miRNA122和miRNA199基因单核苷酸多态性与肝细胞肝癌易感性及预后的关系

发布时间：2018-09-12 11:13

【摘要】：原发性肝癌是全球最常见的恶性肿瘤之一,其中85%～90%属于肝细胞肝癌。我国是肝癌高发国家,全世界约50%的肝癌新发和死亡病例发生在中国。肝细胞肝癌恶性程度高、预后差,肝癌的临床表现十分隐匿,大部分患者往往确诊时已到了中晚期,因而延误了手术治疗的最佳时机。肝细胞肝癌患者即使进行肝癌切除术,两年的复发率可高达80%,术后五年的生存率约为10%～20%,晚期患者的五年生存率则低于3%。广西是我国肝癌高发区之一,死亡率高居广西肿瘤死因谱首位,严重危害人们健康和生命。研究结果表明,肝细胞肝癌发生发展是环境暴露因素和个体遗传因素共同作用的结果。但迄今为止,肝细胞肝癌最重要病因、预后影响因素及发病机制仍不十分明确。大量研究表明,个体遗传变异对疾病发生、临床特征及预后具有重要影响。单核苷酸多态性(single nucleotide polymorphism, SNPs)是决定个体遗传变异的重要方面,是目前应用最广的遗传标记物。目前,已有研究表明,编码基因SNPs与多种恶性肿瘤发生、发展、临床特征及预后密切相关。但是非编码基因多态性与肝细胞肝癌易感性及预后的关联研究鲜见报道。microRNA(miRNA)是一类内源性非编码单链小分子RNA,并以高度保守的方式参与调节下游靶基因的表达。miRNA主要通过与靶mRNA3’-非翻译区(3’-UTR)的靶序列完全或部分互补结合,在转录后水平降解mRNA或抑制其翻译,从而负性调控基因表达。miRNA既可以起到癌基因的作用,表现为在肿瘤中表达上调,也可以起到抑癌基因的作用,表现为在肿瘤中表达下调,并与多种恶性肿瘤密切相关。miRNA-122是肝脏中含量最丰富的microRNA,在肝脏发育过程中呈持续表达,且广泛参与生理及病理过程。在生理状态下,miRNA-122可参与肝脏分化、发育和代谢,肝细胞免疫应答以及肝细胞表型等生理活动过程。而在病理状态下,miRNA-122可促进丙型肝炎病毒(HCV)在肝细胞内复制,并与肝细胞肝癌发生、发展密切相关。miRNA-199是近年来发现的具有抑癌基因样功能的重要调控因子。研究表明,miRNA-199在肝细胞肝癌的发生、发展过程中起重要作用。miRNA-199在人正常肝脏高表达,而在人肝癌中普遍显著降低,并与肝细胞肝癌患者的生存期降低显著相关。亦有研究发现,miRNA-199可通过负调控原癌基因c-Met及其下游的效应因子ERK2而影响肿瘤细胞的侵袭和转移。近年研究表明,miRNA相关SNPs可通过影响miRNA的成熟过程、表达水平及与靶基因的识别及结合等,使miRNA调控网络发生异常,从而影响肿瘤的发生、发展及预后。miRNA相关SNPs主要包括miRNA自身基因SNPs、生物合成通路相关基因SNPs及miRNA靶基因中的SNPs。而由于成熟miRNA的分子长度较短,所以位于自身基因中的SNPs极可能影响该miRNA分子的成熟过程及下游功能,一系列基因组关联研究及体外实验的证实了这一假设。鉴于miRNA-122和miRNA-199在肝细胞肝癌过程中的作用,因此,推测位于miRNA-122和miRNA-199自身基因SNPs可能通过影响相应miRNA的调控网络,从而影响肝细胞肝癌发病风险、临床特征及预后。本研究将分两部分,第一部分采用病例对照研究方法探讨miRNA-122和miRNA-199基因SNPs与肝细胞肝癌遗传易感性的关系,第二部分收集肝细胞肝癌病例相关临床资料,并对肝癌根治术病例进行随访研究,探讨miRNA-122和niRNA-199基因SNPs与肝细胞肝癌临床特征及肝癌根治术后预后的关系。第一部分miRNA-122和miRNA-199基因单核苷酸多态性与肝细胞肝癌遗传易感性的关联研究目的探讨miRNA-122 rs9966765、rs1135519、rs17669及miRNA-199rs74723057和rs12120556,5个位点SNPs与广西地区人群肝细胞肝癌遗传易感性的关系。并分析上述多态位点基因与基因、基因与环境因素的交互作用在肝细胞肝癌易感性中的作用。方法(1)本研究采用以医院为基础的较大样本病例-对照研究设计方法。所有研究对象均为从2007年6月到2011年6月,来自广西医科大学第一附属医院、附属肿瘤医院和广西中医学院第一附属医院同意参与调查、经组织学病理确诊,未接受化疗和放射治疗的原发性肝细胞肝癌新发病例,共收集1050例；对照组选白同期住院的脊柱骨科、创伤外科、美容整形外科及眼科的非肿瘤患者,按年龄、性别匹配,共收集对照组1079例。采用调查问卷收集研究对象的基本情况、既往史、个人史、家族史、饮酒史和吸烟史等。采集研究对象外周血2 ml,采用Sequenom MassArray基因分型系统对候选的5个多态位点进行因分型检测。(2)miRNA-122、miRNA-199基因SNPs的选择利用dbSNP数据库,按照预先设定的频率标准筛选上述SNPs。本研究设定的频率标准是：候选SNPs最小等位基因频率(MAF)为0.05且各SNPs之间LD(r2)小于0.8。(3)采用卡方检验比较病例组和对照组一般人口学资料。采用非条件Logistic回归比较各基因型在肝细胞肝癌患者和对照组之间分布频率的差异。采用多因素Logistic回归分析基因-基因及基因-环境的交互作用。结果(1) miRNA-122基因rs1135519位点野生纯合子TT在病例和对照组中的分布频率分别为68.65%、73.14%,突变杂合子TC在两组中的分布频率分别为28.37%、25.09%,突变纯合子CC在两组的分布频率分别为2.98%、1.77%,经卡方检验TT、TC、CC三种基因型在病例和对照组中的分布具有统计学差异(P=0.022)。调整年龄、性别、吸烟、饮酒以及HBV感染等影响因素,携带突变基因型CC可显著增加肝细胞肝癌患病风险(OR=2.708,95% CI:1.154-6.356)o但是未发现miRNA-122基因rs9966765、rs17669和miRNA-199基因rs74723057、rs12120556多态性位点与肝细胞肝癌患病风险相关。(2)分层分析结果显示,miRNA-122基因rs9966765 GC/CC、 rs1135519 TC/CC位点基因型可增加较大年纪(47岁,平均年龄)个体罹患肝细胞肝癌的风险(调整OR=1.565,95%CI:1.063-2.303; OR=1.534, 95%CI:1.043-2.257); miRNA-122基因rs17669位点AG/GG基因型可增加较大年纪(47岁,平均年龄)个体罹患肝细胞肝癌的风险(OR=1.482,95%CI：1.003-2.192),并且可增加女性肝细胞肝癌的患病风险(OR=2.240,95%CI：1.035-4.484)。(3)基因-环境因素交互作用分析结果显示,miRNA-122 rs17669位点AG基因型与饮酒在肝细胞肝癌发病中存在交互作用(P=0.045)；合并AG/GG基因型发现,G等位基因与饮酒在肝细胞肝癌发病过程中存在显著交互作用(P=0.044),但是未发现miRNA-122 rs9966765、rs1135519和miRNA-199基因rs74723057、rs12120556位点多态性与饮酒存在交互作用。