【作者】 张东红；

【导师】 崔巍；

【作者基本信息】 北京协和医学院 ， 临床检验诊断学， 2014， 博士

【摘要】 研究背景：食管癌是世界范围内常见的,且具有明显地区聚集性的恶性肿瘤。在我国,食管癌预后较差,其死亡率一直居高不下,现位居所有肿瘤死亡原因的第四位,同样具有地域分布特征。广东汕头地区是我国唯一沿海食管癌高发区。我们前期研究表明该地区食管癌患者癌组织中人乳头瘤病毒(Human papillomavirus, HPV)感染率在50-80%之间,并且以高危型HPV感染为主,但HPV感染对食管癌的致病机理和预后状况尚不清楚。端粒和端粒酶是染色体末端一种维持染色体完整和稳定的特殊结构,能防止染色体DNA降解、末端融合缺失和非正常重组。研究表明,端粒长度的进行性缩短和端粒酶的激活在肿瘤细胞的恶性转化和进展中起着重要作用。而端粒长度和端粒酶的表达均与端粒酶相关基因或亚端粒的DNA甲基化调控有关。因此,从端粒酶的表观遗传学修饰的角度探讨HPV感染与食管癌的相关性有助于食管癌病因学和食管癌患者预后的研究。研究目的与假说：目的：从端粒酶相关基因或亚端粒的DNA甲基化的角度分析HPV和端粒长度的相关性,以及对食管癌预后的影响,以期能为食管癌的病因学研究和预后分析提供一定的理论依据。假说：食管癌细胞中高危型HPV持续感染→HPV DNA断裂,E6/7癌基因整合入宿主基因组DNA的“脆性部位”→HPVE6/7癌蛋白稳定持续的表达→端粒酶相关基因(hTERT和TRF2)或亚端粒的DNA甲基化改变→食管癌中端粒长度的变化→增加癌细胞的恶性转化和永生化→影响食管癌患者预后。研究方法：通过构建分别含有HPV-16,-18和-58型E6/7和E2基因片段的质粒DNA,建立稳定且灵敏的绝对实时定量PCR技术(quantitative real time PCR, qPCR),对70例食管癌患者中的癌组织及其配对的癌旁正常食管组织,以及60例正常人的食管粘膜组织中的高危型HPV-16,-18和-58型感染的病毒含量(E6/7基因拷贝数)和整合量(E2基因和E6/7基因拷贝数的比值)进行定量检测；采用免疫组化(immunohistochemistry, IHC)技术检测食管癌中HPV16E6蛋白的表达情况；以HEK293细胞DNA为标准品,采用实时定量PCR技术建立端粒长度的定量检测法方法,以检测食管癌和正常食管组织中的端粒长度；采用甲基化特异性PCR (methylation-specific PCR, MSP)技术检测食管癌中端粒酶相关基因人端粒酶催化亚单位(human reverse-transcriptase telomerase,端粒重复序列结合因子(Telomeric repeat-binding factor2, TRF2)和亚端粒DNA甲基化。采用SPSS16.0软件统计分析高危型HPV感染,端粒长度与DNA甲基化水平的三者之间相关性,以及对食管癌预后的影响。研究结果：(1)对构建的系列浓度HPV E6/7和E2基因标准品——含有HPV-16E6/7、HPV-16E2, HPV-18E6/7、HPV-18E2、HPV-58E6/7和HPV58E2基因片段质粒DNA,进行定量PCR扩增,其特异性较好。六种基因标准曲线的R2分别为0.9945、0.991、0.995、0.9976、0.9879和0.999,经相关性检验发现六个标准曲线均具有显著的相关性(P<0.05)。同时,E6/7和E2基因分别在三种型别HPV的扩增效率相一致。(2)食管癌组织中HPV-16,-18和-58病毒含量变异均较大,但三者之间无显著性差别。在食管癌组织中,三种型别HPV合并感染率和感染量均显著性高于正常食管组织(50.00%vs.33.33%, P=0.045;2.55±3.19vs.0.55±0.57copies/cell,P=0.021)。同时,HPV DNA的混合状态或整合状态在食管癌和正常食管中均较高(91.43%vs.85.00%),但在食管癌中的整合状态比正常食管组织中显著性增高(F=17.832,P=0.001)。高危型HPV DNA的整合量与肿瘤分级呈正相关(r=3.753,P=0.033),但与年龄、性别、吸烟、饮酒、家族史和淋巴结转移无关。HPV病毒含量与临床资料均无显著相关性。(3)免疫组化检测发现HPV16主要定位于食管癌细胞浆,HPV16蛋白在食管癌组织中的阳性率为59.26%,并随着肿瘤分级的增加而增加(r=0.301,P=0.027)。(4)从正常食管,到食管癌旁正常组织再到食管癌中,端粒长度依次缩短[4.77(4.28-5.33,),4.37(3.89-5.09)和4.27(3.79-4.57)kb](P趋势性检验<0.01)。(5)HPV或高危型HPV仅能延长食管癌组织中的端粒长度,而不影响正常食管和癌旁正常组织中的端粒长度。同时在食管癌组织中,端粒长度分别和高危型HPV基因的感染量和整合量呈正相关(r=0.410, P=0.037; r=0.492, P=0.01)。(6)食管癌组织中的端粒长度和癌/癌旁端粒长度比值分别在高危型HPV感染患者、食管癌细胞中HPV含量大于1的患者和HPV整合状态的患者中较长/大。同时,癌/癌旁端粒长度比值在吸烟患者、肿瘤分级为Ⅱ和Ⅲ的患者以及5年内死亡的患者中较大。(7)甲基化特异性PCR检测发现,食管癌中hTERT,7q,9q和XqYq均呈高甲基化状态,而TRF2、17q、18q和21q则呈低甲基化状态。7q基因甲基化水平分别与9q、18q呈正相关。而TRF2基因甲基化水平分别与hTERT、21q呈负相关。(8)相对与HPV阴性的食管癌患者,高危型HPV感染的患者中hTERT、18q和21q基因的甲基化水平较高,而TRF2较低。高危型HPV的整合量与hTERT甲基化水平呈正相关,与TRF2呈正相关。HPV病毒量和hTET、TRF2、7q、9q、17q、18q、21q、和XqYq基因甲基化水平无相关性。(9)相对于食管癌组织中端粒相对长度<0.7的患者,端粒相对长度≥0.7的患者中,hTERT和21q基因的甲基化水平增高,而TRF2则降低。食管癌组织中端粒长度分别与hTERT (r=0.318, P=0.007)和21q (r=0.297, P=0.013)基因甲基化水平呈正相关。(10)采用Kaplan-Meier分析和单因素Cox回归模型分析显示：肿瘤分级为Ⅲ级的食管癌患者,高危型HPV感染的患者以及癌/癌旁端粒长度比值≥0.8的患者预后较差,其生存的相对危险比(95%置信区间)值分别是对照组的3.13(1.26-7.50)、1.93(1.04-3.57)和2.30(1.19-4.46)。而食管癌患者的预后与年龄、性别、吸烟、家族史、淋巴结转移和HPV感染无关。采用多因素Cox回归模型分析显示：只有癌/癌旁端粒长度比值和高危型HPV感染分别是食管癌预后的独立指标,癌/癌旁端粒长度比值越大或高危型HPV感染的食管癌预后越差。研究小结：(1)采用实时定量PCR技术,我们构建了稳定且灵敏的HPV16,-18和-58型病毒绝对含量和整合量,以及相对端粒长度的检测方法；(2)食管癌患者中高危型HPV感染的拷贝数较低,但HPV DNA的高整合率可维持其蛋白的高表达状态；(3)端粒长度在食管癌变过程中进行性缩短,高危型HPV感染可逆转食管癌中端粒的缩短,使端粒长度维持在一定的长度；(4)高危型HPV感染维持端粒长度的过程与hTERT、TRF2和亚端粒21q的DNA甲基化水平有关；(5)高危型HPV感染、食管癌/癌旁端粒长度的比值分别是影响食管癌患者不良预后的独立危险因素。研究结论：食管癌患者肿瘤细胞中高危型HPV DNA的高感染率和整合率使其E6/7癌蛋白持续性高表达,进而可能通过端粒酶相关基因hTERT和TRF2的DNA甲基化水平的改变,影响了端粒酶活性,逆转食管癌中端粒的进行性缩短,增加细胞的恶性转化和永生化,与食管癌患者的不良预后密切相关。更多还原

