高危型人乳头瘤病毒基因组整合的研究进展

高危型人乳头瘤病毒(HPV)基因组整合在宫颈癌等多种肿瘤中被发现。流行病学和实验证据支持高危型HPV基因组整合在宫颈癌等相关肿瘤中起重要作用,其发生可能与宿主染色体不稳定和DNA甲基化相关。近年来发现克隆选择与干扰素通路相关。另外,整合对宿主细胞基因影响和整合位点检测方法方面的研究也取得了重要进展。本文就高危型HPV基因组整合研究的最新进展作一综述。     

人乳头状瘤病毒(HPV)基因整合与宫颈病变

HPV基因整合是启动宫颈癌发生的一个重要事件,高危性人乳头状瘤病毒(high risk human papillomavirus,hr-HPV)基因组整合率和宫颈病变程度密切相关,可能是宫颈癌发生发展过程中的重要标志.HPV基因整合机制研究、整合位点的发现、相关整合通路的探索,对于宫颈癌的精准治疗和预后的判断有着重大的...     

高危型HPV DNA整合导致宫颈癌的作用机制和临床检测进展

高危型人乳头瘤病毒(human papillomavirus,HPV)感染是宫颈癌发生发展的必要因素之一。病毒DNA整合到宿主基因组中被认为是导致宫颈癌最重要的病因。高危型HPV DNA整合往往导致其E1和E2区大部分缺失或中断,E6和E7致癌基因过表达,宿主致癌基因激活和抑癌基因失活。目前研究表明高危型HPV整合可作为优质宫颈病变筛查的预测生物标志物,且其检测的有效方法大都基于荧光原位杂交、实时荧光定量PCR和杂交捕获技术结合Sanger测序法等。本文重点阐述了高危型HPV整合导致宫颈癌的主要机制,描述了宫颈病变筛查标志物以及预防性HPV疫苗研发和推广的研究进展,并综述了高危型HPV DNA整合状态的检测方法。     

新疆维吾尔族妇女宫颈癌组织中乳头瘤病毒16型E6基因的克隆和序列分析

为了分析新疆南部地区维吾尔族妇女宫颈癌组织中HPV16型E6基因结构特点，从中国新疆南部地区维吾尔族妇女宫颈癌活检组织标本中提取DNA，以宫颈癌活检组织标本DNA为模板进行PCR扩增，获得HPV16 E6基因，将其克隆到pUCm-T载体上，并对其进行基因全序列分析.PCR检测结果显示宫颈癌组织中HPV16 E6阳性率为82.35%（14/17）；测序结果显示，新疆株HPV16 E6基因全长456 bp，大小与德国标准株一致.E6基因的第247位碱基发生T→G突变，并由此引起所编码的氨基酸亦发生改变.上述结果表明，中国新疆南部地区维吾尔族妇女宫颈癌患者组织中HPV16 E6的基因结构与德国标准株HPV16 E6基因之间存在差异.     

HPV16 变异体在新疆地区宫颈癌及癌前病变中的分布研究

目的:研究新疆地区HPV16 E6、E7、LCR基因突变情况,分析HPV16变异体在宫颈癌及癌前病变发生发展中的作用。方法:选择HPV16阳性的宫颈癌及癌前病变患者,提取基因组DNA,利用PCR扩增HPV16 DNA E6、E7基因及LCR区核苷酸片段,正反向测序。与HPV16基因序列分析比对,分析核苷酸突变位点。结果:E6基因突变率为80.00%(92/115)主要突变位点T350G(59.78%)、T178G(18.47%);E7突变率为54.78%(63/115),主要突变位点A647G(33.33%)、T846C(26.98%);LCR突变率为23.48%(27/115),主要突变位点为C24T(74.07%)、C13T(25.92%)。维吾尔族T350G突变率较汉族妇女显著升高,而汉族A647G、T846C、C24T突变率显著高于维吾尔族,差异具有统计学意义(P0.05)。维吾尔族宫颈癌组T350G突变率显著高于炎症组(P0.05),且随病变严重程度增加突变率上升,汉族T350G、A647G、T846C、C24T突变率炎症组、宫颈病变组显著高于宫颈癌组(P0.05),维吾尔族C24T突变率炎症组显著高于宫颈癌组(P0.05),差异均具有统计学意义(P0.05)。结论:HPV16E6、E7突变可能与宫颈病变进展有关,T350G突变可能是维吾尔族宫颈癌高发的原因之一。     

