CHRN、CXCL12和TERT-CLPTM1L基因多态性与胃癌患病风险和淋巴结转移的关联研究

发布时间：2018-06-01 01:07

本文选题：胃癌 + 单核苷酸多态性　；参考：《山东大学》2016年博士论文

【摘要】：胃癌是主要的恶性肿瘤之一,2012年全球估计新发胃癌951,600例,病死人数723,100例,东亚地区包括中国是胃癌的高发地区,随着人口老龄化的加剧,胃癌仍是我国最常见的恶性肿瘤之一,给个人及家庭带来了沉重的经济和精神负担。胃癌发病隐匿,早期可无任何症状或只有一些非特异性的消化道症状,早期胃癌诊断率低,临床就诊时,许多患者已发生局部和／或远处转移,进展期胃癌的淋巴结(LN)转移率可达70%左右,严重影响了手术和放化疗等临床抗肿瘤综合治疗的疗效,患者预后生存较差。胃癌的发生和进展是一个多因素参与、多阶段进展的复杂过程,是遗传和环境因素共同作用的结果,遗传易感性和基因-环境交互作用的相关研究,有利于高危个体的筛查和及早干预,最终使患者受益。单核苷酸多态性(single nucleotide polymorphism,SNP)是一种非常常见的遗传变异类型,它是染色体DNA中单个核苷酸转换、颠换等变化所导致的序列多态性,作为限制性片段长度多态性以及微卫星多态性后的新一代遗传标记,SNP在高危个体筛查、易感基因鉴定等方面有着广泛的应用前景。近年来,包括全基因组关联分析(Genome-wide association study, GWAS)在内的关联研究表明,许多单核苷酸多态与肿瘤的易感性有关,最近,一个纳入了824个研究的大荟萃分析发现了一些“高质量”的胃癌易感位点,分析显示11个SNP与胃癌的患病风险显著相关,包括：MUC1 rs2070803, MTX1 rs2075570, PSCA rs2294008, PRKAA1 rs13361707, PLCE1 rs2274223, TGFBR2 rs3087465, PKLR rs3762272, PSCA rs2976392, GSTP1 rs1695, CASP8 rs3834129和TNF rs1799724.不同遗传背景人群的关联研究,有助于发现更多与胃癌相关的重要SNP位点并进一步阐明胃癌的易感机制。近年来欧美人群的全基因组关联研究显示烟碱型乙酰胆碱受体(nicotinic acetylcholine receptor, nAChR)基因簇的多个SNP位点与肺癌的患病风险和吸烟行为相关。吸烟是肺癌、食管癌、胃癌等多种肿瘤的重要危险因素,烟草中的活性物质,如4-(甲基亚硝胺基)-1-(3-吡啶基)-1-丁酮(NNK)和N-亚硝基降烟碱(NNN),可作用于尼古丁乙酰胆碱受体,促进细胞增殖及致瘤性转化。nAChR基因簇内的尼古丁乙酰胆碱受体基因(cholinergic nicotine receptor genes),比如CHRNA3和CHRNA5,分别编码nAChR受体蛋白的不同多肽亚基。目前对nAChR基因簇SNP位点与吸烟相关肿瘤发病风险的研究主要集中在肺癌,对其他吸烟相关肿瘤,如食管癌、胃癌的研究很少。本单位在前期研究中发现,与从基因型相比,CHRNB3 rs 13280604 GG/AG携带者食管鳞癌的患病风险显著升高(OR=1.44, 95%CI:1.26-1.65, P=8.7×10-8)。CHRNB3 rs13280604 (与rs4950连锁)与食管鳞癌发病风险明显关联,但CHRNB3基因的多态位点是否影响其他吸烟相关肿瘤如胃癌的易感性,值得进一步探讨。此外,其他nAChR基因簇多态位点,如CHRNA5 rs667282, CHRNA3 rs3743073,据报道在中国汉族人群中与肺癌风险相关,但这些多态位点与胃癌的关系,尚有待探讨。此外,研究显示,趋化因子12 (chemokine ligand 12, CXCL12)基因3’UTR区域的一个重要多态位点,rs1801157(G801A),可能与CXCL12的表达有关,与G等位基因相比,rs1801157 A等位基因与CXCL12基因的高表达相关。CXCL12,又叫基质细胞衍生因子-1(stromal cell-derived factor-1, SDF-1),通过与趋化因子受体4(chemokine receptor 4, CXCR4))结合,在肿瘤的生长、进展中发挥着重要作用。既往研究发现,CXCR4阳性的胃癌更易发生腹膜种植转移,并且CXCR4在分期晚、有淋巴结转移的胃癌中显著阳性,转移淋巴结中SDF-1 mRNA的水平明显高于正常淋巴结,提示CXCL12/CXCR4在胃癌进展中的重要角色。体外实验还发现,CXCR4在胃癌SGC-7901和MGC-803细胞系中表达,SDF-1促进肿瘤细胞的增殖,且效应呈剂量依赖性。有学者在T3期结直肠癌的研究中观察到,有淋巴结转移的患者rs1801157GA/AA频率高于淋巴结转移阴性者。还有文献报道,CXCL12 G801A多态可能增加乳腺癌和肺癌的患病风险。而且,在采用Kaplan-Meier Plotter在线分析(http://kmplot.com/analvsis/)公共数据库中胃癌CXCL12表达与患者预后生存的关系时,我们发现,与低表达CXCL12的胃癌患者相比,高表达CXCL12者总生存和无进展生存均较差(Affymetrix probe ID:203666_at)。因此,我们假设CXCL12 rs1801157 (G801A)多态可能通过CXCL12/CXCR4通路的作用进而影响胃癌的发生和进展,为验证这个假设,本研究中我们分析了该位点与胃癌发病风险及淋巴结转移的关系。另外,染色体5p15.33区域是调节端粒生物学功能的重要区域,该区域包含两个重要的基因：端粒酶反转录酶(telomerase reverse transcriptase, TERT)和唇腭裂跨膜1样蛋白基因(cleft lip and palate trans-membrane 1-like,CLPTMIL)。端粒酶由端粒酶逆转录酶、端粒酶RNA和端粒酶协同蛋白组成,在端粒的调节及染色体完整性的维持中有着重要作用,端粒酶在许多肿瘤组织包括胃癌中都有表达,而正常细胞在绝大多数时候都不表达或仅少量表达,一些培养的增殖活跃的恶性肿瘤细胞,也呈现出较高的端粒酶活性。多数情况下,常见遗传变异只与特定肿瘤类型风险相关,但包括全基因组关联分析在内的多项研究提示,TERT-CLPTM1L区域的多态位点,比如rs2736098、rs401681,与多种肿瘤的易感性有关。rs401681是CLPTMIL基因内含子区域的一个SNP,最近的荟萃分析显示,携带rs401681[T]等位基因者皮肤黑色素瘤、胰腺癌患病风险增高,但罹患膀胱癌、前列腺癌、肺癌等肿瘤的风险降低。此外,有文献指出,rs401681[C]可能与较短的端粒长度相关。并且,荟萃分析提示,较短的端粒长度与消化道肿瘤患病风险相关,而且短的端粒长度可能预示着较差的预后生存。但是,rs401681多态位点是否影响国人胃癌的发生和进展,尚缺乏相关报道。鉴于以上所述,本课题在前期研究的基础上,采用病例一对照研究方法,收集性别与年龄匹配的胃癌和正常对照外周静脉血标本,共收集了1658例,其中胃癌716例,健康对照942例,提取各研究对象的基因组DNA,设计、合成单核苷酸多态位点CHRNA5 rs667282,CHRNA3 rs3743073,CHRNB3 rs4950,CXCL12 rs1801157,TERT-CLPTM1L rs401681的荧光探针和引物,采用TaqMan探针法在ABI 7500 PCR仪上进行多态位点的检测和基因分型,比较病例组和对照组基因型频率分布差异,并结合问卷调查及病历系统收集的相关资料信息,多元Logistic回归分析各多态位点与胃癌发病风险及淋巴结转移的关系。本研究中,所有研究对象均为中国汉族人群,胃癌患者为病理确认的胃腺癌患者,对照组来自医院查体中心的健康人群,我们采用TaqMan探针法对上述多态位点成功进行了基因分型,对照组中各SNP基因型分布均符合Hardy-Weinberg平衡(P0.05)。