【Abstract】 Background:Esophageal squamous cell carcinoma (ESCC) is a common malignancy with high mortality and ranks as the fourth most frequent cause of cancer death in China as well as in worldwide. The Shantou of eastern Guangdong province, in southern China, is only one of coastal area with the high incidence of esophageal carcinoma (EC). The presence of (Human papillomavirus, HPV), mainly for high risk of HPV (HR-HPV), in cases of ESCC has been reported repeatedly about50-80%from Shantou, China, and other regions with a high incidence of esophageal carcinoma (EC). Thus, the role of high-risk HPV types in the carcinogenesis and prognosis of ESCC remains uncertain.Telomeres and Telomerase, as the special structure at the ends of chromosomes, could maintain the stability, and prevent the degradation, abnormal terminal fusion or recombinant of chromosomal DNA. Telomere shortening and activation, which related with DNA methylation of telomere and subtelomere, is crucial in carcinogenesis and tumor progression. It’s helpful for etiology study of EC to research the relationship between HPV infection and epigenetic mechanism.Aim and Hypothesis:Here, we fully explored the association among HR-HPV infection load and integration, telomere length (TL) and DNA methylation of telomerase related gene and submelomere, as well as their significance in the prognosis of ESCC, so as to provide some supplements to the etiology study of EC.Our hypothesis as following:HR-HPV sustain infection in ESCC→HPV DNA breakage and its E6/7oncogene integration at the "fragile site" of the host chromosomes→The sustain and continuous expression of HPVE6/7oncoprotein→The changes of telomerase related gene (hTERT and TRF2) and subtelomere DNA methylation level→Effect of telomere length→Promotion of the malignant transformation and immortalization→Affect the prognosis of ESCC patients. Method:We established a stable and sensitive methodology of real time PCR to assess the prevalence, viral load and physical status of HR-HPV-16,-18and-58, infection the3most common HR-HPVs, as well as telomere length (LT) in patients with70cases of ESCC tumor (T) tissues and paired of paired nontumor (NT) tissues and50cases of normal esophagus (NE). DNA methylation and HPV-16protein expression were carried out by methylation-specific PCR (MSP) and using immunohistochemistry (IHC) assay, respectively. The prognostic value of TL and DNA methylation in ESCC was analyzed using SPSS16.0soft ware (SPSS Inc., Chicago, IL).Results:(1) All typical amplification plots involved HPV-16,-18and-58types for E2and E6/7plasmid DNA (change in the fluorescent signal versus cycle number). Six of standard curve showed a dynamic linear range for quantification across copy number over the entire range of dilution concentrations on bivariate correlation analysis (P<0.05).(2) Widespread, but no difference of HR-HPV-16,-18and-58vial loads were found. The combined infection rates (50.00%vs.33.33%, P=0.045) and viral load (2.55±3.19vs.0.55±0.57copies/cell, P=0.029) for all HR-HPVs in ESCC patients was significantly higher than that of their corresponding controls. HPV DNA integration and mixed forms were more frequently observed in ESCC patients and in controls (91.43%vs.85.00%). The frequency of HR-HPV integration was higher than that for ESCC controls (F=17.832, P=0.001). The integration level was associated with pathological grade of ESCC (r=3.753, P=0.033). However, no significant associations were found between HPV physical status and patient age or lymph-node metastasis. HPV viral loads were not associated with ESCC patient age, lymph-node metastasis or cancer grade.