《自然-遗传学》:人乳头瘤病毒整合位点图公布

<正>在线发表于《自然-遗传学》上的一项研究公布了宫颈癌的主要致病原因--人乳头瘤病毒(HPV)的病毒整合位点图。病毒整合是病毒将自身DNA插入宿主DNA的过程,这也是感染HPV后导致宫颈癌产生的主要风险因素之一。Ding Ma等人利用高通量病毒整合检测方法在超过100个宫颈癌样本中找到3667个HPV整合位点。他们鉴定出9个基因在至少5个样本中存有整合位点,另外33个基因在至少4个样本中存有整合位点。依据病毒整合到基因中的位置,病毒整合会导致宿主基因表达或丢失或增加,     

HLA-I表达与维吾尔族妇女宫颈癌前病变及HPV16感染的关系研究

目的：观察人白细胞相关抗原I（human leukocyte antigen class I,HLA-I）表达与维吾尔族妇女宫颈癌前病变进程及高危型HPV16的关系。方法：收集维吾尔族妇女宫颈炎、宫颈内上皮瘤样病变（cervical intraepithelial neoplasia,CIN）和宫颈鳞癌患者的石蜡包埋组织标本共148例,提取组织DNA,应用PCR的方法检测HPV阳性及HPV16型别;同时采用免疫组织化学SP法检测HLA-I蛋白表达水平。结果：（1）在维吾尔族妇女中HLA-I抗原在宫颈炎、CINI-II、CINIII、SCC组中阳性表达逐渐减少,差异有统计学意义（P〈0.001）。（2）HLA-I的阳性表达下降趋势与宫颈癌临床分期、组织分化程度和淋巴结转移密切相关。（3）HPV在宫颈炎、CINI-II、CINIII、宫颈癌中的感染率分别为13%、46%、82%、95%,差异有统计学（P〈0.001）。（4）HPV16在宫颈炎、CINI-II、CINIII、宫颈癌中的感染率分别为4%、30%、68%、85%,差异有统计学（P〈0.001）。（5）在HPV16阳性标本中,存在HLA-I表达缺失的占71%（58/82）,HPV16感染与HLA-I表达呈负相关（r=-0.625,P〈0.001）。结论：（1）HLA-I表达缺陷可能是宫颈病变进展的重要标志,对宫颈癌的预测预警提供依据。（2）HPV16感染在宫颈病变的发展过程中起到了极大的促进作用,是一个很强的致癌因素。（3）HPV16感染与HLA-I表达之间的关系对揭示宫颈癌发病机制提供了客观依据。     

我国人乳头瘤病毒（HPV16Z）基因克隆和鉴定

本文作者采用基因克隆手段,以pAt153为载体,从一份山西襄坦宫颈癌高发区宫颈癌患者的手术标本中,成功地克隆到2株与HPV16同源的基因片段。经PstI、KpnI、TaqvI、PvuII等16种限制性内切酶酶谱分析及其部分基因序列的鉴定,证明这是在国内首次克姓到一株分子量约为8．0kb完整的HPV16型全序列DNA及一株分子量为5．4kb的HPV6基因片段。经实验证明：该基因片段E6、E7及部分LI基因丢失,在750单核苷酸处发生变异,产生一新的BamHI酶切位点。该完整的HPV16基因组被命名为HPV16Z,HPV16基因片段被命名为HPV16F。用新分离到的HPV16Z作分子探针,检测襄坦337份宫颈癌及阴道活检标本的HPV16型同源序列的结果显示,慢性阴道炎阳性率为17．28％（14／81）;宫颈炎为11．89％（17／143）;宫颈癌前病变46．81％（22／47）;宫颈癌为72．73％（48／66）。证明山西宫颈癌高发区宫颈癌前病变及宫颈癌组织中主要为HPV16Z感染。     

