Wnt信号相关胞外拮抗因子遗传变异与胃癌易感性的关联性及相关功能学研究

发布时间：2018-05-28 17:32

本文选题：胃癌 + 遗传变异　；参考：《南京医科大学》2017年博士论文

【摘要】：背景与目的:我国是胃癌的高负担国家。据最新统计数据显示,胃癌在我国男性肿瘤发病率中位居第二位,仅次于肺癌;在女性中位列第三位,仅次于乳腺癌和肺癌。尽管早期胃癌经手术治疗后,五年生存率达到90%左右,但早期胃癌往往缺乏典型临床症状,大多数患者确诊时已进入中晚期,错过最佳治疗时期,导致胃癌在人群中的死亡率较高(胃癌占肿瘤死因的第二位)。阐明影响胃癌发生相关危险因素,并积极采取三级预防控制措施对防治胃癌发生具有重要的公共卫生学意义。胃癌是一种与环境密切相关的肿瘤。幽门螺旋杆菌感染、腌制食品、硝酸盐、多环芳烃化合物、烟草与酒精等暴露都被证实与胃癌的发生有关。近年来,有越来越多的研究表明,处于相同的环境暴露下,不同遗传背景的个体之间胃癌的易感性也存在差异,说明遗传因素也是影响胃癌发生的重要因素。这种遗传性差异在人群中更多的表现为单核苷酸多态性(SNP)。全基因组关联研究(GWAS)是一种高效的用于筛选遗传易感位点的研究策略,相关胃癌GWAS 发现 3q13.31(rs9841504)、5p13.1(rs13361707)区域是中国人群胃癌的易感区域。但GWAS阳性位点大多位于无生物学功能的区域,对此存在的统计学关联仍然难以解释;此外GWAS通常采用较高的统计学检验水准(P10-7),假阴性的出现难以避免。与此同时,基于候选基因的研究策略研究胃癌的易感性,尤其是对关键致癌/抑癌基因的易感性研究在阐述胃癌的发生发展过程具有十分重要的作用。肿瘤细胞干性特征模型表现为具有自我更新的潜能,也用于解释许多肿瘤表型。尽管肿瘤干性概念仍有争议,但Wnt信号在正常和肿瘤干细胞功能方面的重要作用已得到普遍的认同。相对于前体细胞而言,干细胞具有更高的增生能力,且寿命延长,这样就有更多的机会累积遗传以及表观遗传变异,从而导致肿瘤的发生。在利用Wnt信号完成自我更新和修复的组织中,当信号出现异常激活,肿瘤就会产生,这一点在结直肠癌中已被充分证实。虽然Wnt信号在胃癌发生中的作用机制研究没有结直肠癌中那么地全面和深入,但是在胃癌中普遍存在该信号的异常激活,并与肿瘤进展和转移有关。和其他重要的信号通路一样,Wnt信号被一系列拮抗因子和激动因子调节以维持动态平衡。Dickkopfs(DKKs)和 secreted frizzled-related proteins(sFRPs)是Wnt信号通路中重要的胞外抑制因子,分别可以与信号受体(LRP)和配体(Wnt)结合,通过阻碍配体-受体复合体形成的方式下调Wnt信号。基因遗传变异,尤其是潜在功能区域变异与肿瘤发生风险关系已得到越来越多的研究者关注。在肾癌、膀胱癌以及乳腺癌中发现DKKs和sFRPs遗传变异(SNP)与肿瘤发生风险有显著性关联。但DKKs和sFRPs遗传变异与胃癌的发生风险有无关联,目前尚未见报道。本研究拟采用病例-对照研究设计等方法探讨DKKs与sFRPs基因功能区域多态性与胃癌的风险关系以及相关遗传变异可能的生物学作用。同时,利用癌症基因图谱 The Cancer Genome Altas(TCGA)胃腺癌项目(TCGA-STAD)公开的RNA-seq数据初步探讨DKKs与sFRPs在胃癌发生发展中的作用,并通过胃癌新发病例组织标本进行验证。本研究可以从概率论因果观的角度为胃癌发病分子机理研究提供更多的理论依据。研究对象与方法:本研究设计首先采用基于医院的病例-对照研究。病例组为经组织病理学确诊的原发性胃癌;患者来源于江苏省中医院;对照来源于江苏省中医院体检中心,排除癌症、重大器质性病变、严重消化系统疾病。收集时间均为2008年1月到2012年7月。以DKK(1-4)以及sFRP1为候选基因,利用NCBI(https://www.ncbi.nlm.nih.gov/projects/SNP/)筛选位于潜在功能区域(5' 非翻译区、外显子以及3'非翻译区),且在北京汉族人群(Han Chinese in Beijing,HCB)最小等位基因频率10%的位点。对于位于3'非翻译区位点,利用在线SNP功能分析预测网站 SNPinfo(http://snpinfo.niehs.nih.gov/snpinfo/snpfunc.htm)选择吉布斯自由能(Energy)绝对值大于20的miRNA;构建pGL3-promoter-SNP重组质粒,合成miRNA模拟物(mimics)及阴性对照miRNA(negative control)。将包含不同等位基因的质粒及内对照质粒PRL-SV40瞬时转染至人胃癌细胞株MGC803中同时共转染miRNA模拟物或阴性对照miRNA,使用双荧光素酶报告基因检测试剂盒检测各组萤火虫/海肾荧光相对比值,探讨miRNA与SNP位点间的调控关系。我们基于癌症基因图谱The Cancer Genome Altas(TCGA)(https://gdc-portal.nci.nih.gov/),TCGA-STAD 项目 RNA-seq 数据,并联合患者人口学信息、临床病理学特征探讨DKKs和sFRP1在胃癌与癌旁组织样本中mRNA表达量的差异以及mRNA表达对预后的影响,并利用胃癌新发病例组织样本进行验证,初步推测DKKs和sFRP1在胃癌发生和预后中潜在的生物学功能。病例组和对照组间人口学特征比较采用t检验和χ2检验;拟合优度χ2检验用于分析各SNP在对照人群中是否符合Hardy-Weinberg平衡;多因素Logistic回归模型用于计算年龄与性别调整的比值比(OR值)及其95%可信区间;采用Bonferroni法进行组内多重比较校正;采用基于χ2分布的Q检验估计各亚组间的异质性;PHASE2.0用于单倍型的构建;Quanto software用于统计学把握度的计算;TCGA数据整合和转换采用Perl脚本。运用SPSS21.0(International Business Machines Corp.,IBM)进行数据管理,SAS 9.1.3(SAS Institute,Inc.,Cary,NC,USA)软件进行统计分析。研究结果:1、本研究在DKKs中共筛选出6个SNP位点:rs2241529(AG)、rs3733635(TC)、rs17037102(GA)、rs419764(CT)、rs3206824(GA)、rs2073664(CT)。对这六个SNP位点在病例组和对照组中分别进行基因分型,并没有发现两组间存在显著性的差异(P0.05)。2、sFRP1中筛选出2个位于3'非翻译区的SNP位点rs1127379和rs10088390。sFRP1rs1127379三种基因型(AA、AG、GG)在对照组中的分布频率为37.1%、46.1%以及16.8%;在病例组中为42.7%、46.3%以及11.0%。经双侧χ2检验,发现这三种基因型在病例组与对照组中的分布有显著的统计学差异(χ2=7.652,P=0.022)。经调整年龄性别后,Logistic回归模型分析发现与AA基因型相比较,携带GG基因型与胃癌风险的降低显著相关(OR=0.53,95%CI:0.35-0.81);同时,与携带A等位基因的个体(AA/AG)相比,隐性模型亦与胃癌风险的降低显著关联(OR=0.55,95%CI:0.37-0.81)。