HPV16型E6、E7变异与宫颈癌发生发展的研究进展

宫颈癌是全球15 ～44岁女性中第二常见的恶性肿瘤,每年的死亡人数约为265 653人,在中国,宫颈癌的发生率及死亡率仍较高.高危型人乳头状瘤病毒(HPV)持续感染是宫颈癌前病变及宫颈癌发生的必要条件,HPV16是最常见的高危人乳头瘤病毒.HPV16编码的E6和E7蛋白在HPV相关的肿瘤中起关键作用.近年来的研究揭示了HPV16 E6、E7基因的变异引起氨基酸变化可影响E6、E7蛋白与p53、pRb 的结合,进而与宿主细胞恶性转化相关.本文将对近年来HPV16 E6、E7变异在宫颈癌发生发展中的作用作一综述.     

宫颈癌组织中人乳头瘤病毒HPV16型基因组同源序列的检测

本文报道应用Southern blot核酸分子杂交技术,以标记的国内HPV16型基因组分子为探针,共检测22例宫颈癌、4例宫颈不典型增生、6例宫颈慢性炎症,5例宫颈内膜息肉,6例正常宫颈上皮组织中的HPVl6型基因组同源序列。杂交结果表明,宫颈癌组织中HPV16型基因组同源序列阳性率为36.4％(相关序列阳性率为59.1％),非癌组织和正常宫颈上皮组织均为阴性,P<0.05具有显著性差异。同时还观察到宫颈癌病理组织学特征和组织细胞体外培养CPE与HPV16型基因组同源序列检出率呈正相关,P<0.001。更进一步证明宫颈癌发病与HPV感染密切相关。并提出HPV感染的初筛检测方法。     

宫颈癌及其癌前病变组织中HPV16 E6、HPV16 E7蛋白的表达及其意义

目的 探讨宫颈癌及其癌前病变组织中HPV16E6、E7蛋白的表达及其意义。方法 采用免疫组化SP法对15例正常宫颈、25例宫颈上皮内瘤变（C1N）以及61例浸润性宫颈癌组织进行了HPVl6E6、HPVl6E7蛋白表达的检测。结果在正常宫颈、CINI～Ⅱ、CINⅢ及浸润性宫颈癌中，HPVl6E6蛋白的阳性表达率分别为0（0／15）、7．14％（1／14）、36．36％（4／11）、59．02％（36／61）；CINⅢ和浸润性宫颈癌中HPVl6E6蛋白阳性表达率明显高于正常宫颈组织和CINI～Ⅱ（P〈0．05）；在高、中、低分化宫颈癌中，HPVl6E6蛋白阳性表达率分别为45．45％（5／11）、77．78％（14／18）、53．13％（17／32）；HPVl6E6蛋白在不同分化程度的宫颈癌组织中的阳性表达率有显著性差异（P〈0．05），但HPVl6E6蛋白表达与官颈癌组织分化程度无明显相关性（ys=0．123），HPVl6E6蛋白阳性表达率与宫颈癌临床分期和淋巴结转移无关（P〉0．05）。HPVl6E7蛋白在正常宫颈上皮、CINI-Ⅱ、CIN Ⅲ及浸润性富颈癌组织中的阳性表达率分别为20．00％（3／15）、42．86％（6／14）、63．64％（7／11）、57．38％（35／61），HPVl6E7蛋白在不同宫颈组织中的阳性表达率无明显差异（P=0．05）；HPVl6E7蛋白的表达与富颈癌临床分期、淋巴结转移和组织分化均无关（P〉0．05）。结论 HPVl6E6蛋白的检测有可能作为宫颈癌前病变转归的指标。     

宫颈癌HPV16 型L1基因的克隆和序列分析

目的 获取人乳头瘤病毒16(HPV16)晚期表达基因L1，为HPV感染的检测和诊断奠定基础。方法 收集宫颈癌组织标本，设计一对L1基因特异引物，用PCR方法从宫颈癌组织中获得L1基因片段，构建人克隆载体pGEM—T，双向测定插入片段序列并进行序列分析。结果 这一株HPV16型L1蛋白序列，与既往报道的L1序列高度同源。结论 构建的重组质粒为进一步研究L1蛋白的表达及其免疫学创造了条件。     

