【摘要】：原發(fā)性肝癌是全球最常見的惡性腫瘤之一,其中85%～90%屬于肝細胞肝癌。我國是肝癌高發(fā)國家,全世界約50%的肝癌新發(fā)和死亡病例發(fā)生在中國。肝細胞肝癌惡性程度高、預后差,肝癌的臨床表現(xiàn)十分隱匿,大部分患者往往確診時已到了中晚期,因而延誤了手術治療的最佳時機。肝細胞肝癌患者即使進行肝癌切除術,兩年的復發(fā)率可高達80%,術后五年的生存率約為10%～20%,晚期患者的五年生存率則低于3%。廣西是我國肝癌高發(fā)區(qū)之一,死亡率高居廣西腫瘤死因譜首位,嚴重危害人們健康和生命。研究結果表明,肝細胞肝癌發(fā)生發(fā)展是環(huán)境暴露因素和個體遺傳因素共同作用的結果。但迄今為止,肝細胞肝癌最重要病因、預后影響因素及發(fā)病機制仍不十分明確。大量研究表明,個體遺傳變異對疾病發(fā)生、臨床特征及預后具有重要影響。單核苷酸多態(tài)性(single nucleotide polymorphism, SNPs)是決定個體遺傳變異的重要方面,是目前應用最廣的遺傳標記物。目前,已有研究表明,編碼基因SNPs與多種惡性腫瘤發(fā)生、發(fā)展、臨床特征及預后密切相關。但是非編碼基因多態(tài)性與肝細胞肝癌易感性及預后的關聯(lián)研究鮮見報道。microRNA(miRNA)是一類內源性非編碼單鏈小分子RNA,并以高度保守的方式參與調節(jié)下游靶基因的表達。miRNA主要通過與靶mRNA3’-非翻譯區(qū)(3’-UTR)的靶序列完全或部分互補結合,在轉錄后水平降解mRNA或抑制其翻譯,從而負性調控基因表達。miRNA既可以起到癌基因的作用,表現(xiàn)為在腫瘤中表達上調,也可以起到抑癌基因的作用,表現(xiàn)為在腫瘤中表達下調,并與多種惡性腫瘤密切相關。miRNA-122是肝臟中含量最豐富的microRNA,在肝臟發(fā)育過程中呈持續(xù)表達,且廣泛參與生理及病理過程。在生理狀態(tài)下,miRNA-122可參與肝臟分化、發(fā)育和代謝,肝細胞免疫應答以及肝細胞表型等生理活動過程。而在病理狀態(tài)下,miRNA-122可促進丙型肝炎病毒(HCV)在肝細胞內復制,并與肝細胞肝癌發(fā)生、發(fā)展密切相關。miRNA-199是近年來發(fā)現(xiàn)的具有抑癌基因樣功能的重要調控因子。研究表明,miRNA-199在肝細胞肝癌的發(fā)生、發(fā)展過程中起重要作用。miRNA-199在人正常肝臟高表達,而在人肝癌中普遍顯著降低,并與肝細胞肝癌患者的生存期降低顯著相關。亦有研究發(fā)現(xiàn),miRNA-199可通過負調控原癌基因c-Met及其下游的效應因子ERK2而影響腫瘤細胞的侵襲和轉移。近年研究表明,miRNA相關SNPs可通過影響miRNA的成熟過程、表達水平及與靶基因的識別及結合等,使miRNA調控網絡發(fā)生異常,從而影響腫瘤的發(fā)生、發(fā)展及預后。miRNA相關SNPs主要包括miRNA自身基因SNPs、生物合成通路相關基因SNPs及miRNA靶基因中的SNPs。而由于成熟miRNA的分子長度較短,所以位于自身基因中的SNPs極可能影響該miRNA分子的成熟過程及下游功能,一系列基因組關聯(lián)研究及體外實驗的證實了這一假設。鑒于miRNA-122和miRNA-199在肝細胞肝癌過程中的作用,因此,推測位于miRNA-122和miRNA-199自身基因SNPs可能通過影響相應miRNA的調控網絡,從而影響肝細胞肝癌發(fā)病風險、臨床特征及預后。本研究將分兩部分,第一部分采用病例對照研究方法探討miRNA-122和miRNA-199基因SNPs與肝細胞肝癌遺傳易感性的關系,第二部分收集肝細胞肝癌病例相關臨床資料,并對肝癌根治術病例進行隨訪研究,探討miRNA-122和niRNA-199基因SNPs與肝細胞肝癌臨床特征及肝癌根治術后預后的關系。第一部分miRNA-122和miRNA-199基因單核苷酸多態(tài)性與肝細胞肝癌遺傳易感性的關聯(lián)研究目的探討miRNA-122 rs9966765、rs1135519、rs17669及miRNA-199rs74723057和rs12120556,5個位點SNPs與廣西地區(qū)人群肝細胞肝癌遺傳易感性的關系。