(4)基因-基因交互作用分析结果显示,miRNA-122基因rs9966765、rs1135519、rs17669和miRNA-199基因rs74723057、rs12120556多态性位点之间不存在SNP-SNP交互作用。结论本研究结果表明,miRNA-122基因SNPs可能影响肝细胞肝癌遗传易感性,rs1135519位点多态性可能是广西地区人群肝细胞肝癌独立危险因素,rs17669位点多态性与饮酒交互作用影响肝细胞肝癌发病风险。第二部分miRNA-122和miRNA-199基因单核苷酸多态性与肝细胞肝癌临床特征及肝癌根治术后预后的关系目的探讨广西地区人群miRNA-122基因rs9966765、rs1135519、rs17669和miRNA-199基因rs74723057、rs12120556五个多态性位点SNPs与肝细胞肝癌临床特征及肝癌根治术后整体生存及无复发生存时间的关系,为肝细胞肝癌的预后评价提供理论依据。方法收集2007年2月至2009年2月在广西医科大学第一附属医院和广西医科大学附属肿瘤医院就诊和治疗的肝细胞肝癌新发病例276例,包括接受根治性切除术或非手术治疗的肝细胞肝癌患者。通过查阅住院病历详细记录患者的一般情况、确诊时间、治疗方式、影像学检查结果等临床特征数据及AFP水平、总胆红素及白蛋白等实验室检查结果。并对纳入本研究的接受肝癌根治术后的肝细胞肝癌确诊病例进行电话随访,收集并详细记录患者存活、复发、转移及死亡情况等情况,共收集到资料完整病例179例。采用Sequenom MassArray技术对上述候选位点进行基因分型检测,应用EpiData3.0对所有资料进行双录入和一致性检查,并运用SPSS13.0对数据进行统计学分析。以卡方检验或确切概率法比较各基因型分布频率与肝细胞肝癌临床特征的关系,用乘积极限法(Kaplan-Meier-method)绘制生存曲线,对数秩检验(Log-rank test)比较上述位点不同基因型携带者无复发生存时间、总生存时间的差异,多因素Cox比例风险回归模型分析位于miRNA-122和miRNA-199基因上五个SNPs与肝细胞肝癌临床特征及肝癌根治术后整体生存及无复发生存的关系。所有统计检验均为双侧概率检验,检验水准α=0.05。结果(1) miRNA-122和miRNA-199基因5个SNPs各位点基因型与肝细胞肝癌患者临床特征,包括大体分型、肿瘤直径大小、肿瘤位置、肿瘤个数、术前肝硬化、门脉癌栓、脉管侵犯情况、被膜侵犯以及血清中AFP、总胆红素、白蛋白水平等均无统计学意义(P0.05)。(2)K-M单因素分析结果显示,miRNA-122和miRNA-199基因5个SNPs各位点基因型与肝细胞肝癌患者总生存时间和无复发生存时间均无统计学关联(P0.05)。(3)校正年龄性别吸烟饮酒和HBV感染因素的Cox回归分析结果显示,miRNA-122基因rs1135519位点CC基因型与肝细胞癌根治术后总体生存时间无统计学关联,但是可延长肝癌根治术后无复发生存时间(校正HR=0.192,95%CI 0.043-0.858)。(4)纳入全部可能影响因素的Cox回归分析结果显示,影响肝癌细胞肝癌总体生存时间的因素有血清白蛋白、肿瘤数目、脉管侵犯以及miRNA-122基因rs1135519位点TC基因型,而影响肝癌根治术后无复发生存时间的因素有AFP水平、肿瘤大体分型、被膜侵犯以及miRNA-199基因rs74723057位点CG基因型。结论miRNA-122基因rs9966765、rs1135519、rs17669和miRNA-199基因rs74723057、rs12120556五个多态性位点基因多态性与肝细胞肝癌临床特征无统计学关联,miRNA-122基因rs1135519位点基因多态性可影响肝癌根治术后总体生存时间,而miRNA-199基因rs74723057位点多态性则与肝癌根治术后无复发生存时间相关。
[Abstract]:Primary hepatocellular carcinoma (PHC) is one of the most common malignant tumors in the world, 85%-90% of which belong to hepatocellular carcinoma (HCC). China is a country with a high incidence of HCC. About 50% of new cases and deaths of HCC worldwide occur in China. Even after hepatectomy, the two-year recurrence rate of hepatocellular carcinoma can be as high as 80%, the five-year survival rate after surgery is about 10%-20%, and the five-year survival rate of advanced patients is less than 3%. Guangxi is one of the high-incidence areas of hepatocellular carcinoma in China, with a high mortality rate ranking first in the cancer death spectrum of Guangxi. The results show that the occurrence and development of hepatocellular carcinoma is the result of environmental exposure factors and individual genetic factors. But up to now, the most important etiology, prognostic factors and pathogenesis of hepatocellular carcinoma are still not very clear. Single nucleotide polymorphism (SNPs) is an important aspect of determining individual genetic variation and is the most widely used genetic marker at present. MicroRNAs (microRNAs) are a class of endogenous non-coding single-stranded small RNAs that participate in the regulation of downstream target gene expression in a highly conserved manner. MiRNA-122 is the most abundant m in the liver. MicroRNAs, which are continuously expressed during liver development, are involved in a wide range of physiological and