宫颈癌细胞系中HPV16 E6、E7及E6/E7基因对STK31基因甲基化状态及表达的影响

背景与目的：丝氨酸/苏氨酸蛋白激酶31(serine/threonine kinases 31,STK31)基因在人类多种癌症中扮演重要角色,且STK31基因的表达受其启动子及第一外显子区甲基化状态的影响;病毒感染与肿瘤组织中某些抑癌基因启动子区高甲基化有关。本研究旨在探讨宫颈癌细胞系中HPV16 E6、E7及E6/E7癌基因对STK31基因甲基化状态及表达的影响,以及不同种类甲基转移酶(DNA methyltransferases,DNMTs)基因在STK31基因甲基化中的潜在作用。方法：构建外源性HPV16 E6、E7以及E6/E7基因共表达慢病毒,分别感染人乳头瘤病毒(human papillomavirus,HPV)阴性宫颈癌细胞系HT-3及C33A,获得稳定转染的细胞系;采用亚硫酸氢盐基因组测序法(bisulfite genomic sequencing,BGS)和甲基化特异性PCR (methylation-specific PCR,MSP)检测3种HPV阳性宫颈癌细胞系HeLa、SiHa和CasKi以及HPV阴性宫颈癌细胞系HT-3和C33A转染前后STK31基因的甲基化状态;RT-PCR及蛋白［质］印迹法(Western blot)检测上述宫颈癌细胞系中STK31基因的表达以及DNMT1、DNMT2、DNMT3a、DNMT3b和DNMT3L基因在HPV16转染前后宫颈癌细胞系及HPV阳性、HPV阴性宫颈癌组织中的表达情况。结果：外源性HPV16 E6、E7以及E6/E7基因可在HPV阴性宫颈癌细胞系中稳定表达。3种HPV阳性细胞系HeLa、SiHa和CasKi中STK31基因呈低甲基化状态,STK31 mRNA及蛋白质表达阳性;2种HPV阴性细胞系HT-3、C33A中STK31基因则表现为高甲基化状态,STK31 mRNA及蛋白质表达缺失;与未感染慢病毒HT-3和C33A细胞系比较,外源性HPV16 E7以及E6/E7表达的HT-3和C33A细胞系STK31基因甲基化程度降低,其mRNA及蛋白质重新表达。DNMT1、DNMT3a和DNMT3b基因在HT-3E6/E7和C33AE6/E7细胞系中mRNA水平分别高于HT-3空载细胞系和C33A空载细胞系,差异有统计学意义(P<0.001)。DNMT1、DNMT3a和DNMT3b基因的mRNA水平在HPV16阳性宫颈癌组织中的表达高于其在HPV阴性宫颈癌组织中的表达,差异有统计学意义(t=5.997,P<0.001;t=6.743,P<0.001;t=7.926,P<0.001)。DNMT2在HT-3E6/E7和C33AE6/E7细胞系中mRNA表达水平分别低于HT-3空载细胞系和C33A空载细胞系,差异有统计学意义(t=7.451,P<0.001;t=2.451,P<0.05);DNMT2基因转录水平在HPV16阳性宫颈癌组织中低于HPV阴性宫颈癌组织(t=9.134,P<0.001)。DNMT3LmRNA表达水平在宫颈癌细胞系转染前后及HPV阴阳性宫颈癌组织中的差异无统计学意义(P>0.05)。结论：HPV感染可导致STK31基因启动子及第1外显子区甲基化状态降低,低甲基化状态促进该基因表达。STK31基因的表达受其启动子及第1外显子区甲基化状态的调控。HPV16 E7、E6/E7基因可能通过影响DNMT2的表达参与调控癌基因STK31基因启动子及第1外显子区甲基化状态。     