并分析上述多態(tài)位點基因與基因、基因與環(huán)境因素的交互作用在肝細胞肝癌易感性中的作用。方法(1)本研究采用以醫(yī)院為基礎的較大樣本病例-對照研究設計方法。所有研究對象均為從2007年6月到2011年6月,來自廣西醫(yī)科大學第一附屬醫(yī)院、附屬腫瘤醫(yī)院和廣西中醫(yī)學院第一附屬醫(yī)院同意參與調查、經組織學病理確診,未接受化療和放射治療的原發(fā)性肝細胞肝癌新發(fā)病例,共收集1050例；對照組選白同期住院的脊柱骨科、創(chuàng)傷外科、美容整形外科及眼科的非腫瘤患者,按年齡、性別匹配,共收集對照組1079例。采用調查問卷收集研究對象的基本情況、既往史、個人史、家族史、飲酒史和吸煙史等。采集研究對象外周血2 ml,采用Sequenom MassArray基因分型系統(tǒng)對候選的5個多態(tài)位點進行因分型檢測。(2)miRNA-122、miRNA-199基因SNPs的選擇利用dbSNP數(shù)據庫,按照預先設定的頻率標準篩選上述SNPs。本研究設定的頻率標準是：候選SNPs最小等位基因頻率(MAF)為0.05且各SNPs之間LD(r2)小于0.8。(3)采用卡方檢驗比較病例組和對照組一般人口學資料。采用非條件Logistic回歸比較各基因型在肝細胞肝癌患者和對照組之間分布頻率的差異。采用多因素Logistic回歸分析基因-基因及基因-環(huán)境的交互作用。結果(1) miRNA-122基因rs1135519位點野生純合子TT在病例和對照組中的分布頻率分別為68.65%、73.14%,突變雜合子TC在兩組中的分布頻率分別為28.37%、25.09%,突變純合子CC在兩組的分布頻率分別為2.98%、1.77%,經卡方檢驗TT、TC、CC三種基因型在病例和對照組中的分布具有統(tǒng)計學差異(P=0.022)。調整年齡、性別、吸煙、飲酒以及HBV感染等影響因素,攜帶突變基因型CC可顯著增加肝細胞肝癌患病風險(OR=2.708,95% CI:1.154-6.356)o但是未發(fā)現(xiàn)miRNA-122基因rs9966765、rs17669和miRNA-199基因rs74723057、rs12120556多態(tài)性位點與肝細胞肝癌患病風險相關。(2)分層分析結果顯示,miRNA-122基因rs9966765 GC/CC、 rs1135519 TC/CC位點基因型可增加較大年紀(47歲,平均年齡)個體罹患肝細胞肝癌的風險(調整OR=1.565,95%CI:1.063-2.303; OR=1.534, 95%CI:1.043-2.257); miRNA-122基因rs17669位點AG/GG基因型可增加較大年紀(47歲,平均年齡)個體罹患肝細胞肝癌的風險(OR=1.482,95%CI：1.003-2.192),并且可增加女性肝細胞肝癌的患病風險(OR=2.240,95%CI：1.035-4.484)。(3)基因-環(huán)境因素交互作用分析結果顯示,miRNA-122 rs17669位點AG基因型與飲酒在肝細胞肝癌發(fā)病中存在交互作用(P=0.045)；合并AG/GG基因型發(fā)現(xiàn),G等位基因與飲酒在肝細胞肝癌發(fā)病過程中存在顯著交互作用(P=0.044),但是未發(fā)現(xiàn)miRNA-122 rs9966765、rs1135519和miRNA-199基因rs74723057、rs12120556位點多態(tài)性與飲酒存在交互作用。(4)基因-基因交互作用分析結果顯示,miRNA-122基因rs9966765、rs1135519、rs17669和miRNA-199基因rs74723057、rs12120556多態(tài)性位點之間不存在SNP-SNP交互作用。結論本研究結果表明,miRNA-122基因SNPs可能影響肝細胞肝癌遺傳易感性,rs1135519位點多態(tài)性可能是廣西地區(qū)人群肝細胞肝癌獨立危險因素,rs17669位點多態(tài)性與飲酒交互作用影響肝細胞肝癌發(fā)病風險。