活性,G风险等位基因削弱了该蛋白对下游重要底物Src的去磷酸化调节作用,从而可能在结直肠癌发病中发挥作用。研究创新：采用生物信息学、两阶段人群研究结合蛋白功能实验的综合分析策略,首次在中国人群中系统性探讨了经典PTPs基因家族的遗传错义多态与结直肠癌发病风险的关联,同时深入分析了相关基因位点与吸烟或饮酒的交互作用,并通过蛋白功能实验为发现的人群关联提供了一定的分子生物学支持证据。
[Abstract]:Colorectal cancer is the fourth largest malignant tumor in the world. The number of new cases of colorectal cancer in China is estimated to be up to 250 thousand annually, and the incidence of colorectal cancer has been increasing in recent years. The prevention and control of colorectal cancer has been urgent. A large number of epidemiological studies and molecular functional experiments show that colorectal cancer is caused by a long-term common effect of heredity and environment. The environmental risk factors that are now revealed include tobacco, alcohol, high fat diet, and lack of exercise. But among people exposed to the same or similar environment, only a few individuals eventually progressed to colorectal cancer, suggesting that genetic factors also play an important role in the susceptibility of colorectal cancer. With the rapid development of the genome association and candidate gene research in colorectal cancer, many susceptible regions and loci have been identified, but it is still not enough to fill the gap in the genetic mechanism of colorectal cancer. It is urgent for us to dig into the potential susceptible loci from more angles and help build the genetic susceptibility map of the disease.
Protein phosphorylation is a very important post-translational modification of eukaryotic cells, which almost participates in all aspects of cell life activities. The modification regulation system is mainly composed of protein kinase (catalytic phosphorylation), protein phosphatase (catalytic dephosphorylation) and substrate protein. Abnormal phosphorylation can lead to the decrease of protein stability. Abnormal functional activity, which affects cell proliferation, differentiation and apoptosis, and eventually participates in the disease. By integrating the data of genomics and phosphorylation proteomics, more and more phosphorylation related gene mutations have been found to be associated with tumors, and the genetic variation on the kinase, phosphatase and substrate molecules can be significantly affected. At present, the research on the cancer of the population is sensitive. The research on the kinase and tumor is now mature, while the related research of phosphatase / substrate and tumor has achieved certain results, but most of our understanding of it remains at the exploratory stage, and more population studies and functional experiments are needed to clarify. A case control population study and bioinformatics analysis were used to explore the association between the genetic variation associated with protein phosphorylation and the risk of colorectal cancer in Chinese people.
Part I phosphorylation related genetic variants ADD1-rs4963 and susceptibility to colorectal cancer
Background: ADD1 (alpha -adducin) protein is an important part of the cytoskeleton and plays an important role in cell proliferation, exercise and communication. Previous studies have shown that the ADD1 gene may have potential effects on the occurrence of tumors. Recent studies have shown that the phosphorylation related missense polymorphic rs4963 (Ser586Cys) and non cardia in this gene are located in this gene. The risk of gastric cancer is related to the risk of gastrointestinal cancer.
Objective: To explore the association between ADD1-rs4963 and the risk of colorectal cancer in Chinese people, and to analyze the potential impact of gene environmental interaction on the susceptibility of colorectal cancer.
Methods: a hospital based case-control study was used to detect ADD1-rs4963 genotypes in 1054 cases of primary colorectal cancer and 1128 healthy controls by mainstream TaqMan typing. The basic characteristics of the case group and the control were evaluated by Pearson's chi 2 test and independent sample t test. Distribution (including age, sex, smoking and drinking). Using the ratio of disease ratio (OR) and 95% confidence interval (95%CI) as the effect index, the association between ADD1-rs4963 and the risk of colorectal cancer was analyzed by unconditional logistic regression, and the interaction of ADD1 gene and smoking (or drinking) exposure.
Results: the allele frequency and genotype frequency of ADD1-rs4963 were statistically different in both the colorectal cancer case group and the healthy control group (the allele frequency comparison: P=0.011; genotype frequency comparison: P=0.028). The CG genotype and the GG gene were compared with the CC genotype after the correction of age, sex, smoking and drinking factors. The risk of colorectal cancer was increased by 25% (OR=1.25,95%CI=1.02-1.55, P=0.036) and 35% (OR=1.35,95%CI=1.06.1.72, P=0.015), and the dominant model showed that the risk of colorectal cancer in CG and GG genotypes was 1.28 times that of the CC genotype (OR=1.28,95%CI= 1.05-1.57, P=0.014). The risk of colorectal cancer increased by 0.16 times the G allele (OR=1.16,95%CI=1.03-1.31, P=0.016). The above analysis suggested that the locus was significantly related to the susceptibility to colorectal cancer. We further explored the interaction between ADD1 gene and smoking / drinking, but we did not find the positive results.