(3) IHC assay indicated that HPV-16protein expression, located mainly in the cytoplasm of cancer cells, was detected in59.26%of ESCC tissues and was positively correlated with pathological grade of cancer (r=0.301, P=0.027) in ESCC patients.(4) TL decreased from normal esophagus mucosa, adjacent NT tissue to T tissue [4.77(4.28-5.33),4.37(3.89-5.09) and4.27(3.79-4.57) kb].(5) T-TL was longer in ESCC samples positive for HPV (represented as low-risk HPV and HR-HPV) or HR-HPV. T-TL was positively correlated with viral load (r=0.410, P=0.037) and integration (r=0.492, P=0.01).(6) Relatively long T-TL and high T/NT-TL ratio were frequently found with high viral load and integrated status. T/NT-TL was higher in ESCC patients with smoking, HPV infection or tumor grade III. T/NT-TL ratio was higher for surviving than non-surviving patients.(7) Using MSP method, we found a hyper-methylation ratio (≥60%) for hTERT,7q,9q and XqYq but a hypo-methylation ratio (<40%) for TRF2,17q, and21q. we also found the concordant methylation level of7q and9q (r=0.304, P=0.011) and18q (r=0.311, P=0.009). The methylation level of hTERT and21q was negatively correlated with that of TRF2(r=-0.281, P=0.025, and r=-0.251, P=0.036, respectively).(8) HR-HPV positivity increased the levels of hTERT,18q and21q methylation and decreased that of TFR2methylation. Furthermore, HR-HPV integration was positively correlated with hTERT methylation (r=0.413, P=0.036) and positively with TRF2methylation (r=0.453, P=0.02). However, HR-HPV viral load did not affect the ratio of hTERT, TRF2and subtelomere DNA methylation.(9) High hTERT and21q methylation was more prevalent with long than short T-TL and was positively correlated with T-TL.(10) Kaplan-Meier analysis and Univariate Cox regression analysis revealed significantly worse outcomes for patients with cancer grade Ⅲ (PHR=0.013), HR-HPV infection (PHR=0.037) and high T/NT-TL ratio (PHR=0.014). Multivariate survival analysis with a Cox proportional-hazards model revealed HR-HPV infection and high T/NT-TL ratio as the poor prognostic factors of survival, independent of age, gender, smoking, alcohol drinking, lymph node metastasis and cancer grade. Summary:(1) We established a stable and sensitive methodology of real time PCR to assess the viral load and physical status of HR-HPV-16,-18and-58DNA, as well as the relative telomere length.(2) A relatively low HR-HPV copy number infection in ESCC is unlikely to play an essential a role in the carcinogenesis of ESCC. However, the mainly presentation of the physical status could sustain the high expression of the HR-HPV types in ESCC, which strongly suggests a possible role of oncogenic HPV infection in carcinogenesis of ESCC.(3) TL gradually decreased in the process of esophageal carcinogenesis. HR-HPV infection could reverse the shortening of LT and maintain the TL in a critical length.(4) DNA methylation levels of hTERT, TRF2and21q were participated in the maintenance of LT by HR-HPV infection.(5) HR-HPV infection and high T/NT-TL ratio was the dependent of prognostic factors for the poor survival of ESCC.Conclusion:HR-HPV integration and expression related to telomere elongation and DNA methylation of hTERT and TRF2, may be a potential biomarker of prognosis in ESCC.更多还原