我们发现,CHRNB3 rs4950在病例组和对照组中基因型分布有显著统计学差异(P=0.006),其TT CT.CC基因型频率在病例组中分别为54.6%、39.8%和5.6%,在对照组中分别为62.1%、34.1%和3.8%。Logistic回归分析显示,在校正了性别、年龄、吸烟和饮酒因素后,SNP rs4950 CT/CC基因型携带者胃癌患病风险明显高于TT基因型者(CT/CC vs. TT:OR=1.346,95% CI=1.101-1.645, P=0.004; CT vs. TT:OR=1.311,95% CI=1.065-1.614,P=0.011:CC vs.TT:OR=1.688,95% CI=1.050一2.715,P=0.031).通过亚组分层分析我们发现,在男性组、非饮酒组中,携带CHRNB3 rs4950 CT/CC基因型者罹患胃癌的风险明显增加,CT/CC基因型在吸烟组和不吸烟组均增加胃癌的患病风险,在年龄分层中,小于等于60岁和大于60岁者发病风险相似。而对于CHRNA5 rs667282,CHRNA3 rs3743073,CXCL12 rs1801157及TERT-CLPTM1L rs401681,病例组和对照组基因型分布无明显差异(P值分别为0.565、0.541、0.604和0.532),Logistic回归也未发现这四个多态位点与胃癌易感性相关。此外,在有淋巴结转移资料的630例胃癌患者中,我们分析了各多态位点与胃癌淋巴结(LN)转移风险的关系,我们发现,在胃癌淋巴结转移组和无淋巴结转移组中,CHRNA5 rs667282,CHRNA3 rs3743073,CHRNB3 rs4950及TERT-CLPTM1L rs401681基因型频率分布无统计学差异,但是,CXCL12 rs1801157多态在淋巴结转移组和无淋巴结转移组中基因型分布有明显差异,其GG、GA、AA基因型频率在两组中分别为47.9%、43.6%、8.6%和58.8%、35.2%、6.0%,与CXCL12 rs1801157 GG基因型相比GA、GA/AA基因型携带者胃癌淋巴结转移风险增高GA vs.GG OR=1.521,95% CI=1.080-2.143,P=0.016；GA/AA vs.GG OR=1.555,95% CI=1.121-2.156,P=0.008),在调整了年龄、性别、吸烟及饮酒状态后,该多态位点与胃癌淋巴结转移风险的相关性仍然显著(GA vs.GG OR=1.506,95% CI=1.067-2.126,P=0.020；GA/AA vs.GG OR=1.555,95% CI=1.119-2.162, P=0.009；A vs.G OR=1.410,95% CI=1.084-1.834,P=0.010)。综上所述,通过包含1658例研究对象的病例一对照研究,我们发现CHRNB3 rs4950 CT/CC基因型显著增加中国北方汉族人群胃癌的发病风险。另外,CXCL12 rs1801157多态虽然不影响胃癌的易感性,但其GA/AA基因型携带者胃癌淋巴结转移风险明显增加。本研究提示SNP在高风险个体筛查中的潜在应用价值,但是,SNP关联研究仍需要大样本、不同遗传背景人群的验证以及后续的功能学实验研究。
[Abstract]:Gastric cancer is one of the main malignant tumors. In 2012, 951600 cases of new gastric cancer were estimated, 723100 cases were dead, the region of East Asia including China was the high incidence of gastric cancer. With the increasing aging of the population, gastric cancer was still one of the most common malignant tumors in China, which brought heavy economic and spiritual burden to individuals and families. There is no symptoms or only some nonspecific symptoms of digestive tract in the early stage. The diagnosis rate of early gastric cancer is low. In clinical treatment, many patients have local and / or distant metastasis. The lymph node (LN) transfer rate of advanced gastric cancer is about 70%, which seriously affects the curative effect of comprehensive antitumor treatment such as surgery and radiotherapy and chemotherapy. The prognosis of the patients is poor. The occurrence and progress of gastric cancer is a multi factor participation, the complex process of multistage progress is the result of the combination of genetic and environmental factors. Genetic susceptibility and gene environment interaction are related to the screening and early intervention of high-risk individuals, and the patients benefit from single nucleotide polymorphisms. (single nucleotide polymorphism, SNP) is a very common genetic variation type, which is the sequence polymorphism caused by the change of single nucleotide transformation and transformation in chromosome DNA, as a new generation marker after restriction fragment length polymorphism and microsatellite polymorphism, SNP in high-risk individuals screening, susceptibility gene identification, and so on. In recent years, association studies, including Genome-wide association study (GWAS), have shown that many single nucleotide polymorphisms are associated with tumor susceptibility. Recently, a large meta analysis included in 824 studies found some "high quality" gastric