pathological processes. Under physiological conditions, microRNAs-122 can participate in the process of liver differentiation, development and metabolism, hepatocyte immune response and hepatocyte phenotype. Under pathological conditions, microRNAs-122 can promote hepatitis C virus (HCV) replication in hepatocytes. MiRNA-199 is an important regulatory factor with tumor suppressor gene-like function found in recent years. Studies have shown that microRNA-199 plays an important role in the development of hepatocellular carcinoma. MiRNA-199 is highly expressed in human normal liver, but is generally significantly reduced in human hepatocellular carcinoma and is associated with hepatocytes. It has also been found that microRNAs-199 can negatively regulate the oncogene c-Met and its downstream effector ERK2 to influence the invasion and metastasis of tumor cells. MicroRNA-related SNPs mainly include microRNA-related gene SNPs, biosynthetic pathway-related gene SNPs, and microRNA target gene SNPs. Because of the short molecular length of mature microRNAs, SNPs located in their own genes are likely to affect the maturation of the microRNA molecule. This hypothesis is supported by a series of genomic association studies and in vitro experiments. In view of the role of microNA-122 and microNA-199 in hepatocellular carcinoma, it is speculated that SNPs located in microNA-122 and microNA-199 may influence the risk of hepatocellular carcinoma by affecting the regulatory network of the corresponding microNAs. This study will be divided into two parts, the first part of the case-control study to explore the relationship between the genetic susceptibility of hepatocellular carcinoma (HCC) and microRNA-122 and microRNA-199 SNPs, the second part of the collection of hepatocellular carcinoma case-related clinical data, and follow-up study of patients undergoing radical hepatectomy, to explore the relationship between microRNA-122 and niRNA-19. The relationship between SNPs of 9 gene and clinical characteristics of hepatocellular carcinoma and prognosis after radical hepatectomy. Part I Correlation between single nucleotide polymorphisms of microRNA-122 and microRNA-199 gene and genetic susceptibility to hepatocellular carcinoma Objective To explore the relationship between SNPs of microNA-122 rs9966765, rs1135519, rs17669, microRNA-199rs74723057 and rs12120556, 5 loci and Guangxi region. The relationship between genetic susceptibility to HCC and the interaction between genes and environmental factors was analyzed. Methods (1) A large hospital-based case-control study design was used in this study. All subjects were from June 2007 to February 2007. In June 2011, 1050 new cases of primary hepatocellular carcinoma (PHC) were collected from the First Affiliated Hospital of Guangxi Medical University, Tumor Affiliated Hospital and the First Affiliated Hospital of Guangxi College of Traditional Chinese Medicine. The control group was selected to be hospitalized at the same time in the Department of Spinal Orthopedics and Traumatic Surgery. 