p53和HPV—16、18E6蛋白在宫颈癌的表达

目的探讨宫颈癌中抑癌基因p53蛋白与HPV-16、18癌基因E6蛋白的表达及其意义.方法采用SP免疫组织化学方法,检测68例宫颈癌和8例慢性宫颈炎上皮组织中p53和HPV-16、18E6蛋白的表达.结果68例宫颈癌中有17例p53蛋白和60例HPV-16、18E6蛋白呈阳性表达,阳性率分别为25.0%和88.2%.p53蛋白阳性表达与组织学类型有关,其阳性率在宫颈腺癌中为83.3%和宫颈鳞癌中为17.2%,有极显著性差异(P＜0.01);p53蛋白表达也与临床分期相关,该蛋白阳性表达率随着临床分期上升而增高.p53表达同肿瘤分化程度,淋巴结转移无关(P＞0.05).HPV-16、18E6蛋白在宫颈癌组织中的表达同肿瘤分化程度、淋巴结转移、组织学类型和临床分期无关(P＞0.05).在慢性宫颈炎上皮组织中均无p53和HPV-16、18E6蛋白表达.结论p53蛋白阴性和弱阳性表达以及HPV-16、18E6蛋白过度表达与宫颈癌发生有关,但在宫颈腺癌和临床分期升高的宫颈癌组织中,p53蛋白阳性表达的增强可能是p53基因突变的结果,或者与其他细胞蛋白结合,阻止了HPV-16、18E6蛋白对p53蛋白的降解作用,使p53蛋白稳定性增加和阳性表达水平提高.     

HPV16-E6-DNA病毒载量对预测宫颈癌患者生存状况的影响

目的：探讨宫颈癌组织中人乳头瘤病毒16型致癌基因E6（HPV16-E6-DNA）的病毒载量对预测宫颈癌患者生存状况的影响。方法选取126例宫颈癌患者为研究对象,提取宫颈癌组织DNA,利用荧光定量PCR技术检测宫颈癌组织中HPV16-E6-DNA病毒载量,根据检测的HPV16-E6-DNA载量将患者分为低载量组（﹤106 copy/ng）与高载量组（≥106 copy/ng）。采用Kaplan-Meier法计算两组患者的生存率,采用Cox回归对影响患者5年生存率的因素进行分析。结果比较两组患者间一般资料特征,年龄、FIGO分期及淋巴结转移情况,差异有统计学意义（P﹤0.05）。Kaplan-Meier生存率分析显示,低载量组患者1年、3年及5年生存率分别为95.67%、89.32%和74.29%,高载量组患者1年、3年及5年生存率分别为89.09%、62.60%和51.37%,低载量组高于高载量组,两组差异均有统计学意义（P﹤0.01）。Cox多因素回归分析结果显示,HPV16-E6-DNA病毒载量及淋巴结转移是影响宫颈癌患者预后的主要因素。结论 HPV16-E6-DNA病毒载量与宫颈癌患者的远期预后密切相关,可作为术后宫颈癌患者预后的预测因子。     

宫颈癌组织中人乳头瘤病毒16E6基因突变分析

背景与目的：高危型人乳头瘤病毒（humanpa pillomavirus,HPV）以16型最为常见,HPV16E6是宫颈癌主要的致癌基因之一,特定的E6突变是宫颈癌发生的主要因素之一。本研究主要观察兰州地区宫颈癌组织中是否存在E6突变,并探讨了突变与宫颈癌发生的关系。方法：以23例宫颈癌手术切除标本及5例正常宫颈组织的DNA作为模板,PCR扩增HPV-16E6基因201-523位,PCR产物直接测序．分析其HPV16E6基因的突变规律。结果：PCR扩增结果表明5例正常宫颈组织中HPV16E6阳性率为0％（0／5）,宫颈癌组织中HPV16E6阳性率为82．61％（19／23）,对18例样本PCR产物的测序和序列分析结果表明,6例（33．33％）E6基因与原型相同,12例（66．67％）E6基因发生突变,其中11例（61．11％）发生了350G突变。同时,在1例样本（5．56％）发现了249G突变。结论：兰州地区宫颈癌组织中存在着非常高的HPV感染率,且多数HP116E6发生了突变。     