cancer susceptibility loci. The correlation between the 11 SNP is associated with the risk of gastric cancer, including MUC1 rs2070803, MTX1 rs2075570, PSCA rs2294008, PRKAA1 rs13361707, PLCE1 rs2274223. More important SNP loci associated with gastric cancer and further elucidate the susceptibility mechanism of gastric cancer. In recent years, all genomic association studies in European and American populations have shown that the multiple SNP loci of the nicotinic acetylcholine receptor (nicotinic acetylcholine receptor, nAChR) gene cluster are related to the risk of lung cancer and smoking behavior. Smoking is a lung cancer and esophageal cancer. An important risk factor for a variety of cancers such as gastric cancer, such as 4- (methyl nitrosamine) -1- (3- pyridyl) -1- butanone (NNK) and N- nitronicotine (NNN), can act on nicotine acetylcholine receptor, promote cell proliferation and tumorigenic.NAChR gene cluster of nicotinic acetylcholine receptor gene (cholinergic NIC). Otine receptor genes), such as CHRNA3 and CHRNA5, encode the different peptide subunits of the nAChR receptor protein, respectively. The current studies on the risk of nAChR gene cluster SNP sites and smoking related tumors are mainly concentrated in lung cancer, and there are few studies on other smoking related tumors, such as esophageal cancer, and gastric cancer. The risk of esophageal squamous cell carcinoma in CHRNB3 RS 13280604 GG/AG carriers increased significantly (OR=1.44, 95%CI:1.26-1.65, P=8.7 x 10-8).CHRNB3 rs13280604 (and rs4950 linkage), which was significantly associated with the risk of esophageal squamous cell carcinoma, but the polymorphic loci of the CHRNB3 gene were worthy of the susceptibility to other smoking related tumors, such as gastric cancer. In addition, other polymorphic loci of nAChR gene cluster, such as CHRNA5 rs667282 and CHRNA3 rs3743073, are reported to be associated with lung cancer risk in Chinese Han population, but the relationship between these polymorphic loci and gastric cancer remains to be discussed. Furthermore, the study shows that chemokine 12 (chemokine ligand 12, CXCL12) gene is an important part of the 3 'UTR region. The polymorphic loci, rs1801157 (G801A), may be related to the expression of CXCL12. Compared with the G allele, the rs1801157 A allele is associated with the high expression of the CXCL12 gene, also called the matrix cell derivative -1 (stromal cell-derived factor-1), by combining with chemokine receptor 4 (G). Previous studies have shown that CXCR4 positive gastric cancer is more likely to occur in peritoneal metastasis, and CXCR4 is significantly positive in gastric cancer with lymph node metastasis in late stages, and the level of SDF-1 mRNA in the metastatic lymph nodes is significantly higher than that in the normal lymph nodes, suggesting that CXCL12/CXCR4 plays an important role in the progression of gastric cancer. It was also found that CXCR4 was expressed in the SGC-7901 and MGC-803 cell lines of gastric cancer, and SDF-1 promoted the proliferation of tumor cells, and the effect was dose-dependent. In the study of T3 stage colorectal cancer, some scholars have observed that the frequency of rs1801157GA/AA in patients with lymph node metastasis is higher than that of lymph node metastases. There is also a literature report that the CXCL12 G801A polymorphism is possible. Increase