基于研究的样本含量,检测到OR=0.53的统计学把握度达到91.66%,OR=0.55时的统计学把握度为88.66%。把握度计算相关参数设置为:rs1127379的G等位基因频率为0.4530,胃癌中国人群患病率估计(31.8/100000),遗传模型则为隐性模型。3、对胃癌的病理类型和原发部位进行分层分析,rs1127379这种保护性作用在肠型胃癌(OR=0.53,95%CI:0.34-0.84)与非贲门型胃癌中(OR=0.52,95%CI:0.33-0.82)仍然存在的。而在弥漫型胃癌与贲门癌中则未发现这种显著性关联(P0.05)。4、根据年龄、性别、吸烟和饮酒状况进行分层,进一步探讨分析rs1127379位点的风险效应。研究发现无论是高龄组还是低龄组,携带GG基因型的个体与携带A等位基因的个体相比,均与胃癌风险的降低显著相关(低龄组:调整OR=0.50,95%CI:0.26-0.94;高龄组:调整 OR=0.57,95%CI:0.35-0.94),且两者之间同质性较好(异质性检验P=0.66)。在男性中,携带GG基因型个体与胃癌风险的降低显著相关(调整OR=0.46,95%CI:0.29-0.73);在饮酒者中,与胃癌风险的下降具有显著性的关联(调整OR=0.26,95%CI:0.08-0.88);在吸烟者中,GG基因型亦与胃癌风险的降低具有显著的关联(调整OR=0.21,95%CI:0.09-0.47)。5、sFRP13'非翻译区SNPrs10088390三种基因型CC、CG、GG在病例和对照组的分布分别为35.1%、47.0%、17.9%以及37.0%、46.9%、16.1%。两组之间分布经双侧χ2检验无显著性统计学差异(P=0.708)。6、利用PHASE 2.0将sFRP1两个位于3'非翻译区的SNP位点rs1 127379与rs10088390构建单倍型。结果推断出4种单倍型,分别是Ars1127379Crs10088390,Ars1127379Grs10088390,Grs1127379Grs10088390 以及 Grs1127379Crs10088390。与频数最多的 A rs1127379C rs10088390 相比,A rs1127379Grs10088390 与胃癌风险升高具有显著关联(OR=2.54,95%CI:1.76-3.67)。7、通过SNPinfo预测网站发现miRNA-1182可特异性地与sFPR1 rs1127379位点A等位基因结合,且吉布斯自由能绝对值大于20。采用双荧光报告基因实验探讨miRNA-1182与rs1127379 AG位点间的调控关系。结果显示sFRP1 rs1127379 AG改变可影响miRNA1182对其的调控作用。8、癌症基因图谱TCGA 27对胃癌与癌旁组织mRNA表达差异比较发现DKK2 mRNA在胃癌组织中的表达水平显著地高于相应的癌旁组织(P=0.007);DKK4mRNA在胃癌组织中的表达水平则显著地低于相应的癌旁组织(P0.0001);DKK1和DKK3 mRNA表达水平在癌组织与癌旁组织中无明显差异(P0.05)。生存分析发现DKK1低表达组的五年生存率显著的高于高表达组(P=0.0086)。sFRP1mRNA在胃癌组织中的表达水平显著地低于相应的癌旁组织(P0.0001),利用7对胃癌新发病例对sFRP1 mRNA在胃癌和癌旁组织中的差异进行验证,并未发现两组间存在显著差异(P0.05)。研究结论:①DKKs潜在功能性SNP位点(rs2241529(AG)、rs3733635(TC)、rs17037102(GA)、rs419764(CT)、rs3206824(GA)、rs2073664(CT))可能与胃癌的发生风险无显著性关联,同时未发现这些SNP位点与人口学特征、胃癌病理分型以及发病部位有关;②sFRP1 rs10088390(CG)多态与胃癌发病风险没有显著性关联;③sFRP1rs1127379GG基因型与肠型胃癌以及非贲门胃癌的发生风险有关,且该位点改变可影响miRNA-1182对其的调控水平;④TCGA的研究结果表明DKKs与sFRP1在胃癌发生发展中起一定的生物学作用。然而,sFRP1可能抑制胃癌的作用在7对新发胃癌组织中并未体现,提示其具有种族异质性。Wnt信号拮抗因子中的遗传易感性位点与胃癌的发生风险有关,对其进行研究有助于我们进一步认识信号拮抗因子在肿瘤发生发展中的作用,并为肿瘤高危人群预防策略提供生物标志物。
[Abstract]:Background and objective: China is the country of high burden of gastric cancer. According to the latest statistics, gastric cancer ranks the second highest in the incidence of male cancer in China, second only to lung cancer; it ranks third among women, second only to breast cancer and lung cancer. Although the five year survival rate of early gastric cancer is about 90%, but early gastric cancer is often lacking. For the typical clinical symptoms, most patients have entered the middle and late stages of diagnosis and miss the best period of treatment, which leads to the high mortality rate of gastric cancer in the population (second of the causes of cancer death). It illustrates the risk factors affecting the occurrence of gastric cancer and actively adopt the three level prevention and control measures to prevent and control the occurrence of gastric cancer. Gastric cancer is a kind of cancer closely related to the environment. Helicobacter pylori infection, pickled food, nitrate, polycyclic aromatic hydrocarbons, tobacco and alcohol have been proved to be related to the occurrence of gastric cancer. In recent years, more and more studies have shown that under the same