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HPV16型E6基因变异分析

目的:新疆维吾尔族妇女宫颈癌的发生主要与人乳头状瘤病毒16(HPV16)感染相关,特定的HPV16 E6突变株具有更高的致癌危险性.本研究通过检测维吾尔族妇女宫颈癌组织中HPV16 E6基因突变情况,探讨其与维吾尔族妇女宫颈癌高发的关系.方法:从140例维吾尔族妇女宫颈癌石蜡包埋组织中提取DNA作为模板,PCR 扩增HPV16 E6 全长基因,PCR 产物直接测序,进行突变分析.结果:本组维吾尔族妇女宫颈癌组织中HPV16的阳性率是73.6%(103/140),91例E6基因测序及分析结果表明,维吾尔族妇女宫颈癌组织中存在HPV16 E6变异株,其中49例(53.8%)分离株发生L83V突变,4例(4.39%)发生D25E突变,2例(2.20%)发生D64E/L83V突变,36例(39.6%)与野生型相同.结论:新疆维吾尔族妇女宫颈癌患者HPV16 E6基因发生变异,主要以L83V突变为主,HPV16变异株在维吾尔族妇女宫颈癌患者中的分布可能与维吾尔族妇女宫颈癌高发存在一定关系.     

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。     

山东青岛地区宫颈癌组织中ＨＰＶ１６型Ｅ６和Ｅ２基因突变分析

目的　通过分析山东青岛地区宫颈组织中ＨＰＶ１６型Ｅ６和Ｅ２基因突变情况,探讨其与该地区宫颈癌的关系。方法　从１０４例青岛地区宫颈疾病组织中提取ＤＮＡ作为模板,采用聚合酶链式反应（ＰＣＲ）技术筛选出高危型ＨＰＶ及ＨＰＶ１６阳性标本,扩增出ＨＰＶ１６型Ｅ６、Ｅ２全长基因,ＰＣＲ产物纯化后测序,与德国ＨＰＶ标准株进行比对分析。结果　宫颈组织中高危型ＨＰＶ阳性率为９３.２７％（９７／１０４）,ＨＰＶ１６阳性率为６９.２３％（７２／１０４）。ＨＰＶ１６阳性标本中扩增出Ｅ６基因３７例,有５例与标准株序列相同,３２例存在突变,其中２５例突变型别为Ｔ１７８Ｇ或Ｔ１７８Ａ（Ｄ２５Ｅ）。在Ｅ２基因全序列测序中,２３例均存在Ｃ３６８４Ａ（Ｔ-Ｋ）,１４例同时存在Ｔ３５２４Ｃ、Ｃ３６８４Ａ（Ｔ-Ｋ）和Ｃ３７８７Ａ（Ｄ-Ｅ）,９例同时存在Ａ２９２６Ｇ、Ｃ３１５９Ａ（Ｔ-Ｋ）、Ｇ３２４９Ａ（Ｒ-Ｑ）、Ｔ３３８４Ｃ（Ｉ-T）、Ｃ３４１０Ｔ（Ｐ-Ｓ）和Ｃ３６８４Ａ（Ｔ-Ｋ）。结论　山东青岛地区宫颈癌患者ＨＰＶ１６型Ｅ６、Ｅ２基因与德国标准株比较存在多处变异,Ｅ６与Ｅ２基因突变可能存在相关性。     

宫颈癌组织中HPV16E6序列多态性及同源性分析

目的探讨广东地区宫颈癌组织中HPV16肿瘤相关性抗原E6基因序列的多态性及同源性。方法采用通用引物PCR直接测序法对宫颈癌标本中的HPV分型,从含有HPV16型的标本中采用自行设计的多重引物通过巢式PCR扩增出HPV16E6,经DNA序列测定法检测其基因变异,进而分析其同源性。结果50例宫颈癌组织HPV-DNA的检出率为78%,其中HPV16和HPV18型混合感染18例,单纯HPV16型感染15例。含有HPV16型的标本34例中扩增出HPV16E625例。其中178位核苷酸变异较大,变异率为72%,其相应氨基酸均由天冬氨酸变为谷氨酸。结论HPV16E6DNA序列发生碱基替换的区域主要在氨基端94~241位,羧基端相对保守,未见变异。广东地区宫颈癌组织中HPV16E6的热点突变为Nt178。     