HPV检测在宫颈癌、宫颈癌前病变及宫颈炎患者中的临床应用

目的:研究人类乳头瘤病毒(HPV)检测在宫颈癌、宫颈癌前病变及宫颈炎患者中的临床鉴别价值。方法:选取2013年5月到2015年5月我院确诊的宫颈病变临床资料320例,根据病理诊断结果将患者分为宫颈癌组(59例)、癌前病变组(138例)以及宫颈炎组(123例),检测并比较三组HPV阳性检出率、HPV相对含量以及HPV-DNA亚型情况。结果:宫颈炎组HPV阳性检出率、HPV相对含量及HPV-DNA各亚型阳性率均显著低于癌前病变组和宫颈癌组,且癌前病变组均显著低于宫颈癌组,比较差异具有统计学意义(P0.05)。结论:HPV有助于鉴别宫颈癌、宫颈癌前病变及宫颈炎,对宫颈病变治疗和预后具有评价作用。     

HPV16/18感染导致宫颈癌的分子机制研究

目的探讨高危型HPV16/18感染对宫颈组织中抑癌基因Rap1GAP的影响,揭示HPV16/18感染导致宫颈癌的分子机制。方法收集因子宫肌瘤手术切除的正常宫颈组织20例和宫颈癌组织20例。检测HPV16/18感染情况后,Trizol法提取组织总RNA,RT-PCR检测组织中Rap1GAP mRNA;提取基因组DNA,PCR检测Rap1GAP第11和19号外显子(E11、E19)。SPSS 18.0软件进行统计学分析。结果 (1)正常宫颈组织中,不论是否HPV16/18感染,均能检出Rap1GAP mRNA,检出率为100%。宫颈癌组织中,HPV16/18感染标本Rap1GAP mRNA的检出率为0%,未感染标本检出率为60%,HPV16/18感染和Rap1GAP mRNA检出率呈负相关(P0.05)。(2)正常宫颈组织和宫颈癌中E11、E19的检出率均分别为100%、90%,两组比较差异无统计学意义(P0.05)。宫颈癌HPV16/18感染标本E11和E19的检出率均为87%,未感染标本检出率均为100%,两组比较差异无统计学意义(P0.05)。结论 HPV16/18感染下调Rap1GAP mRNA表达可能是其导致宫颈癌的机制之一。     

Analysis of the physical state of different human papillomavirus DNAs in intraepithelial and invasive cervical neoplasm.

The integration of human papillomavirus (HPV) DNA into the human genome has been generally accepted as a characteristic of malignant lesions. To gain a better understanding of this phenomenon, genomic DNA from 181 cervical biopsy specimens was isolated and analyzed for HPV type and physical state of the HPV genome. These specimens represented the full spectrum of cervical disease, from condyloma to invasive carcinoma. Discrimination between integrated and episomal HPV DNA was accomplished by the detection of HPV-human DNA junction fragments on Southern blots. In most cases in which ambiguous Southern blot results were obtained, the specimens were reanalyzed by two-dimensional gel electrophoresis. Of the 100 biopsy specimens of cervical intraepithelial neoplasia analyzed, only 3 showed integrated HPV DNA, in contrast to 56 (81%) of 69 cervical carcinomas (P less than 0.001) showing integrated HPV DNA. Of the 40 carcinomas containing HPV 16 DNA, 29 (72%) had integrated HPV DNA, of which 8 (20%) also had episomal HPV DNA. In 11 (27%) cancers, only episomal HPV 16 DNA was detected. All 23 HPV 18-containing carcinomas had integrated HPV DNA, and 1 also had episomal HPV 18 DNA. The difference between HPV types 16 and 18 with respect to frequency of integration was statistically significant (P less than 0.01). The results of this study indicate that detectable integration of HPV DNA, regardless of type, occurs infrequently in cervical intraepithelial neoplasia. The absence of HPV 16 DNA integration in some carcinomas implies that integration is not always required for malignant progression. In contrast, the consistent integration of HPV 18 DNA in all cervical cancers examined may be related to its greater transforming efficiency in vitro and its reported clinical association with more aggressive cervical cancers.     