1079 non-neoplastic patients in cosmetic surgery and ophthalmology were enrolled in the control group according to age and sex matching. (2) Selection of SNPs of microRNAs-122 and microRNAs-199 genes was screened using dbSNP database according to preset frequency criteria. The frequency criteria set in this study were: the minimum allele frequency (MAF) of candidate SNPs was 0.05 and LD (r2) between SNPs was less than 0.8. (3) Chi-square test was used to compare the case group and control group. Results (1) MiRNA-122 gene rs1135519 wild homozygote TT was found in both cases and controls. The distribution frequencies of TC were 68.65%, 73.14%, 28.37% and 25.09% respectively. The distribution frequencies of CC in the two groups were 2.98% and 1.77% respectively. The distribution of TT, TC and CC genotypes in the two groups was statistically different (P = 0.022). Age, sex, smoking and drinking were adjusted. The risk of hepatocellular carcinoma (OR = 2.708, 95% CI: 1.154-6.356) o was significantly increased by carrying the mutant genotype CC, but no association was found between the polymorphisms of the microRNA-122 gene rs9966765, rs17669, and microRNA-199 gene rs74723057, rs12120556 and the risk of hepatocellular carcinoma (2) The stratified analysis showed that microRNA-122 gene rs9966765, rs17669 and microRNA-199 gene rs74723057 were associated with the risk of hepatocellular carcinoma. A-122 gene rs9966765 GC/CC, rs1135519 TC/CC genotype increased the risk of HCC in older individuals (adjusted OR = 1.565, 95% CI: 1.063-2.303; OR = 1.534, 95% CI: 1.043-2.257); MiNA-122 gene rs17669 AG/GG genotype increased the risk of HCC in older individuals (adjusted OR = 1.565, 95% CI: 1.063-2.303; OR = 1.534, 95% CI: 1.043-2.257); Risk of HCC (OR = 1.482,95% CI: 1.003-2.192) and increased risk of HCC in women (OR = 2.240,95% CI: 1.035-4.484). (3) Gene-environment interaction analysis showed that the AG genotype at the microRNA122 rs17669 locus interacted with alcohol consumption in HCC (P = 0.045); Genotype analysis showed that G allele had significant interaction with alcohol intake (P = 0.044), but no interaction was found between microRNAs-122 rs9966765, rs1135519 and microRNAs-199 gene rs74723057, rs12120556 polymorphism and alcohol intake. (4) Gene-gene interaction analysis showed that microRNAs-122 gene rs9966. 765, rs1135519, rs17669, and the rs74723057, rs12120556 polymorphisms of the microNA-199 gene did not show SNP-SNP interaction. Conclusion The results suggest that the SNPs of the microNA-122 gene may affect the genetic susceptibility to hepatocellular carcinoma, and the rs1135519 polymorphism may be an independent risk factor for hepatocellular carcinoma in Guangxi population, and rs17669 may be an independent risk factor. The interaction between polymorphism and alcohol consumption affects the risk of hepatocellular carcinoma (HCC). Part 2: The relationship between the single nucleotide polymorphisms of microRNA-122 and microNA-199 genes and the clinical characteristics of HCC and the prognosis of HCC after radical hepatectomy Objective To investigate the relationship between the polymorphisms of microNA-122 genes rs9966765, rs1135519, rs17669 