人乳头瘤病毒16型转化基因在不同阶段宫颈上皮病变组织中的分布及基因变异特点

目的 研究人乳头瘤病毒(HPV)16 E6、E7和E5基因在湖北地区不同阶段宫颈上皮病变患者组织中的分布以及E6、E7基闪的变异特点.方法 从124例宫颈癌、17例宫颈上皮内瘤变(CIN) Ⅰ+CINⅡ级、23例CIN Ⅲ级和36例慢性宫颈炎患者活检或手术切除标本中提取组织DNA,用HPV16 E6、E7和E5特异性引物进行PCR扩增,对部分扩增的 E6 和 E7 产物片段进行测序分析.结果 在官颈炎、CIN Ⅰ+CINⅡ级、CINⅢ级和宫颈癌组织中,E6基因的阳性率分别为25.0%、29.4%、60.9%和76.6%;E7基因的阳性率分别为16.7%、41.2%、43.5%和61.3%:E5 基因的阳性率分别为5.6%、5.9%、30.4%和40.3%.E6、E7和E5基因在不同阶段宫颈上皮病变组织中的阳性率差异均有统计学意义(均P<0.01).在80例官颈癌测序组织中,有47例发生E6基因178位点的T→C突变,突变率为58.8%,相应氨基酸由天冬氨酸(Asp)改变为谷氨酸(Glu);而在20例宫颈炎和22例CINⅠ～Ⅲ级测序组织中,E6基因178位点的突变率分别为25.0%和31.8%.在30例宫颈癌测序组织中,有21例发生E7基因647位点的A-G突变,突变率为70.0%,相应氨基酸由天冬酰胺(Asn)改变为丝氨酸(Ser);而在20例宫颈炎和22例CINⅠ～Ⅲ级测序组织中,E7基因647位点的突变率分别为35.0%和40.9%.结论 HPV16 E6、E7和E5基因与宫颈癌的发生和发展有高度的相关性.但E5基因在不同阶段官颈上皮病变中可能存在不同程度的缺失.中国湖北地区流行的HPV16病毒株可能为HPV16亚洲型变异株.     

人乳头瘤病毒感染和p53基因突变与宫颈癌的关系

目的：探讨人乳头瘤病毒１６ 和１８ 型及抑癌基因ｐ５３ 突变对宫颈的致癌作用以及 Ｈ Ｐ Ｖ 感染与ｐ５３ 基因突变的相关性。方法：采用聚合酶链反应（ Ｐ Ｃ Ｒ） 技术和限制性酶切片段多态性分析（ Ｒ Ｆ Ｌ Ｐ） 技术对３４ 例原发性宫颈癌组织及３０ 例正常宫颈组织 Ｈ Ｐ Ｖ１６ ,１８ 型 Ｄ Ｎ Ａ 及抑癌基因ｐ５３ 的突变进行了检测。结果： Ｈ Ｐ Ｖ１６ ,１８ Ｄ Ｎ Ａ 在宫颈癌的总阳性率为６４７ ％ （２２／３４） ,正常宫颈组织只有６７ ％ 阳性,８例宫颈癌组织出现ｐ５３ 基因第６ 外显子突变,其中２ 例为 Ｈ Ｐ Ｖ１６ Ｄ Ｎ Ａ 阳性、１ 例 Ｈ Ｐ Ｖ１８ Ｄ Ｎ Ａ 阳性。结论：宫颈癌的发病与 Ｈ Ｐ Ｖ 感染及ｐ５３ 基因突变有关,宫颈癌组织中ｐ５３ 基因突变与 Ｈ Ｐ Ｖ 感染无关。     

HPV16-E6/E7 Oncogene Mutation and p53 Expression among Indonesian Women with Cervical Cancer