第二部分miRNA-122和miRNA-199基因單核苷酸多態(tài)性與肝細胞肝癌臨床特征及肝癌根治術后預后的關系目的探討廣西地區(qū)人群miRNA-122基因rs9966765、rs1135519、rs17669和miRNA-199基因rs74723057、rs12120556五個多態(tài)性位點SNPs與肝細胞肝癌臨床特征及肝癌根治術后整體生存及無復發(fā)生存時間的關系,為肝細胞肝癌的預后評價提供理論依據。方法收集2007年2月至2009年2月在廣西醫(yī)科大學第一附屬醫(yī)院和廣西醫(yī)科大學附屬腫瘤醫(yī)院就診和治療的肝細胞肝癌新發(fā)病例276例,包括接受根治性切除術或非手術治療的肝細胞肝癌患者。通過查閱住院病歷詳細記錄患者的一般情況、確診時間、治療方式、影像學檢查結果等臨床特征數(shù)據及AFP水平、總膽紅素及白蛋白等實驗室檢查結果。并對納入本研究的接受肝癌根治術后的肝細胞肝癌確診病例進行電話隨訪,收集并詳細記錄患者存活、復發(fā)、轉移及死亡情況等情況,共收集到資料完整病例179例。采用Sequenom MassArray技術對上述候選位點進行基因分型檢測,應用EpiData3.0對所有資料進行雙錄入和一致性檢查,并運用SPSS13.0對數(shù)據進行統(tǒng)計學分析。以卡方檢驗或確切概率法比較各基因型分布頻率與肝細胞肝癌臨床特征的關系,用乘積極限法(Kaplan-Meier-method)繪制生存曲線,對數(shù)秩檢驗(Log-rank test)比較上述位點不同基因型攜帶者無復發(fā)生存時間、總生存時間的差異,多因素Cox比例風險回歸模型分析位于miRNA-122和miRNA-199基因上五個SNPs與肝細胞肝癌臨床特征及肝癌根治術后整體生存及無復發(fā)生存的關系。所有統(tǒng)計檢驗均為雙側概率檢驗,檢驗水準α=0.05。結果(1) miRNA-122和miRNA-199基因5個SNPs各位點基因型與肝細胞肝癌患者臨床特征,包括大體分型、腫瘤直徑大小、腫瘤位置、腫瘤個數(shù)、術前肝硬化、門脈癌栓、脈管侵犯情況、被膜侵犯以及血清中AFP、總膽紅素、白蛋白水平等均無統(tǒng)計學意義(P0.05)。(2)K-M單因素分析結果顯示,miRNA-122和miRNA-199基因5個SNPs各位點基因型與肝細胞肝癌患者總生存時間和無復發(fā)生存時間均無統(tǒng)計學關聯(lián)(P0.05)。(3)校正年齡性別吸煙飲酒和HBV感染因素的Cox回歸分析結果顯示,miRNA-122基因rs1135519位點CC基因型與肝細胞癌根治術后總體生存時間無統(tǒng)計學關聯(lián),但是可延長肝癌根治術后無復發(fā)生存時間(校正HR=0.192,95%CI 0.043-0.858)。(4)納入全部可能影響因素的Cox回歸分析結果顯示,影響肝癌細胞肝癌總體生存時間的因素有血清白蛋白、腫瘤數(shù)目、脈管侵犯以及miRNA-122基因rs1135519位點TC基因型,而影響肝癌根治術后無復發(fā)生存時間的因素有AFP水平、腫瘤大體分型、被膜侵犯以及miRNA-199基因rs74723057位點CG基因型。結論miRNA-122基因rs9966765、rs1135519、rs17669和miRNA-199基因rs74723057、rs12120556五個多態(tài)性位點基因多態(tài)性與肝細胞肝癌臨床特征無統(tǒng)計學關聯(lián),miRNA-122基因rs1135519位點基因多態(tài)性可影響肝癌根治術后總體生存時間,而miRNA-199基因rs74723057位點多態(tài)性則與肝癌根治術后無復發(fā)生存時間相關。
[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.
【學位授予單位】：廣西醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R735.7

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