新疆维吾尔族妇女宫颈癌组织中HPV16型E6基因突变分析

背景与目的：高危型人乳头状瘤病毒16和18(human papillomavirus type16 and 18,HPV16,HPV18)是宫颈癌主要病因之一,尤其以HPV16最为常见,其中HPV16E6是主要癌基因之一。在一些地区,特定的E6基因突变株是宫颈癌发生的危险因素。新疆南部维吾尔族聚居区足宫颈癌高发区,我们已在前期的研究中发现该地区HPV16E6基因发生突变。本研究旨在检测该突变在新疆南部维吾尔族妇女宫颈癌组织中的分布规律,并探讨其与该地区宫颈癌高发的关系。方法：从35例中国新疆南部维吾尔族妇女宫颈癌活检标本中提取组织DNA作为模板,PCR扩增HPV16E6全长基因,PCR产物直接测序或克隆后测序,分析新疆维吾尔族妇女宫颈癌组织中HPV16E6基因的突变。结果：PCR检测结果表明宫颈癌组织中HPV16E6阳性率为82．86％(29／35);26例中E6分离片段的测序和序列分析表明,15例(57．69％)分离株E6基因与原型相同,另有11例(42．31％)E6基因突变,其中9例(34．62％)分离株发生了L83V突变,2例(7．69％)分离株发生rL83V／D63E突变。结论：中国新疆南部地区HPV16E6基囚发生变异,其原型和变异型在该地区维吾尔族宫颈癌患者巾的分布规律可能与该地区宫颈癌高发存在一定关系。     

特异性siRNA抑制宫颈癌细胞CaSKi中HPV16 E6基因的表达

目的：研究RNAi技术抑制宫颈癌细胞CaSKi中HPV16 E6癌基因的表达以及对细胞生物学特性的影响。方法：构建真核细胞表达特异性shRNA的pGENESIL-1/E6（PS6）重组质粒,脂质体转染细胞后用半定量RT-PCR技术检测CaSKi细胞中E6mRNA水平的变化,然后用western blotting检测RNA干扰前后p53和p21蛋白的表达来验证RNAi效应。用透射电子显微镜观察CaSKi/PS6细胞形态变化;应用流式细胞仪检测细胞的凋亡和细胞周期的变化,最后利用细胞计数和裸鼠成瘤实验测定CaSKi/PS6细胞增殖能力。结果：成功构建了真核细胞表达特异性siRNA的PS6重组质粒;在RNA干扰24、48和72小时后CaSKi/PS6细胞中E6mRNA分别下降了21.50%、52.60%和65.60%;CaSKi/PS6细胞出现凋亡的特征性改变;RNA干扰24h、48h和72h后CaSKi/PS6细胞的凋亡率分别为27.94%、31.95%和56.63%,细胞生长周期停滞于S期。结论：应用RNA干扰技术能有效地、特异地抑制宫颈癌细胞中HPV16 E6基因的表达,为RNA干扰应用于研究宫颈癌发病分子机制、临床治疗和干预性预防提供了新的方法。     

Identification of novel immunogenic human leukocyte antigen-A 2402-binding epitopes of human papillomavirus type 16 E7 for immunotherapy against human cervical cancer

BACKGROUND:

A study was undertaken to identify new immunogenic human leukocyte antigen (HLA)‐A*2402‐restricted epitopes from human papillomavirus (HPV) type 16 E7 protein for immunotherapy against cervical cancer.

METHODS:

Synthetic overlapping peptides were screened by measuring the frequency of CD8⁺ cytotoxic T lymphocytes (CTLs) producing intracellular interferon‐γ (IFN‐γ) using flow cytometry and were validated in SiHa cells with a Cr release cytotoxicity assay. In vivo antitumor effects of peptide‐sensitized peripheral blood mononuclear cells (PBMCs) and isolated CD8⁺ CTLs were evaluated using BALB/c nude mice with SiHa cell xenotransplants.

RESULTS:

Among 14 overlapping 15‐amino acid peptides, E7_61‐75(CDSTLRLCVQSTHVD) and E7_67‐81(LCVQSTHVDIRTLED) induced significantly higher IFN‐γ production (P < .05) and showed higher in vitro cytotoxicity against SiHa cells than did cells sensitized with the negative control. To determine the exact HLA‐A*2402‐restricted epitopes, a total of 25 overlapping 9‐ or 10‐amino acid peptides spanning E7_61‐75 and E7_67‐81 were synthesized. E7_61‐69(CDSTLRLCV) and E7_67‐76(LCVQSTHVDI) induced significantly greater IFN‐γ production as well as increased in vitro cytotoxicity against SiHa cells compared with those of other peptides and the negative control (P < .01), and the antitumor effects of these peptide‐sensitized PBMCs were induced by CD8⁺ CTLs. E7_61‐69‐sensitized and E7_67‐76‐sensitized PBMCs and isolated CD8⁺ CTLs showed a much greater suppression of tumor growth in vivo compared with that of control groups treated with PBS (P < .01). The authors also confirmed the synergistic antitumor effect of cisplatin followed by E7_67‐76‐sensitized PBMCs in vivo.