Objective: To characterize HPV16 E6/E7 mutation and its association with p53 expression among Indonesian women with cervical cancer. Methods: This is a cross-sectional study involving 31 Indonesian women with pathologically proven cervical cancer and HPV16 infection. Data about the clinical characteristics of the study population were obtained from the medical records. Biopsy specimen of the cervical cancer mass from each study participant was obtained for DNA isolation. The ORFs of E6 and E7 genes were amplified using specific primer designed according to K02718/HPV16R gene sequence obtained from GenBank. Sequencing was performed using software program MEGA10. HPV16 E6 and E7 prototype sequences for nucleotide alignment (HPv16. P, GenBank Access code: NC_001526) was selected from European variant. The sequence of nucleotide and amino acid was aligned using software program BioEdit. p53 expression was evaluated through immunohistochemistry and quantified using immunoreactivity score (IRS). Results: Twelve subjects (38.7%) present with E6 and E7 mutation. Median age, parity, stage and histologic type of the tumour did not associate with E6/E7 mutation. E6 and E7 mutation rate was 25.8% (8/31) and 12.9% (4/31), respectively. Seven single nucleotide changes were identified within the E6 and E7 oncogenes, including four non-synonymous and three synonymous mutations. E6 T27C was the most prevalent mutation (16.1%). Nonsynonymous mutations were more prevalent within E7 gene (9.6%) (N29T, N29S, and R77C). Median IRS did not differ between HPV16-E6/E7 variants and wildtype (p value = 0.990). There was no association between E6/E7 mutations and p53 expression in Indonesian women with cervical cancer (PR 1.4, 95% CI: 0.29-6.77, p value = 0.704). Conclusions: HPV16 E6 mutation was more prevalent than E7 mutation among Indonesian women. There was no association between E6/E7 mutation and p53 expression level.     

荧光偏振法检测高危HPV16型E6基因350位(T→G)突变

目的:检测宫颈癌人类乳头瘤病毒(HPV)16 E6基因350位(T→G)点突变情况.方法:应用TDI -FP方法检测宫颈癌患者HPV16F6基因350位(T→G)点突变情况.TDI - FP技术是一种在聚合酶链反应的基础上具备液相探针杂交和碱基掺入三重特异反应的检测新方法,具有极高的特异性和敏感性.结果:应用TDI - FP法检测宫颈癌组织中HPV16型E6基因350位(T→G)突变率为6.06％,HPV16 E6基因350位突变与对照组相比无统计学差异(P=0.603.P＞0.05).结论:本地区宫颈癌患者中HPV16型E6 350位(T→G)很少发生突变,有别于欧洲的HPVI6型E6 350位(T→G)高突变.     

半定量PCR检测子宫颈组织中人乳头瘤病毒16型DNA的研究

目的 :探讨子宫颈组织中人乳头瘤病毒 16型 (HPV16 )E6基因的含量与疾病严重程度的关系。方法 :用半定量PCR检测 2 0例慢性宫颈炎 ,6例宫颈非典型增生 ,18例宫颈癌组织中HPV16E6基因的含量。结果 :宫颈非典型增生及宫颈癌组织中E6基因的含量显著高于慢性宫颈炎 (P <0 0 1) ;宫颈癌及宫颈非典型增生的差异无显著性 (P >0 0 5 )。结论 :HPV16E6基因的含量随着宫颈疾病严重程度增加而增加 ,定量检测HPV16DNA的含量可能作为监测宫颈癌高危人群的一种方法     

四川地区宫颈癌HPV33、52和58型E6基因突变分析

目的:调查我国四川地区妇女宫颈癌组织中HPV33、52和58型的感染率及E6基因结构特点,探讨其与宫颈癌发生的关系。方法:采用PCR技术,对四川地区2004年60例宫颈癌患者癌组织DNA进行HPV检测。结果:获得HPV52和HPV58型各4例,检出率为6．7％;HPV33型1例,检出率为1．7％。对所获得的各例 HPV33、52和58 E6基因测序分析。结论:与Genbank标准株相比,四川地区发现的HPV33和HPV52 E6基因有多处突变,与日本发现的HPV33,52突变株相近。     

湖北地区HPV16 E7和E5基因突变与宫颈 病变的相关性

 摘要：目的分析湖北地区宫颈病变组织中HPV16 E7和E5基因序列变异及该变异与宫颈病变的相关关系。方 法对HPV16 E7和E5 DNA阳性的20例正常宫颈、30例CIN Ⅰ~Ⅱ、30例CINⅢ和35例宫颈鳞癌的基因片段进行 纯化测序,检测基因变异。结果除了HPV16 E7和E5原型序列以外,HPV16 E7最常见变异为A647G和T846C的 联合变异株,该联合变异株频率在正常宫颈、CIN Ⅰ~Ⅱ、CINⅢ和鳞癌组中突变率分别为25%、30%、 53.3%、62.9%;HPV16 E5最常见变异为A3979C +A4042G联合变异株,联合变异株在正常宫颈、CIN Ⅰ~Ⅱ 、CINⅢ组和鳞癌组中突变率分别为30%、33.3%、56.7%、71.4%;HPV16 E7和E5联合突变株在宫颈病变各 阶段差异有统计学意义。结论HPV16 E7.A647G／T846C和HPV16 E5.A4042G／A3979C联合变异株为中国湖北 地区宫颈病变中流行的变异株,此变异株与宫颈病变程度呈正相关。     