environmental exposure, individuals with different genetic backgrounds have gastric cancer. There are also differences in susceptibility, indicating that genetic factors are also an important factor affecting the occurrence of gastric cancer. The genetic diversity is more characterized by single nucleotide polymorphisms (SNP). The whole genome association study (GWAS) is a highly efficient research strategy for screening genetic susceptibility loci, and related gastric cancer GWAS is found to be 3q13.31 (rs9841504 The 5p13.1 (rs13361707) region is a susceptible region of gastric cancer in Chinese population. But most of the GWAS positive loci are located in the area without biological function, and the statistical correlation is still difficult to explain. In addition, the GWAS usually uses a higher statistical test level (P10-7), and the appearance of the false negative is difficult to avoid. The study of the susceptibility to gastric cancer, especially the susceptibility to the key carcinogenic / tumor suppressor genes, plays an important role in explaining the development of gastric cancer. The tumor cell dry feature model shows the potential of self renewal and is also used to explain many tumor forms. Although the concept of tumor stem is still controversial, Wnt The important role of signal in normal and tumor stem cell function has been widely recognized. Compared with the precursor cells, the stem cells have higher proliferative capacity and longer life span, so there are more opportunities for accumulation of heredity and epigenetic variation, resulting in swelling of the tumor. The use of Wnt signal to complete self renewal and In the repaired tissues, when the signal is abnormal activation, the tumor is produced, which has been fully confirmed in the colorectal cancer. Although the mechanism of the Wnt signal in the occurrence of gastric cancer is not so comprehensive and deep in colorectal cancer, the abnormal activation of the signal is common in the gastric cancer, and it is associated with the progression and metastasis of the tumor. As with other important signaling pathways, Wnt signals are regulated by a series of antagonistic and excitant factors to maintain dynamic equilibrium.Dickkopfs (DKKs) and secreted frizzled-related proteins (sFRPs), an important extracellular inhibitory factor in the Wnt signaling pathway, which can be combined with the signal receptor (LRP) and ligand (Wnt), by blocking the ligand - The way the receptor complex is formed is down-regulation of Wnt signals. Genetic variations, especially potential functional regional variations and the risk of cancer, have attracted more and more attention. There is a significant association between the DKKs and sFRPs genetic variations (SNP) in renal, bladder and breast cancers (SNP). But DKKs and sFRPs inheritance There is no correlation between the variation and the risk of gastric cancer. This study intends to use case control study design to explore the relationship between the DKKs and sFRPs gene functional regional polymorphism and the risk of gastric cancer, as well as the