the risk of breast cancer and lung cancer. Moreover, we found that the total survival and progression free survival of those with high expression of CXCL12 were poorer than those with low expression of CXCL12 in the Kaplan-Meier Plotter online analysis (http://kmplot.com/analvsis/) public database. Metrix probe ID:203666_at). Therefore, we hypothesized that the CXCL12 rs1801157 (G801A) polymorphism may affect the occurrence and progress of gastric cancer through the role of the CXCL12/CXCR4 pathway. In order to verify this hypothesis, we have analyzed the relationship between the site and the risk of gastric cancer and lymph node metastasis. In addition, the chromosome 5p15.33 area is a regulation. An important region of telomere biological function, which contains two important genes: the telomerase reverse transcriptase (telomerase reverse transcriptase, TERT) and the cleft lip and palate transmembrane 1 protein gene (cleft lip and palate trans-membrane 1-like, CLPTMIL). Telomerase is composed of telomerase reverse transcriptase, telomerase RNA and telomerase synergetic protein group It plays an important role in the regulation of telomere and the maintenance of the integrity of chromosomes. Telomerase is expressed in many tumor tissues, including gastric cancer, while normal cells are not expressed or only a small amount of expression in most cases. Some of the cultured malignant tumor cells also exhibit higher telomerase activity. The common genetic variation is only associated with a specific tumor type risk, but a number of studies, including full genome association analysis, suggest that the polymorphic loci in the TERT-CLPTM1L region, such as rs2736098, rs401681, are associated with a variety of tumor susceptibility,.Rs401681 is a SNP in the intron of the CLPTMIL gene, and recent meta-analysis shows that the polymorphic loci of the CLPTMIL gene are carried. The risk of rs401681[T] allele skin melanoma and pancreatic cancer is higher, but the risk of bladder, prostate and lung cancer is lower. In addition, the literature suggests that rs401681[C] may be associated with shorter telomere length. And a meta-analysis suggests that shorter telomere length is associated with the risk of gastrointestinal cancer, and Short telomere length may indicate poor prognosis. However, there are no reports on whether rs401681 polymorphic loci affect the occurrence and progression of human gastric cancer. A total of 1658 cases were collected, including 716 cases of gastric cancer and 942 healthy controls. The genomic DNA of each study object was extracted, designed to synthesize the single nucleotide polymorphic loci CHRNA5 rs667282, CHRNA3 rs3743073, CHRNB3 rs4950, CXCL12 rs1801157, TERT-CLPTM1L rs401681 fluorescent probes and primers. The TaqMan probe method was used on the 7500 detector. The polymorphism loci were detected and genotyping, and the difference of genotype frequency distribution between the case group and the control group was compared, and the correlation data collected by the questionnaire survey and the medical record system were combined with the multiple Logistic regression analysis of the polymorphic loci and the relationship between the risk of gastric cancer and the lymph node metastasis. All the research subjects were Chinese Han. In the population, gastric cancer patients were confirmed as gastric adenocarcinoma, and