宫颈癌患者HPV16型E6蛋白的表达纯化及血清抗体检测

背景与目的:人乳头瘤病毒16型(humanpapillomavirustype16,HPV16)是宫颈癌组织中最常见的高危HPV,其相应蛋白的血清抗体与宫颈癌的发生发展相关。本研究构建HPV16E6重组表达载体并表达纯化获得HPV16E6重组蛋白,用于检测不同人群血清相应抗体,初步探讨本地区HPV16E6血清抗体反应与宫颈癌的相关性。方法:将HPV16E6基因与pRSET-A融合表达载体连接,获得E6表达重组体,转化大肠杆菌BL21(DE3)并用异丙基硫代-$-D-半乳糖苷(isopropylthio-$-D-galactoside,IPTG)诱导表达。表达的包涵体变性后经Ni柱纯化,复性并经活性鉴定后,用以包被ELISA板,检测正常女性、慢性宫颈炎患者和宫颈癌患者血清抗体。同时采用荧光偏振方法分型检测宫颈癌组织HPVDNA。结果:pRSET-16E6表达重组体的工程菌经IPTG诱导后可表达Mr24×103的HPV16E6组氨酸融合蛋白,表达量占菌体蛋白的22.3%。表达形式为包涵体,重组蛋白纯度达95%以上,其活性经ELISA法证实。80例正常女性、46例慢性宫颈炎和32例宫颈癌患者血清抗体阳性率分别为5.0%、6.5%和31.2%,宫颈癌患者HPV16E6血清抗体阳性率显著高于正常人(P<0.002)及慢性宫颈炎患者(P<0.01),而正常人与慢性宫颈炎患者间的差异无显著性。32例宫颈癌患者癌组织中,HPVDNA阳性率90.6%,HPV16DNA阳性率46.9%。HPV16DNA阳性组血清HPV16E6抗体阳性率(46.7%)高于阴性组(17.6%),但两组间的差异无显著性(P>0.05)。结论:在pRSET-A/BL21中表达获得的HPV16E6融合蛋白,可用于宫颈癌相关HPV的血清学研究;宫颈癌患者HPV16E6血清抗体阳性率明显高于正常人和慢性宫颈炎患者。     

Nested Multiplex PCR Based Detection of Human Papillomavirusin Cervical Carcinoma Patients of North- East India

Background: Persistent infection of one or more of about 15 high-risk human papillomaviruses (HR-HPVs),most commonly HPV types 16/18, has a significant role in cervical cancer initiation and progression. There arelimited data available from north-east India about HPV prevalence though this region has high incidence ratesof cervical cancer. The aim of this study was to investigate the HPV genotypes prevalent in cervical cancerpatients of north-east India. Materials and Methods: We analyzed 107 cervical cancer patient samples. Nestedmultiplex PCR assays were employed for detection of 13 high risk and 5 low risk HPV types. Results: HPV wasconfirmed in 105 samples. The presence of 6 ‘carcinogenic’ HPV types, HPV-16 (88%), -18 (15%), -31(4%) ,-45(3%), -59 (4%), -58(1%), and one non carcinogenic, HPV-6/11 (6%), was recorded. Among various demographicand clinical factors only tumour stage showed a statistically significant association with HPV type infection(P=0.019). Conclusions: We suggest that the most prevalent genotype is HPV-16 followed by HPV-18 in cervicalcarcinoma patients of the north-eastern region of India. Advanced tumour stage may be associated with increasedpossibility of harbouring multiple HPV genotypes.     

Elderly Japanese women with cervical carcinoma show higher proportions of both intermediate-risk human papillomavirus types and p53 mutations

The p53 mutation has been found only in 0-6% of cervical carcinomas. In light of recent studies demonstrating that mutation of p53 gene was found in over 20% of the patients with vulvar carcinoma, a disease of elderly women and a known human papillomavirus (HPV)-related malignancy, we analysed mutation of the p53 gene in 46 women with cervical carcinomas at the age of 60 or more (mean; 71 years, range; 60-96 years). The presence of HPV and its type were analysed by polymerase chain reaction (PCR)-based assay using the consensus primers for L1 region. Mutation of the p53 gene was analysed by PCR-based single-strand conformation polymorphism and DNA sequencing technique. Point mutation of the p53 gene was detected in 5 out of 46 (11%) cervical carcinomas: 1 of 17 (6%) samples associated with high-risk HPVs (HPV 16 and HPV 18) and 4 of 27 samples (15%) with intermediate-risk HPVs (P= 0.36) whereas no mutation was found in 2 HPV negative cases. The mutated residues resided in the selective sequence known as a DNA-binding domain. The immunohistochemistry revealed the overexpression in cancer tissues positive for p53 mutation. All of the observed mutations of the p53 gene were transition type, suggesting that the mutation may be caused by endogenous mutagenesis. Although falling short of statistical significance reduces the strength of the conclusion, data presented here imply that p53 gene mutation, particularly along with intermediate-risk HPV types, may constitute one pathogenetic factor in cervical carcinoma affecting elderly women.     

