Aptima法人乳头瘤病毒E6/E7 mRNA检测在子宫颈癌筛查中的流行病学分析及其诊断价值

目的 通过Aptima法检测人乳头瘤病毒(HPV)E6/E7 mRNA,研究其分型和定量检测结果在不同年龄和子宫颈活检中的分布情况,探讨其在子宫颈病变中的诊断价值.方法 选取2019年1月至2020年1月于郑州大学第三附属医院行HPV E6/E7 mRNA检测的患者为研究对象,收集HPVE6/E7 mRNA阳性患者的年...     

Human papillomavirus type 16 E2 and E6/E7 variants

OBJECTIVES: Polymorphisms in human papillomavirus (HPV) type 16 have been shown to be related to geographic areas and are broadly classified as European (E), African (Af), Asian (As), or Asian-American (AA). Certain variants have been reported as being more likely to cause cervical disease; our objectives were to identify new HPV16 polymorphisms, to determine the linkage of the E2 and E6/E7 regions and to determine the minimum sequence necessary to classify variants. METHODS: We sequenced the complete E2, E6, and E7 regions in all HPV16-positive cervical samples identified in a case-control study of pre-invasive cervical disease. RESULTS: In the 100 samples analyzed, only one new polymorphism was identified, a synonymous change, T3205A, in region E2. The frequency distribution of variants in the sample set was 37 European prototypes and 27 E-G350, 16 AA, 5 Af1, 2 Af2, 8 E-C109G, 3 E-G131G, and 2 As. As shown by others, region E7 varied much less than E6 and E2. CONCLUSIONS: In each case, E2 changes were linked to the expected E6/E7 changes, and there was no evidence for recombination. The linkage between E2 and E6/E7 allows variant classification to be based on a short E6 sequence (nt 109-350).     

Polymorphism in the E6 gene of human papillomavirus type 16 in the cervical tissues of Korean women

The aim of this study was to identify sequence variants in the HPV 16 E6 gene in Korean women and to examine the possible association between these sequence variants and cervical cancer development. We examined the HPV 16 DNA of 215 patients with no cervical disease (NCD) (n = 105) or with cervical neoplasia (n = 110) [cervical intraepithelial neoplasia (CIN), n = 61; invasive cervical carcinoma (ICC), n = 49] using the nested polymerase chain reaction (PCR) and PCR-directed sequencing methods. Fifty-four (NCD, n = 10; CIN, n = 17; ICC, n = 27) of the 215 samples contained HPV 16 E6 DNA, but only two (7.4%) of 27 ICC samples had prototype sequences. The most frequently found variation was D25E (in NCD, n = 8, 80%; in CIN, n = 9, 52.9%; in ICC, n = 23, 85.2%). This is a rare variation in western countries. No significance difference was found between the frequencies of D25E variation in cancerous and non-cancerous lesions. Among the 11 kinds of variants identified, four variants were novel and have been registered with GenBank. This study demonstrates that the D25 variant is the most prevalent E6 genomic variant type in Korean population. However, it was not found to be associated with an increased risk of ICC.     

腺病毒伴随病毒表达载体表达人乳头状瘤病毒16型E6/E7基因的构建及应用

目的　为了了解人乳头状瘤病毒 (Humanpapillomavirus ,HPV) 1 6型的E6 /E7基因在细胞恶性转化中所起的作用 ,利用腺病毒伴随病毒载体 (AAVHelper -FreeSystem)构建和表达人乳头状瘤病毒 1 6型E6 /E7基因。方法　在pLXSN1 6E6E7质粒中经PCR扩增回收HPV 1 6E6E7基因片段 ,连接于T载体上进行测序 ,将正确的HPV 1 6E6E7插入pAAV -IRES -hrGFP质粒 ,协同pAAV -RC质粒和pHelper质粒共转染HEK 2 93细胞 ,包装表达HPV 1 6E6E7基因的重组腺病毒伴随病毒 ,收获病毒 ,并检测病毒的感染效率。结果　在包装细胞系HEK 2 93细胞中能形成较高感染效率的腺病毒伴随病毒 ,激光共聚焦检测可发现HEK 2 93细胞内有绿色荧光蛋白表达 ,HEK 2 93细胞经PCR可扩增出特异性的HPV 1 6E6E7基因片段 ,经流式细胞仪检测重组病毒的感染效率为71 3%。结论　携带人乳头状瘤病毒 1 6型E6E7基因的腺病毒伴随病毒可感染细胞 ,并在细胞内表达 ,可望用于宫颈癌病因学的研究     