the control group was from the healthy population of the hospital check-up center. We used the TaqMan probe to genotyping the polymorphic loci successfully. The distribution of SNP genotypes in the control group was consistent with the Hardy-Weinberg balance (P0.05). We found that CHRNB3 rs4950 was in the case group and the control group. The genotype distribution in the group was significantly different (P=0.006), and the TT CT.CC genotype frequency was 54.6%, 39.8% and 5.6% in the case group. The 62.1%, 34.1% and 3.8%.Logistic regression analysis in the control group showed the risk of gastric cancer in the SNP rs4950 CT/CC genotype carriers after the correction of sex, age, smoking and drinking factors. It was significantly higher than the TT genotype (CT/CC vs. TT:OR=1.346,95% CI=1.101-1.645, P=0.004; CT vs. TT:OR=1.311,95% CI=1.065-1.614, P=0.011:CC vs.TT:OR=1.688,95%). We found that the risk of gastric cancer in the male, non drinking group and the non drinkers group Significantly increased, CT/CC genotype increased the risk of gastric cancer in both smoking and non smoking groups. In age stratification, the risk of onset was similar to those aged 60 and 60 years old. For CHRNA5 rs667282, CHRNA3 rs3743073, CXCL12 rs1801157 and TERT-CLPTM1L rs401681, there was no significant difference in genotype distribution between the case group and the control group (P values, respectively. For 0.565,0.541,0.604 and 0.532), the four polymorphic loci were not found to be associated with the susceptibility to gastric cancer in Logistic regression. In addition, in 630 cases of gastric cancer with lymph node metastasis, we analyzed the relationship between the polymorphic loci and the risk of lymph node (LN) metastasis of gastric cancer. We found that the lymph node metastasis and lymph node metastasis groups were in the gastric cancer. The frequency distribution of CHRNA5 rs667282, CHRNA3 rs3743073, CHRNB3 rs4950 and TERT-CLPTM1L rs401681 genotype was not statistically different, but the polymorphism of CXCL12 rs1801157 polymorphism was significantly different in the lymph node metastasis group and the non lymph node metastasis group. The frequency of GG, GA, and genotype was 47.9%, 43.6%, 8.6% and 58.8%, 35.2%, respectively, in the two groups. 6%, compared with the CXCL12 rs1801157 GG genotype GA, the risk of lymph node metastasis of gastric cancer in the GA/AA genotype carriers increased GA vs.GG OR=1.521,95% CI=1.080-2.143, P=0.016, GA/AA vs.GG, and after adjusting the age, sex, smoking and drinking state, the polymorphic loci were associated with the risk of lymph node metastasis of gastric cancer. The correlation is still significant (GA vs.GG OR=1.506,95% CI=1.067-2.126, P=0.020; GA/AA vs.GG OR=1.555,95% CI=1.119-2.162, P=0.009; A vs.G). The risk of gastric cancer in the Han population. In addition, although CXCL12 rs1801157 polymorphism does not affect the susceptibility of gastric cancer, the risk of lymph node metastasis in the GA/AA genotype carriers is significantly increased. This study suggests the potential application value of SNP in high-risk individual screening, but the SNP study still needs large samples and different genetic backgrounds. Verification and subsequent functional experimental research.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.2

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