Cervical carcinoma in Southern Mexico: Human papillomavirus and cofactors

Background: This study was conducted to determine human papillomavirus (HPV) types in women with cervical cancer (CC) and normal cervical cytology in the Southern region of Mexico, and to know the contribution of HPV types and cofactors in cervical cancer etiology. Methods: A case-control study was performed in 133 women with CC and 256 controls. HPV detection was done by MY09/11 and GP5+/GP6+ PCR systems and typing by restriction fragment length polymorphism or DNA sequencing. Results: HPV was found in 100% of CC and 35.5% of controls. The genotype distribution in CC was: HPV 16 (66.8%), 18 (9%), 31 (7.5%), 45 (4.5%), 58 (3.7%), 69 (3%), 52 (1.6%), 6, 11, 33, 56, and 67 (0.8% each). Among controls, HPV 33 followed by HPV 16 were the most frequent. Cervical cancer was associated with HPV 16 (OR = 573.5), HPV 18 (OR = 804.4), and undetermined risk HPV (types 67 and 69) (OR = 434.3). Age at first intercourse <16 years (OR = 9.6) and ≥3 births (OR = 16) were significant risk factors for CC. Conclusions: HPV 16, by far, is the most frequent type in CC, HPV 16 and 18 are responsible for 75.8% of the CC cases and high-risk HPV for 94.7%, which is useful data to take into account in vaccination programs. HPV 33 is the most frequent type in controls and high-risk HPV are more common than low-risk HPV.     

RXRα deletion and E6E7 oncogene expression are sufficient to induce cervical malignant lesions in vivo

Cervical cancer is the second leading cause of cancer deaths among women worldwide. High-Risk-Human Papillomaviruses (HR-HPVs) play an important etiologic role in the development of carcinoma of the uterine cervix. However, host factors are important in determining the outcome of genital HPV infection as most cervical precancerous lesions containing HR-HPVs do not progress to invasive carcinomas. Retinoids, acting through nuclear receptors (RARs, RXRs), play a crucial role in cervix development and homeostasis regulating growth and differentiation of a wide variety of cell types; indeed, they can inhibit cell proliferation, and induce cell differentiation or apoptotic cell death. Here we introduce a mouse model that develops spontaneously malignant cervical lesions allowing the study of the cooperative effect between HPV16E6E7 expression and the lack of RXRα in cervical cancer development. This model could be useful to study multistep carcinogenesis of uterine cervix tissue and might improve chemopreventive and chemotherapeutic strategies for this neoplasia.     

Pathogenetic significance of p53 and c-Ki-ras gene mutations and human papillomavirus dna integration in adenocarcinoma of the uterine cervix and uterine isthmus

The pathogenetic significance of p53 and c-Ki-ras gene mutations and genomic integration of human papillomavirus (HPV) DNA was examined in surgically resected specimens of adenocarcinomas of the uterine cervix and isthmus using polymerase chain reaction (PCR), single-strand-conformation polymorphism and Southern blotting analysis. Among 25 cervical adeno-carcinomas, p53 gene mutations between exons 5 and 8 were detected in 32%, and the incidence of these mutations was higher in cases at advanced clinical stages and with high grades of nuclear and structural atypia both in endocervical and in endometrioid types. HPV DNA type 16 or 18 in cervical adenocarcinomas was detected in 35% of cases by PCR and in 29% by Southern blotting, and, in contrast to the p53 mutations, the majority of cases with the HPV DNA were at a relatively early clinical stage with low-grade histological atypia. c-Ki-ras gene mutation was detected in only 4% of cervical adenocarcinomas. Among 8 isthmus adenocarcinomas, the incidence of p53 and c-Ki-ras gene mutations, and the presence and integration of HPV DNA type 16 or 18 were 38%, 50%, 57% and 25% respectively. The pattern of p53 mutations differed between isthmus and cervical adenocarcinomas: all of the mutations in the former were one-base substitutions of the transition type, whereas in the latter nearly half of the mutations were of the transversion type. Among cervical adenocarcinomas, p53 mutations between exons 5 and 8 were indicated as being mostly involved in the pathogenesis and development of biologically aggressive tumors, whereas HPV type 16 or 18 infection appeared to be involved in less aggressive cases. In isthmus adenocarcinoma, c-Ki-ras gene mutation, apart from p53 mutation and HPV-type-16 or ?18 infection, appeared to be involved frequently in cancer development.     