and microNA-199 genes rs74723057, rs121205 in Guangxi population. Methods From February 2007 to February 2009, 565 SNPs were collected from the First Affiliated Hospital of Guangxi Medical University and the Cancer Hospital Affiliated to Guangxi Medical University. And 276 new cases of HCC were treated, including patients undergoing radical hepatectomy or non-surgical treatment. The general condition, diagnosis time, treatment modality, imaging findings, AFP levels, total bilirubin and albumin were recorded in detail by consulting the medical records of the patients. Results. A total of 179 cases of HCC were collected and recorded in detail. Sequenom Mass Array technique was used to detect the genotyping of these candidate sites and E. PiData 3.0 was used to double-entry and consistency test, and SPSS 13.0 was used to analyze the data. The relationship between genotype distribution frequency and clinical characteristics of hepatocellular carcinoma was compared by Chi-square test or exact probability method. The survival curve was drawn by Kaplan-Meier-method and logarithmic rank test. Multivariate Cox proportional hazard regression model was used to analyze the relationship between the five SNPs located on the microRNA-122 and microRNA-199 genes and the clinical features of hepatocellular carcinoma and the overall survival and non-recurrence survival after radical hepatectomy. Results (1) Genotypes of the five SNPs of the microRNA-122 and microNA-199 genes were associated with clinical features of HCC patients, including gross typing, tumor size, tumor location, tumor number, preoperative cirrhosis, portal vein tumor thrombus, vascular invasion, membrane invasion and serum AFP, total bilirubin, albumin. The results of K-M univariate analysis showed that there was no significant correlation between the genotypes of the five SNPs of the microRNA-122 and microRNA-199 genes and the total survival time and recurrence-free survival time of hepatocellular carcinoma patients (P 0.05). (3) Cox regression analysis of adjusted age-sex factors of smoking, drinking and HBV infection showed that The CC genotype at rs1135519 of the microRNA-122 gene was not significantly associated with the overall survival time of HCC patients after radical hepatectomy, but it could prolong the non-recurrence survival time (corrected HR = 0.192, 95% CI 0.043-0.858). (4) The Cox regression analysis of all possible influencing factors showed that influencing the overall survival time of HCC cells. There were serum albumin, tumor number, vascular invasion, and TC genotype at rs1135519 of the microRNA122 gene. AFP level, tumor gross typing, capsular invasion and CG genotype at rs74723057 of the microRNA122 gene were the main factors influencing the survival time of patients without recurrence after radical hepatectomy. The five polymorphisms of rs74723057 and rs12120556 in 669 and microRNA-199 genes were not associated with the clinical features of hepatocellular carcinoma.
【学位授予单位】：广西医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.7

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