possible biological effects of the related genetic variation. At the same time, the cancer gene map The Cancer Genome Altas (TCGA) stomach is used. Adenocarcinoma project (TCGA-STAD) open RNA-seq data preliminarily explore the role of DKKs and sFRPs in the development of gastric cancer, and verify the tissue specimens of new cases of gastric cancer. This study can provide more theoretical basis for the study of molecular mechanism of gastric cancer from the perspective of probability theory. First, a case control study based on a hospital was used. The case group was a histologically confirmed primary gastric cancer; the patients were derived from Jiangsu Province Traditional Chinese Medicine Hospital; the controls were derived from the medical center of the hospital, excluding cancer, major organic lesions, and serious digestive diseases. The collection time was from January 2008 to July 2012. DKK (1-4) And sFRP1 as a candidate gene, using NCBI (https://www.ncbi.nlm.nih.gov/projects/SNP/) screening in potential functional regions (5'untranslated region, exon and 3' non translation region), and in the Beijing Han population (Han Chinese in Beijing, HCB) minimum allele frequency 10%. For the 3'non translation site, the use of online SNP work SNPinfo (http://snpinfo.niehs.nih.gov/snpinfo/snpfunc.htm) can be used to select the miRNA of the absolute value of Gibbs free energy (Energy) greater than 20; construct pGL3-promoter-SNP recombinant plasmid, synthesize miRNA analogue (mimics) and negative control miRNA (negative control). Plasmid containing different alleles and PRL-S plasmid PRL-S V40 transiently transfected to human gastric cancer cell line MGC803 co transfected miRNA analogue or negative control miRNA, using double luciferase reporter gene detection kit to detect the relative fluorescence ratio of firefly / sea kidney, and explore the regulatory relationship between miRNA and SNP loci. We based on the cancer gene map The Cancer Genome Altas (TCGA) (https:/) /gdc-portal.nci.nih.gov/), TCGA-STAD project RNA-seq data, combined with demographic information, and clinicopathological features, to explore the difference of mRNA expression in the samples of gastric cancer and para cancer tissue and the effect of mRNA expression on the prognosis of the gastric carcinoma and sFRP1, and to validate the new case group samples of gastric cancer, and preliminarily speculate that DKKs and sFRP1 are in the form of sFRP1. The potential biological function in the occurrence and prognosis of gastric cancer. The demographic characteristics of the case group and the control group were compared with the t test and the chi 2 test. The goodness of fit chi 2 test was used to analyze whether the SNP was in the Hardy-Weinberg balance in the control population; the multifactor Logistic regression model was used to calculate the ratio Ratio of age to sex adjustment (OR value) and its 95 % confidence interval; Bonferroni method is used to carry out multiple comparison correction in group; Q test based on x 2 distribution is used to estimate the heterogeneity among subgroups; PHASE2.0 