Molecular variants of human papillomavirus type 16 and risk for cervical neoplasia in South Africa

Non-European variants of human papillomavirus (HPV) type 16 are generally associated with a greater risk of cervical neoplasia than European prototype variants. We investigated whether this association would persist in a population in which non-European HPV 16 variants were more common. We sequenced HPV 16 isolates in cervical samples collected from 93 Black South African women enrolled in a cervical cancer screening study and examined associations between cervical neoplasia identified though colposcopy with cervical biopsy and the specific HPV 16 variant identified. The European prototype variant (EP) was the most commonly identified variant in this population (47% of all isolates), but African variants (Af-1 and Af-2) were also quite common (41% of all isolates). In contrast to previous studies, we found no evidence that non-European variants were associated with an increased risk of neoplasia. Rather, most of the HPV 16-associated cancers were found in association with EP (71% of 14 cases). In this setting where African HPV 16 variants were common, no increased risk for cervical neoplasia was found among women with these variants compared with other HPV 16 variants.     

人乳头状瘤病毒16型E6、E7基因转染的人宫颈上皮永生化细胞系的建立及鉴定

目的 建立HPV16 E6、E7基因转染的胎儿宫颈上皮永生化细胞系，为体外研究宫颈上皮HPV感染及其癌变机制提供模型。方法 探索胎儿宫颈上皮细胞原代培养的方法，用逆转录病毒法将HPVl6E6、E7基因转染原代培养的胎儿宫颈上皮细胞，筛选出稳定表达的细胞系，进行传代培养；应用RT-PCR检测细胞中HPV16E6、E7基因的表达，用绘制细胞生长曲线、形态学观察、软琼脂集落形成实验、流式细胞术、Transwell Insert体外侵袭实验和裸鼠成瘤实验等方法对永生化的胎儿宫颈上皮细胞进行鉴定。结果 采用组织块培养法。选用Ker-SFM无血清培养基培养出纯度和生长状态良好的胎儿宫颈上皮原代细胞，可以利用高滴度的逆转录病毒作为载体，感染原代细胞，成功地建立一株永生化的胎儿宫颈上皮细胞系。永生化的细胞仍呈多边形，角蛋白阳性，仍维持上皮细胞表型，雌、孕激素受体阴性，染色体核型发生明显变化，但目前尚无软琼脂集落形成能力、不能穿过Matrigel基底膜，不能在裸鼠体内形成肿瘤。结论 转染HPV16E6、E7基因能使胎儿宫颈上皮细胞永生化，永生化细胞仍维持上皮细胞表型，染色体核型发生明显变化，但尚未完全恶性转化。     

青岛地区宫颈癌组织中HPV16多态性分析

目的探讨山东省青岛地区妇女宫颈癌组织中人乳头瘤病毒16型（HPV16）上游调控区（URR）和E6基因序列多态性,病毒主要分支,以及他们与宫颈癌的相关性。方法从43例HPV16阳性宫颈癌组织中提取DNA,扩增URR及E6基因,进行序列测定。通过DNASTAR生物软件进行核苷酸和氨基酸序列的分析。结果所有标本URR测序结果均发生了G7521A位点突变;26例（60.5%）宫颈癌组织中检测到核苷酸位点C24T,A7730C和G7842A的联合突变,为另一突变热点。E6测序结果发现突变热点为T178G（D25E）,突变率为65.1%（28/43）;仅1例T350G突变（L83V）。AS型是最多见的病毒突变类型,占65.1%（28/43）,其次为野生型（E-P）23.3%（10/43）。结论青岛地区妇女宫颈癌组织中HPV16URR突变热点为G7521A以及C24T,A7730C和G7842A的联合突变。HPV16E6突变热点为T178G,这些突变热点可能与宫颈癌发生发展有密切关系。AS和E-P原型是青岛地区宫颈癌组织中两种主要的HPV16分支。     