Physical Status of Human Papillomavirus Integration in Cervical Cancer Is Associated with Treatment Outcome of the Patients Treated with Radiotherapy

Integration of human papillomavirus (HPV) DNA into the host genome is a critical aetiological event in the progression from normal cervix to intraepithelial neoplasm, and finally to invasive cervical cancer. However, there has been little work on how HPV integration status relates to treatment outcome for cervical carcinomas. In the current study, HPV E2 and E6 gene copy numbers were measured in 111 cervical cancer tissues using real-time QPCR. Integration patterns were divided into four groups: single copy-integrated with episomal components (group 1), single copy-integrated without episomal components (group 2), multicopy tandem repetition-integrated (group 3), and low HPV (group 4) groups. A relapse-predicting model was constructed using multivariable Cox proportional hazards model to classify patients into different risk groups for disease-free survival (DFS). The model was internally validated using bootstrap resampling. Oligonucleotide microarray analysis was performed to evaluate gene expression patterns in relation to the different integration groups. DFS rate was inferior in the order of the patients in group 4, group 2/3, and group 1. Multivariate analysis showed that histologic grade, clinical stage group, and integration pattern were significant prognostic factors for poor DFS. The current prognostic model accurately predicted the risk of relapse, with an area under the receiver operating characteristic curve (AUC) of 0.74 (bootstrap corrected, 0.71). In conclusion, these data suggest that HPV integration pattern is a potent prognostic factor for tailored treatment of cervical cancer.     

Short- and long-range cis interactions between integrated HPV genomes and cellular chromatin dysregulate host gene expression in early cervical carcinogenesis

Development of cervical cancer is directly associated with integration of human papillomavirus (HPV) genomes into host chromosomes and subsequent modulation of HPV oncogene expression, which correlates with multi-layered epigenetic changes at the integrated HPV genomes. However, the process of integration itself and dysregulation of host gene expression at sites of integration in our model of HPV16 integrant clone natural selection has remained enigmatic. We now show, using a state-of-the-art ‘HPV integrated site capture’ (HISC) technique, that integration likely occurs through microhomology-mediated repair (MHMR) mechanisms via either a direct process, resulting in host sequence deletion (in our case, partially homozygously) or via a ‘looping’ mechanism by which flanking host regions become amplified. Furthermore, using our ‘HPV16-specific Region Capture Hi-C’ technique, we have determined that chromatin interactions between the integrated virus genome and host chromosomes, both at short- (<500 kbp) and long-range (>500 kbp), appear to drive local host gene dysregulation through the disruption of host:host interactions within (but not exceeding) host structures known as topologically associating domains (TADs). This mechanism of HPV-induced host gene expression modulation indicates that integration of virus genomes near to or within a ‘cancer-causing gene’ is not essential to influence their expression and that these modifications to genome interactions could have a major role in selection of HPV integrants at the early stage of cervical neoplastic progression.     

新疆维吾尔族和汉族妇女宫颈脱落细胞中HPV Ll 壳蛋白的表达比较

目的:研究HPV L1壳蛋白在在新疆维吾尔族和汉族妇女宫颈脱落细胞中的表达情况及差异性。方法:收集2012年9月至2014年3月在新疆维吾尔自治区人民医院妇科门诊就诊或行宫颈癌机会性筛查的新疆维吾尔族和汉族妇女病例1160例,选择其中HPV感染阳性或TCT阳性或两项同时阳性的465例纳入研究队列,通过免疫细胞化学法检测宫颈脱落细胞中HPV L1蛋白的表达情况。结果:新疆维吾尔族与汉族妇女HPV L1壳蛋白的总阳性表达率比较无显著差异(P=0.964);维吾尔族与汉族妇女正常或慢性炎症组、CIN1组、CIN2组、CIN3组和SCC组HPV L1壳蛋白表达的阳性率比较均无显著差异(P=0.988,0.957,0.803,0.892,1.000)。新疆维吾尔族妇女和汉族妇女HPV L1壳蛋白表达的阳性率在C1N1组为最高,高于正常或慢性炎症组及其它高病变组,且HPV L1壳蛋白表达的阳性率随宫颈病变恶性程度的增加而降低,呈负相关(相关系数=-0.687和-0.379,P0.001)。结论:新疆维吾尔族与汉族妇女宫颈脱落细胞中HPV L1壳蛋白的表达不存在民族差异,但其与宫颈病变的恶性程度呈负相关,可能是宫颈病变的保护性因素之一。