is used for haplotype construction; Quanto software is used for statistical assurance calculation; TCGA data integration and conversion uses Perl scripts. International Business Machines Co is used. RP., IBM) for data management, SAS 9.1.3 (SAS Institute, Inc., Cary, NC, USA) software to carry out statistical analysis. The results are as follows: 1 Genotyping, no significant difference was found between the two groups (P0.05).2, and 2 SNP loci rs1127379 and rs10088390.sFRP1rs1127379 genotypes (AA, AG, GG) in the non translated region of 3'(AA, AG, GG) were found in the control group for 37.1%, 46.1% and 16.8% in the control group, and 42.7%, 46.3%, and 11.0%. were examined by bilateral chi 2 in the case group. It was found that the distribution of the three genotypes in the case group and the control group was statistically significant (x 2=7.652, P=0.022). After the adjustment of age, the Logistic regression model found that compared with the AA genotype, the GG genotype was significantly related to the decrease of the risk of gastric cancer (OR=0.53,95%CI:0.35-0.81); meanwhile, the A allele was carried with the A allele. Compared with the individual (AA/AG), the recessive model was also significantly associated with the reduction of the risk of gastric cancer (OR=0.55,95%CI:0.37-0.81). Based on the sample content of the study, the statistical assurance of OR=0.53 was 91.66%, and the statistical assurance of OR=0.55 was set as the G allele frequency of rs1127379 with the frequency of the G allele of rs1127379 was 0.4530. The prevalence of gastric cancer in Chinese population is estimated (31.8/100000), and the genetic model is a recessive model of.3. The pathological types and primary sites of gastric cancer are stratified. The protective effect of rs1127379 is still existing in the intestinal gastric carcinoma (OR=0.53,95%CI:0.34-0.84) and non cardial gastric carcinoma (OR= 0.52,95%CI:0.33-0.82), and in diffuse gastric cancer and in the diffuse type of gastric cancer. This significant association (P0.05).4 was not found in the carcinoma of the cardia. The risk effect of rs1127379 loci was further explored according to age, sex, smoking and alcohol consumption. The study found that the risk of gastric cancer was lower than the risk of gastric cancer compared with the individuals carrying the A allele in both the elderly and the younger groups. Significant correlation (low age group: adjusted OR=0.50,95%CI:0.26-0.94; older age group: adjusting OR=0.57,95%CI:0.35-0.94) and better homogeneity (heterogeneity test P=0.66). In men, GG genotype individuals were significantly associated with the decrease in the risk of gastric cancer (adjusting OR= 0.46,95%CI:0.29-0.73); in drinkers, the risk of gastric cancer decreased Significant association (adjusted OR=0.26,95%CI:0.08-0.88); in smokers, the GG genotype was also significantly associated with the reduction of the risk of gastric cancer (adjusted OR=0.21,95%CI:0.09-0.47).5. The SNPrs10088390 three genotypes of CC, CG, and GG in the non translated region of sFRP13'were 35.1%, 47%, 17.9%, and 37%, 46.9%, 16.1%. two, respectively. There was no significant statistical difference between groups (P=0.708).6, and PHASE 2 was used to construct haplotypes of sFRP1 two SNP loci RS1 127379 in 3'non translation region with rs10088390, and the results were deduced from 4 haplotypes, Ars1127379Crs10088390, Ars1127379Grs10088390, Grs1127379Grs10088390, and Grs1127379Crs100. 