宫颈癌及癌前病变组织中Notch1及HPV16 E6/E7表达的研究

目的 探讨Notch1受体和人乳头瘤病毒16感染在宫颈癌前病变和宫颈癌发生发展中的作用。方法 采用免疫组化SP法检测18例宫颈上皮内瘤变（cervical intraepithelial neoplasia,CIN）和35例宫颈癌标本中Notch1受体及HPV16E6/E7蛋白的表达,以34例正常宫颈组织及慢性宫颈炎组织作为对照。比较各组间Notch1及HPV16E6/E7表达的差异,并分析Notch1与HPV16E6/E7表达的关系。结果 Notch1蛋白在细胞胞浆、胞核及胞膜中表达,HPV16E6/E7在细胞核中表达。从对照组到CIN组到宫颈癌组,Notch1及HPV16E6/E7的表达均逐渐增强（P〈0.01）。Notch1的阳性表达与宫颈癌不同分期、分化程度、淋巴结是否转移无关（P〉0.05）。在宫颈癌组中Notch1与HPV16E6/E7的表达均呈正相关性（P〈0.01）。结论 Notch1表达与HPV16E6/E7感染可能与CIN及宫颈癌的发生密切相关,两者在宫颈癌的发病机制中可能协同发挥作用。     

反义HPV16 E6/E7-EGFP重组质粒的构建及其对宫颈癌SiHa细胞的促凋亡作用

目的:构建HPV16早期基因E6/E7的反义重组质粒,探讨其对SiHa细胞的促凋亡作用。方法:将HPV16E6/E7基因片段反向克隆于真核表达载体pEGFP-C1并转染SiHa细胞,用RT-PCR方法检测转染后SiHa细胞E6、E7基因mRNA的表达,West-ernblot方法检测转染后E6/E7蛋白的表达,流式细胞仪检测转染后细胞的凋亡率。结果:成功构建携带HPV16E6/E7基因反义片段的真核表达载体,转染该质粒后,SiHa细胞E6、E7基因的mRNA和蛋白均明显下调;转染后细胞凋亡率为(59.3±11.3)%,明显高于转染空载体组[(9.4±1.8)%]和未转染组[(2.1±0.4)%](P<0.05)。结论:反义HPV16E6/E7基因可下调宫颈癌细胞中E6/E7癌基因的表达,诱导宫颈癌细胞凋亡,为宫颈癌的基因治疗提供了实验依据。     

Human papillomavirus status in advanced cervical cancer: predictive and prognostic significance for curative radiation treatment

Human papillomavirus (HPV) infection plays a major role in oncogenesis of squamous cell carcinoma of the cervix. This study was performed to investigate if HPV status and E2 gene integrity are prognostic parameters for clinical outcome and predictive for radiation response. Forty women with locally advanced cervical cancer treated with curative radiotherapy were analyzed for HPV infection and E2 gene integrity by multiplex polymerase chain reaction. Statistical analyses were performed for overall survival, disease-free survival (DFS), local progression-free survival, and treatment response (clinical complete remission). Twenty-eight (70%) of 40 carcinomas were HPV positive. The only significant factor for a better overall survival, DFS, and local progression-free survival was HPV positivity (P < 0.02, P= 0.02, and P < 0.05, log-rank, respectively). HPV-positive tumors had a significantly better clinical complete remission (67% vs 33%, P= 0.04, Fisher's exact test). An intact E2 gene region showed a trend for a better DFS (P= 0.1, log-rank). This study reveals HPV as an independent prognostic parameter for outcome and radiation response. Integration of the virus genome into host cell DNA might be a molecular target to determine the treatment response of HPV-positive cancers.     

New variants of E6 and E7 oncogenes of human papillomavirus type 31 identified in Northeastern Brazil

Objective

We sought to characterize E6 and E7 oncogenes genetic variability of HPV-31 isolated from cervical scraping samples of Northeastern Brazilian women.

Methods

E6 and E7 were amplified with specific primers, cloned and sequenced. The sequences obtained were aligned with the GenBank reference sequences with the aim of evaluating the possible genetic variants.

Results

We identified several genetic variants in E6 and E7 sequences from HPV-31 positive women. Three nucleotide changes in E6 were described for the first time in this study. Some nucleotide changes were non-synonymous substitutions.

Conclusion

The knowledge of region/country HPV specific genetic variations is relevant to understand the epidemiology and the development of effective vaccines.     