CONCLUSIONS:

E7_61‐69 and E7_67‐76 were identified as novel HPV type 16 E7 epitopes for HLA‐A*2402, which could be used for immunotherapy against cervical cancer. Cancer 2012. © 2011 American Cancer Society.     

Immortalization of normal human embryonic fibroblasts by introduction of either the human papillomavirus type 16 E6 or E7 gene alone

The ability of the human papillomavirus type 16 (HPV-16) E6 or E7 gene to induce immortalization of normal human embryonic fibroblast WHE-7 cells was examined. WHE-7 cells at 9 population doublings (PD) were infected with retrovirus vectors encoding either HPV-16 E6 or E7 alone or both E6 and E7 (E6/E7). One of 4 isolated clones carrying E6 alone became immortal and is currently at >445 PD. Four of 4 isolated clones carrying E7 alone escaped from crisis and are currently at >330 PD. Three of 5 isolated clones carrying E6/E7 were also immortalized and are currently at >268 PD. The immortal clone carrying E6 only and 2 of the 3 immortal clones carrying E6/E7 expressed a high level of E6 protein, and all the immortal clones carrying E7 alone and the other immortal clone carrying E6/E7 expressed a high level of E7 protein when compared to their mortal or precrisis clones. The immortal clones expressing a high level of E6 or E7 protein were positive for telomerase activity or an alternative mechanism of telomere maintenance, respectively, known as ALT (alternative lengthening of telomeres). All the mortal or precrisis clones were negative for both phenotypes. All the immortal clones exhibited abrogation of G1 arrest after DNA damage by X-ray irradiation. The expression of INK4a protein (p16(INK4a)) was undetectable in the E6-infected mortal and immortal clones, whereas Rb protein (pRb) was hyperphosphorylated only in the immortal clone. The p16(INK4a) protein was overexpressed in all the E7-infected immortal clones and their clones in the pre-crisis period as well as all the E6/E7-infected mortal and immortal clones, but the pRb expression was downregulated in all of these clones. These results demonstrate for the first time to our knowledge that HPV-16 E6 or E7 alone can induce immortalization of normal human embryonic fibroblasts. Inactivation of p16(INK4a)/pRb pathways in combination with activation of a telomere maintenance mechanism is suggested to be necessary for immortalization of normal human embryonic fibroblasts by these viral oncogenes. The susceptibility of human cells to immortalization may be related to the state of differentiation of the cells.     

The role of human papillomavirus type 16 and the fragile histidine triad gene in the outcome of cervical neoplastic lesions

The presence of high-risk human papillomavirus, loss of heterozygosity on chromosome 3p and fragile histidine triad gene expression were assessed as potential markers of cancer and CIN progression in 83 cervical cancers and 74 cervical intraepithelial neoplasia grade 1 lesions. Human papillomavirus type 16 was an indicator of vascular involvement in cancers. Loss of heterozygosity, especially in the fragile histidine triad gene intron 5, was an indicator of high-grade tumours, greater tumour depth and lymph node involvement. Abnormal fragile histidine triad gene expression was more frequent in cervical intraepithelial neoplasia grade 1 lesions with increased risk of disease progression.     