Human Papillomavirus Genotype Distribution and E6/ E7 Oncogene Expression in Turkish Women with Cervical Cytological Findings

Background: Infection with certain human papillomavirus (HPV) genotypes is the most important riskfactor related with cervical cancer. The objective of the present study was to investigate the prevalence of HPVinfection, the distribution of HPV genotypes and HPV E6/E7 oncogene mRNA expression in Turkish women withdifferent cervical cytological findings in Mersin province, Southern Turkey. Materials and Methods: A total of476 cytological samples belonging to women with normal and abnormal cervical Pap smears were enrolled in thestudy. For the detection and genotyping assay, a PCR/direct cycle sequencing approach was used. E6/E7 mRNAexpression of HPV-16, 18, 31, 33, and 45 was determined by type-specific real-time NASBA assay (NucliSENSEasyQ®HPV v1.1). Results: Of the 476 samples, 106 (22.3%) were found to be positive for HPV DNA by PCR.The presence of HPV was significantly more common (p<0.001) in HSIL (6/8, 75%) when compared with LSIL(6/14, 42.9%), ASC-US (22/74, 29.7%) and normal cytology (72/380, 18.9%). The most prevalent genotypes were,in descending order of frequency, HPV genotype 66 (22.6%), 16 (20.8%), 6 (14.2%), 31 (11.3%), 53 (5.7%), and83 (4.7%). HPV E6/E7 oncogene mRNA positivity (12/476, 2.5%) was lower than DNA positivity (38/476, 7.9%).Conclusions: Our data present a wide distribution of HPV genotypes in the analyzed population. HPV genotypes66, 16, 6, 31, 53 and 83 were the predominant types and most of them were potential carcinogenic types. Becauseof the differences between HPV E6/E7 mRNA and DNA positivity, further studies are required to test the roleof mRNA testing in the triage of women with abnormal cervical cytology or follow up of HPV DNA positive andcytology negative. These epidemiological data will be important to determine the future impact of vaccinationon HPV infected women in our region.     

HPV-16 URR突变对病毒早期启动子活性影响的研究

背景与目的:新疆是宫颈癌高发区,该地区宫颈癌高发与人乳头瘤病毒16型(human papillomavirus type 16,HPV-16)感染密切相关.该研究旨在分析新疆地区妇女宫颈病样组织中HPV-16上游调控区(upstream regulatory region,URR)的突变及其功能.方法:以新疆妇女子宫颈上皮非典型增生(cervical intraepithelial neoplasia,CIN)和宫颈癌病样组织标本DNA为模板,PCR扩增HPV-16URR片段,PCR产物经测序比对,筛选代表性的URR突变体构建至pGL3-Basic载体,将其转染Vero细胞,48 h后检测荧光素酶活性,分析URR突变体启动子活性.结果:采用聚合酶链反应(polymerase chain reaction,PCR)获得了55个HPV-16URR DNA片段,测序及序列分析发现44个突变位点,其中nt7192(G→T)、nt7433(-→T)、nt7435(C→G)和nt7863(A→-)4个位点的突变为所有序列共有,nt7520(G→A)位点的突变存在于54个样品中,剩余39个位点的突变存在于不同样品中.根据突变的位置、频率和程度,筛选出9个URR突变体分别克隆至pGL3-Basic中荧光素酶基因前并转染Vero细胞.荧光素酶活性分析表明,不同URR突变体的启动子活性差异较大,来源于宫颈癌的URR突变体启动子活性显著高于来源于CIN的URR突变体(P<0.01),部分宫颈癌URR突变体的启动子活性显著高于SiHa和Caski细胞来源的URR参照序列的启动子活性.结论:新疆地区分离的HPV-16 URR发生多位点突变,其中部分突变增强了URR内部启动子的活性,导致HPV-16致癌活性增强.