Cancerous inhibitor of protein phosphatase 2A is overexpressed in cervical cancer and upregulated by human papillomavirus 16 E7 oncoprotein

Objectives

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a recently identified oncoprotein stabilizing c-Myc and promoting cell proliferation and transformation. Here we investigated the role of CIP2A in cervical cancer in vivo and in vitro.

Methods

CIP2A expression was assessed in normal cervical, cervical intraepithelial neoplasia (CIN) I to III and cervical cancer tissues by immunohistochemistry and RT-PCR. Cell growth was explored by cell proliferation assay, colony formation assay and anchorage-independent growth in soft agar after inhibition of CIP2A by siRNA in HeLa, SiHa and Caski cells. Crosstalk of CIP2A and HPV16 E7 was investigated by immunohistochemistry in cervical cancer tissues and by real-time PCR and western blot analysis after HPV16 E7 inhibition by siRNA in SiHa cells.

Results

CIP2A was transcribed in 73.3% of cervical cancer tissues (n = 15) but not in normal cervical tissues (n = 8). CIP2A protein was detected in 52.8% of cervical cancer (n = 72) and 12.5% of CIN III tissues (n = 24) but not in normal (n = 15), CIN I (n = 21) or CIN II samples (n = 25). CIP2A protein level was positively associated with HPV16 E7 level in cervical cancer tissues. CIP2A expression was markedly reduced after E7 depletion. Moreover, CIP2A depletion reduced c-Myc protein level and impaired proliferation and growth of cervical cancer cells.

Conclusions

CIP2A is overexpressed in cervical cancer and promotes the malignant growth of cervical cancer cells. Its expression is upregulated by HPV16 E7. Therefore, CIP2A plays an important role in carcinogenisis of cervical cancer and shows promise for the diagnosis and treatment of cervical cancer.     

HPV E6/E7mRNA联合TCT检测对宫颈癌早期病变筛查诊断效能的影响

目的 探讨人乳头瘤病毒(HPV)E6/E7mRNA联合液基薄层细胞学检查(TCT)用于宫颈癌早期病变筛查诊断的价值.方法 选取256例接受宫颈癌早期病变筛查患者,均接受HPVE6/E7mRNA检测、TCT检测及病理检查,以病理结果为金标准,分析HPVE6/E7mRNA、TCT单一检测与联合检测对诊断宫颈病变的价值及效能...     

siRNA对宫颈癌细胞系 HPV16 E6基因的作用

目的 构建并筛选出最有效的HIV16 E6基因特异的小干扰RNA(amall interfering RNA，siRNA)表达载体，观察其对宫颈癌细胞中HIV16 E6基因表达的长期影响，探讨E6基因在宫颈癌发生过程中的分子作用机制，为临床HIV感染及宫颈癌治疗探索新方法。方法 构建Hairpin siRNA质粒，稳定转染宫颈癌SiHa细胞，鉴定转染细胞中的质粒DNA，通过Real-Time RT-PCR检测细胞中HPV16 E6mRNA表达，采用Westem-blot检测p53、p21等蛋白的变化。MTT法(四甲基偶氮唑盐微量酶反应比色法)检测SiHa细胞转染siRNA后细胞增殖曲线。结果 HIV16 E6A hairpin siRNA表达载体转染人宫颈癌SiHa细胞，可以在细胞内长期表达siRNA，有效抑制细胞内HIV16 E6基因的表达。E6A siRNA能抑制细胞生长，作用持续达4个月以上。结论 利用siRNA表达载体抑制整合在细胞中的外源HIV E6病毒癌基因可能是治疗HIV感染和宫颈癌的一种新的理想方法。     

Human papillomavirus,cervical screening and ethical considerations

Human papillomavirus (HPV) is an extremely prevalent virus linked to multiple cancers, but most notably cervical cancer. The cervical screening programme in the UK has developed significantly since it was first introduced in 1964. This, together with the introduction of HPV vaccination, has made a huge difference to the early diagnosis and mortality for cervical cancer patients. Uptake of screening is paramount and this article addresses the barriers to this, and how these might be overcome. Other ethical issues around the topic of HPV, vaccination and screening include discrimination around sexual behaviour and orientation, education, vaccination availability and vertical transmission. This is an area of medicine that continues to develop and evolve as we understand more about HPV and how to tackle it.     