AIM2在宫颈癌组织中的表达及其与HPV16病毒载量的相关性分析

目的:研究AIM2与HPV16病毒载量在宫颈癌及其癌前病变组织中的关系。方法:通过免疫组化检测正常宫颈组织(50例)、LSIL(56例)、HSIL(108例)及宫颈癌组织(145例)中AIM2表达,采用实时荧光定量PCR法(硕世21 HPV 分型定量检测系统,BMRT)检测所有纳入患者HPV16型DNA病毒载量,分析AIM2蛋白表达水平、HPV16病毒载量在宫颈癌及其癌前病变中有无差异以及AIM2蛋白表达水平与HPV16病毒载量的相关性。结果:AIM2在正常宫颈组、LSIL组、HSIL组、宫颈癌组中的阳性表达率分别为12.0%（6/50）、26.8%（15/56）、82.4%（89/108）、84.1%（122/145）,呈逐渐增高趋势,差异有统计学意义(χ2=207.675,P＜0.001)。HPV16在正常宫颈组、LSIL组、HSIL组、宫颈癌组中的阳性表达率分别为16.0%（8/50）、30.4%（17/56）、44.4%（48/108）、47.6%（69/145）,呈逐渐增高趋势,差异有统计学意义(χ2=18.567,P＜0.001)。Spearman相关性分析可得:HPV16病毒载量与宫颈组织病变程度之间呈正相关（r=0.229,P＜0.001）,AIM2表达水平与HPV16病毒载量也呈正相关（r=0.467,P＜0.001）。结论:宫颈病变程度与AIM2蛋白表达水平及HPV16病毒载量密切相关,AIM2蛋白表达水平及HPV16病毒载量随病变程度增加而增加。     

食管鳞癌中人乳头瘤病毒16型E6、E7和细胞特异性增强子片段的检测

目的:检测食管鳞癌组织中人乳头瘤病毒(HPV)16 E6、E7基因和细胞特异性增强子片段(CTSE).方法:采用聚合酶链反应法(PCR)检测40例食管鳞癌和20例正常食管黏膜中HPV16 E6、E7基因和病毒长控制区内(long control region,LCR)的细胞型特异性增强子(cell type specific enhancer,CTSE).结果:在40例食管鳞癌中,HPV16 的E6、E7基因和CTSE片段的检出率分别是37.5%(15/40)、42.5%(17/40)和40%(16/40),20例正常食管黏膜中E6、E7和CTSE的检出率分别为0%(0/20)、0%(0/20)和5%(1/20),两者均存在显著性差异(P<0.05).CTSE片段分别与E6和E7 基因有明显相关性(P<0.05).食管鳞癌中E6 、E7基因及CTSE的检出率在患者不同性别、年龄、肿瘤浸润程度、淋巴结转移和组织学分级肿瘤中差异均无显著性(P>0.05). 结论:E6和E7基因与CTSE 片段共存于HPV16感染的食管鳞癌组织中,三者可能与食管鳞癌的发生和发展有关.     

Methylation of viral and host genes and severity of cervical lesions associated with human papillomavirus type 16

Methylation of human papillomavirus (HPV) and host genes may predict cervical cancer risk. We examined the methylation status of selected sites in HPV16 and human genes in DNA extracted from exfoliated cervical cell samples of 244 women harboring HPV16‐positive cancer or cervical intraepithelial neoplasia (CIN) or negative for intraepithelial lesions or malignancy (NILM). We quantified the methylation of CpG sites in the HPV16 L1 gene (CpG 6367 and 6389) and in the human genes EPB41L3 (CpG 438, 427, 425) and LMX1 (CpG 260, 262, 266, 274) following bisulfite treatment and pyrosequencing. Receiver operating characteristic (ROC) curves were used to analyze the diagnostic utility of methylation level for the different sites and for a joint predictor score. Methylation in all sites significantly increased with lesion severity (p < 0.0001). Area under the curve (AUC) was highest among the CIN2/3 vs. cancer ranging from 0.786 to 0.853 among the different sites. Site‐specific methylation levels strongly discriminated CIN2/3 from NILM/CIN1 and cancer from CIN2/3 (range of odds ratios [OR]: 3.69–12.76, range of lower 95% confidence bounds: 1.03–4.01). When methylation levels were mutually adjusted for each other EPB41L3 was the only independent predictor of CIN2/3 vs. NILM/CIN1 contrasts (OR = 9.94, 95%CI: 2.46–40.27). High methylation levels of viral and host genes are common among precancerous and cancer lesions and can serve as independent risk biomarkers. Methylation of host genes LMX1 and EPB41L3 and of the viral HPV16 L1 sites has the potential to distinguish among precancerous lesions and to distinguish the latter from invasive disease.     