88390. compared with the most frequent A rs1127379C rs10088390, A rs1127379Grs10088390 has a significant association with the risk of gastric cancer (OR=2.54,95%CI:1.76-3.67).7. Through SNPinfo prediction, miRNA-1182 is found to be specifically associated with sFPR1 rs1127379 loci and A alleles, and the absolute value of Gibbs free energy is greater than 20. using double fluores. The regulation relationship between miRNA-1182 and rs1127379 AG loci was investigated by the light report gene experiment. The results showed that the sFRP1 rs1127379 AG change could affect the regulation of miRNA1182 on it, and the cancer gene map TCGA 27 showed that the expression level of DKK2 mRNA in the gastric cancer group was significantly higher than that of the mRNA expression in the para cancer tissues. The expression level of DKK4mRNA in gastric cancer tissue was significantly lower than that of the corresponding para cancerous tissue (P0.0001), and the expression level of DKK1 and DKK3 mRNA was not significantly different between the cancer tissue and the para cancer tissue (P0.05). The survival analysis found that the five year survival rate of the low expression group of DKK1 was significantly higher than that of the high expression group (P=0.0086).SFRP1mRNA in the stomach. The expression level in cancer tissues was significantly lower than that of the corresponding para cancerous tissue (P0.0001). 7 new cases of gastric cancer were used to verify the difference in sFRP1 mRNA in gastric and paracancerous tissues, and there was no significant difference between the two groups (P0.05). Conclusions: (1) DKKs potential active SNP (rs2241529 (AG), rs3733635 (TC), rs17037102 (GA)) S419764 (CT), rs3206824 (GA), rs2073664 (CT)) may have no significant correlation with the risk of gastric cancer, and the SNP loci are not found to be associated with demographic characteristics, pathological types of gastric cancer and the pathogenesis of gastric cancer; (2) sFRP1 rs10088390 (CG) polymorphism is not associated with the risk of gastric cancer; (3) sFRP1rs1127379GG genotypes and intestinal type gastric cancer are not found. It is related to the risk of non cardia gastric cancer and the change of this loci can affect the regulation of miRNA-1182. 4. The results of TCGA study showed that DKKs and sFRP1 played a certain biological role in the development of gastric cancer. However, the effect of sFRP1 on gastric cancer may not be reflected in 7 NEW gastric cancers, suggesting that it has a racial heterogeneity. The genetic susceptibility loci of sex.Wnt signal antagonists are related to the risk of gastric cancer.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.2

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