Human papillomavirus type 18 and other risk factors for cervical cancer in Jakarta, Indonesia

Infection with human papillomavirus (HPV) has now been established as a necessary cause of cervical cancer. Indonesia is a country with a high cervical cancer incidence and with the world's highest prevalence of HPV 18 in cervical cancer. No information exists about the prevalence of HPV 18 or other HPV types in the Indonesian population. We conducted a hospital-based case-control study in Jakarta, Indonesia. A total of 74 cervical carcinoma cases and 209 control women, recruited from the gynecological outpatient clinic of the same hospital, were included. All women were HPV typed by the line probe assay, and interviews were obtained regarding possible risk factors for cervical cancer. HPV was detected in 95.9% of the cases and in 25.4% of the controls. In the control group, 13.4% was infected with a high-risk HPV type. HPV 16 was detected in 35% of the case group and in 1.9% of the control group and HPV 18 was identified in 28% of the case group and in 2.4% of the control group, suggesting that the oncogenic potentials of HPV 16 and HPV 18 in Indonesia are similar. In addition to HPV infection, young age at first intercourse, having a history of more than one sexual partner, and high parity were significant risk factors for cervical cancer. Within the control group, we did not identify determinants of HPV infection. We hypothesize that the high prevalence of HPV 18 in cervical cancer in Indonesia is caused by the high prevalence of HPV 18 in the Indonesian population.     

Extended effects of human papillomavirus 16 E6-specific short hairpin RNA on cervical carcinoma cells

Most cervical carcinomas express high-risk human papillomavirus (HPV) E6 and E7 oncogenes. Small interfering RNA can mediate sequence-specific inhibition of gene expression in mammalian cells. To find a most effective short hairpin RNA (shRNA) for HPV16 E6 messenger RNA (mRNA) and investigate the extended effects of the HPV16 E6 shRNA on cervical carcinoma cells, we stably transfected SiHa cells with four shRNA expression vectors (E6A-D). HPV16 E6A shRNA was found to be the most efficient in our study, which caused the reduction of HPV16 E6 mRNA to 10% in SiHa cells but did not reduce HPV18 E6 mRNA expression in HeLa cells. We subsequently demonstrated that E6A could stably express shRNA and effectively reduce HPV16 E6 and E7 viral genes expression in SiHa cells for more than 4 months. After E6 and E7 repression, there was a dramatic accumulation of p53, p21, and hypophosphorylated pRb proteins in cells. Furthermore, cell proliferation, colony formation ability, tumorigenicity, and in vitro cell invasive capability were suppressed substantially in E6A-transfected cells. These results suggest that the use of shRNA expression vector may be a potential approach for the treatment of persistent HPV infection and HPV-positive cervical carcinoma.     

Human papillomavirus prevalence and predictors for cervical cancer among high-risk women from Rio de Janeiro, Brazil

We assess the prevalence of human papillomavirus (HPV) and cofactors for cervical severe disease, as contribution for vaccine strategies at the right moment in which Brazilian health authorities have approved an anti-HPV vaccine. A case-control study was undertaken with 201 women who attended a public health service with previous abnormal cytology. The HPV status was ascertained by consensus primers My09/11 and typed by 6, 11, 16, 18, 31, 33, 35, and 58 specific primers. Patients diagnosed with high-grade squamous intraepithelial lesions (HSIL) and cervical cancer were referred as cases (n = 84). Patients with normal/inflammatory cervix or carrying benign cervical lesions were included in controls (n = 117). The overall prevalence of HPV infection was 75.6%, with 91.7% among cases. In spite of HPV 16 being the most frequent type (53.3%), 27.6% of infections were attributed to nonvaccine types. High-risk HPV were strongly associated to older women (OR = 6.7). Otherwise, age at the first intercourse (OR = 7.10), three or more parities (OR = 3.05), abortion episodes (OR = 4.80), and smoking (OR = 3.83) conferred a heavy effect in younger women. Among mediators affecting the progress from HSIL to cancer, age played the main role in easing the progression (OR = 1.09, P = 0.002) followed by education level (OR = 4.20, P = 0.066). White ethnia showed to be a protective factor (OR = 0.32, P = 0.055). Predictors from HPV exposure to malignant disease include demographic and behavioral factors. Public policies such as improvement of education and continued prevention campaigns might contribute to reduce this picture. This work also gives background, in identifying a target population, for implementing future vaccine strategies.