Research conclusions: the phosphorylation related genetic variation (rs4963) based on the ADD1 gene significantly affects the risk of colorectal cancer in Chinese population. This study first reports the correlation between the ADD1 gene and the susceptibility to colorectal cancer and further promotes our understanding of the genetic mechanism of colorectal cancer.
Research Innovation: the first exploration of the association between the ADD1 gene and the incidence of colorectal cancer in Chinese population, to a certain extent, has made up for the gap in the study of genetic variation related to protein phosphorylation and the association of tumors in China.
The second part is the genetic missense polymorphism of the classical protein tyrosine phosphatase family and the susceptibility to colorectal cancer.
Background: protein tyrosine phosphatase can antagonize the effect of tyrosine kinase and maintain the phosphorylation of tyrosine protein in cells. It plays a very important role in regulating cell growth, proliferation, apoptosis and movement. It has been reported to be closely related to a variety of human tumors.
Objective: to systematically evaluate the association between the genetic missense polymorphism of the classical protein tyrosine phosphatase gene family and the risk of colorectal cancer in Chinese population; analyze the potential interaction between the candidate loci and smoking / drinking, and further explore the biological function of the positive loci, and analyze it in the pathogenesis of colorectal cancer. The potential effect.
Research methods: first, the genetic missense polymorphism in the classical protein tyrosine phosphatase gene family was excavated by the bioinformatics system. Then the two stage case control study was used to analyze the association between the candidate loci and the incidence of colorectal cancer. The candidate loci were evaluated by the non conditional logistic regression to adjust the common confounding factors. The effects of age, sex, smoking and drinking, as well as the interaction between candidate genes and environmental exposure, and the potential biological effects of positive associated sites were further explored through protein functional experiments.
Results: 25 candidate loci were obtained in the preliminary screening. The first stage population study included 1096 cases of colorectal cancer and 1816 matched controls, of which 2 positive sites were identified, rs3750050 (P-adjusted=2.220 * 10-4, FDR-adjusted= 0.004) on the PTPN12 gene and rs6780013 (P-adjusted=3.645) on PTPN23 gene, respectively. X 10-4, FDR-usted=0.003). We went on to study the 2 loci in 767 cases of colorectal cancer and 1215 matched controls, and found that PTPNl2-rs750050 was successfully verified. The combined analysis showed that rs3750050-G was a risk allele of colorectal cancer, each additional G risk allele, The risk of colorectal cancer will rise by 25% (OR=1.25,95%CI=1.14.1.36, P=8.158 x 10-7). Gene environmental interaction analysis shows that PTPN12-rs3750050 also has a significant interaction with smoking (P smoking =0.013). At the same time, there is a significant increase in the risk of colorectal cancer (OR=1.50,95%CI=1.24.1.83) when there is a GG risk genotype and smoking exposure. One step Western Blot results revealed that the G risk allele of the site could significantly weaken the dephosphorylation of the PTPN12 protein to the downstream Src factor, and may play a role in the pathogenesis of colorectal cancer.
Conclusion: 1.PTPN12 gene is associated with susceptibility to colorectal cancer in Chinese population. Rs3750050-G can significantly increase the risk of colorectal cancer.
There was a significant interaction between 2.PTPN12-rs3750050 and smoking. Exposure to smoking further increased the risk of colorectal cancer in carriers of the risk genotype.
3.PTPN12-rs3750050 affects the activity of the phosphatase of the PTPN12 protein, and the G risk allele weakens the dephosphorylation of the protein, which is an important downstream substrate, Src, and may play a role in the pathogenesis of colorectal cancer.
Research Innovation: the association of the genetic missense polymorphism of the classic PTPs gene family and the risk of colorectal cancer was systematically investigated in Chinese population by bioinformatics, the two stage crowd study combined with the comprehensive analysis of protein function experiments. The protein function test provided some evidence of molecular biology support for the discovery of population association.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.34

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