胃癌组织中HPV16与幽门螺杆菌感染相关性研究

目的:研究胃癌组织中人类乳头瘤病毒16型(HPV 16)感染与幽门螺杆菌感染之间的相关性.方法:运用原位PCR和免疫组化技术分别检测陕西省地区胃癌组织中HPV16 癌基因E6和幽门螺杆菌(Hp).结果:在40例胃癌组织(GC)中HPV16 E6的阳性率为27.5%(11/40) 而在40例癌旁正常组织(GANM)中未检测到;GC组中HPV16 E6 阳性率明显高于GANM组(P=0.0004);贲门癌中HPV16 的感染率明显高于非贲门癌 (P=0.0136);胃癌中HPV16与幽门螺杆菌感染无明显相关性(P=0.0829).HPV16与胃癌患者的性别、年龄、肿瘤浸润、淋巴结转移以及病理分级均无明显相关性(P>0.05).结论:本研究结果提示,在胃癌的发生过程中,HPV16可能不依赖或者不与幽门螺杆菌协作而发挥作用.     

Risk factors and survival by HPV‐16 E6 and E7 antibody status in human papillomavirus positive head and neck cancer

High‐risk human papillomavirus types (HPV‐HR) are associated with head and neck cancer (HNC) risk and better survival. Most patients with HPV‐HR DNA‐positive tumors develop anti‐HPV E6/E7 antibodies; however, it is unclear whether those who mount an immune response have similar risk factors or clinical outcomes as those who do not. HPV‐16 DNA tumor‐positive HNC cases were evaluated for HPV‐16 E6 and E7 antibodies using a GST capture ELISA system. Among 57 HPV‐16 DNA tumor‐positive HNC cases, 67% were detected with HPV‐16 E6 and/or E7 antibodies. Male gender (76% vs. 42%, p = 0.02), younger age (63% vs. 16%, p = 0.001) but not tobacco or alcohol were associated with E6 and/or E7 seropositivity. Seropositivity was associated more often with late stage (76%), poor grade (65%), positive nodes (82%). and in the oropharynx (82%), Median disease‐specific and recurrence‐free survival were longer in E6 and/or E7 seropositive compared to E6/E7‐negative cases (2.2 years vs. 1.4 years, both outcomes), although results were not statistically significant. When examined jointly with p16 expression, E6 and/or E7‐positive/p16‐positive cases had better disease‐specific (2.1 years vs. 1.1 years, p = 0.06) and recurrence‐free (2.3 years vs. 1.1 years, p = 0.03) survival compared to E6‐/E7‐/p16‐ cases. These findings suggest there are 2 distinct HNC patient groups with HPV DNA‐positive tumors, distinguishable by E6 and/or E7 antibody status. Differences in antibody status are associated with distinct risk factors and clinical outcomes. This information can be available as a simple blood test at initial presentation, before the removal of tissue through biopsy or surgery.     

Human papillomavirus type 16 E7 oncoprotein mediates CCNA1 promoter methylation

Human papillomavirus (HPV) oncoproteins drive distinctive promoter methylation patterns in cancer. However, the underlying mechanism remains to be elucidated. Cyclin A1 (CCNA1) promoter methylation is strongly associated with HPV‐associated cancer. CCNA1 methylation is found in HPV‐associated cervical cancers, as well as in head and neck squamous cell cancer. Numerous pieces of evidence suggest that E7 may drive CCNA1 methylation. First, the CCNA1 promoter is methylated in HPV‐positive epithelial lesions after transformation. Second, the CCNA1 promoter is methylated at a high level when HPV is integrated into the human genome. Finally, E7 has been shown to interact with DNA methyltransferase 1 (Dnmt1). Here, we sought to determine the mechanism by which E7 increases methylation in cervical cancer by using CCNA1 as a gene model. We investigated whether E7 induces CCNA1 promoter methylation, resulting in the loss of expression. Using both E7 knockdown and overexpression approaches in SiHa and C33a cells, our data showed that CCNA1 promoter methylation decreases with a corresponding increase in expression in E7 siRNA‐transfected cells. By contrast, CCNA1 promoter methylation was augmented with a corresponding reduction in expression in E7‐overexpressing cells. To confirm whether the binding of the E7–Dnmt1 complex to the CCNA1 promoter induced methylation and loss of expression, ChIP assays were carried out in E7‐, del CR3‐E7 and vector control‐overexpressing C33a cells. The data showed that E7 induced CCNA1 methylation by forming a complex with Dnmt1 at the CCNA1 promoter, resulting in the subsequent reduction of expression in cancers. It is interesting to further explore the genome‐wide mechanism of E7 oncoprotein‐mediated DNA methylation.