Correlation analysis of human papillomavirus E6/E7 mRNA detection with diagnosis,prognosis and recurrence risk in patients with cervical epithelioma

BACKGROUND Cervical intraepithelial neoplasia(CIN)is an important precursor of cervical cancer.Early detection and treatment can reduce the incidence of cervical cancer.AIM To investigate the detection rate of human papillomavirus(HPV)E6/E7 mRNA in cervical tissue of patients with different types of epithelial cell neoplasia(CIN)and its relationship with CIN progression and diagnosis.METHODS One hundred women with HPV infection detected by cervical exfoliation cytology between January 2022 and January 2023 were retrospectively selected.These patients were graded CIN based on colposcopy and cervical pathology.The positive expression rates of HPV E6/E7 mRNA and HPV[polymerase chain reaction(PCR)-reverse dot crossing]were compared among all groups.Patients with HPV E6/E7 mRNA expression in the grade 1 CIN group were followed up for 1 yr.The relationship between atypical squamous epithelium and high malignant epithelial neoplasia was investigated by univariate and multivariate analysis.RESULTS The diagnostic sensitivity,specificity,and sensitivity of PCR-reverse point hybrid ization technology for secondary CIN were 70.41%,70.66%,and 0.714,respectively.Sensitivity and specificity for secondary CIN were 752%and 7853%,respectively,the area under the curve value was 0.789.Logistic Multifactorial model analysis revealed that the HPV positive rates and the HPV E6/E7 mRNA positive rates were independent risk factors of CIN grade I(P<0.05).In CIN grade I patients with positive for HPV E6/E7 mRNA,in its orientation to grade CIN patients,in its orientation to grade CIN patients,at 69.2%,compared with patients negative for HPV E6/E7 mRNA(30.8%),significant difference(P<0.05).CONCLUSION HPV E6/E7 mRNA and HPV(PCR-reverse dot hybrid)positive expression have a close relationship with CINgrade disease progression and is an independent risk factor for high-grade CIN lesions.

Sequence analysis of human papillomavirus type 16 E6E7 gene from cervical carcinoma biopsies of Chinese patients in Shandong province

Objective To study the structure specificity of HPV16E6E7 gene from cervical carcinoma biopsies of patients in Shandong province. Methods The tissue DNAs were extracted from cervical carcinoma biopsies of 14 patients of Chinese from Shandong province. By PCR method using HPV multiple primers, the HPV types were identified in cervical carcinoma tissues, Using the tissue DNA of the 2 cases with the infection of HPV16 type only as templates, HPV16E6E7 gene was amplified by PCR respectively, then inserted the HPV16E6E7 gene into pALTER I vector, and obtained the recombinant plasmid pALTER HPV16E6E7. The constructs were sequenced using Sanger method, and then compared with the sequence of HPV16E6E7 gene of the prototype. Results Sequencing results showed that HPV16E6E7 gene in the 2 cases had sequence diversity from that of the prototype, and a total of five nucleotide exchanges were detected, four of these led to amino acid exchanges. Conclusion There are structure differences between the HPV16E6E7 of Chinese and that of the prototype.

The Analysis of Human Papillomavirus Type 16 E6/E7 Genetic Variability in Jingjiang, Jiangsu Province, China

Human papillomavirus 16 (HPV-16) is a major high-risk type causing cervical cancer. The E6 and E7 genes in HPV-16 are the major virulent genes in HPV, and we wanted to investigate the polymorphism of E6 and E7 genes of HPV-16 originated from Jingjiang, Jiangsu province. In research, HPV-16 sample was collected, and the E6 and E7 genes fragments were amplified by PCR assay. The sequences of E6 and E7 genes were used for phylogenetic analysis by Mega 5.0 software. By comparison, the major mutations of E6 gene were T178G (41.67%) and G658A (17%), as well as C58G, T61A and G188C (14%), T61C and C656A (11%). The mutation of E7 gene was mainly C491A and T935A (23%);G514C, G937C and G519C (11%). The 68 nucleotide site deficiency (69.4%) of E6 and 535 nucleotide site deficiency (23%) of E7 were dominant. In our study, we did not find the relevance between the E6 and E7 genes mutations and pathologic severity. But we think the E6 and E7 genes mutation of HPV-16 might affect vaccine prevention and treatment effect.

Apoptosis of HeLa cells inhibited by human papillomavirus type 16 E6 protein interacting with hDaxx

Objective: To study the interaction of human papillomavirus type 16 (HPV16 E6) protein and human death domain associated protein (hDaxx) and its effect on apoptosis of HeLa cells to provide the experimental basis for exploring the oncogenic mechanism of HPV16 E6 protein. Methods: Recombinant vector of pGADT7/E6 or pGBKT7/hDaxx was con- structed. The interaction of E6 protein and hDaxx was detected by yeast two-hybrid system. Their expression in yeast was detected by Western blotting. The eukaryotic plasmids of E6 and hDaxx were co-transfected into HeLa cells. Apoptosis was induced by 5-FU. The apoptotic rate was measured by flow cytometry (FCM). Results: E6 protein had intracellular interaction with hDaxx. The apoptotic rate was rising with the increase in the transfection quantity of pcDNA3.1 (-) / hDaxx in pcDNA3.1 (-) /E6 and pcDNA3.1 (-) / hDaxx co-transfected cells. The difference was significant ( P < 0. 01). Conclusion: There is intracellular interaction between HPV16 E6 protein and hDaxx. The over-expression of hDaxx can increase the sensitivity of E6 protein positive HeLa cells to 5-FU. The effect was in a dose dependent manner. HPV16 E6 protein inhibited the apoptosis of HeLa cells by interacting with hDaxx.

Transient over-expression of human papillomavirus type 16 E6 protein down-regulate the secretion of TNF-αor IL-1β LPS-induced from macrophages

In order to provide the experimental basis for the further studies on the oncogenic mechanism of the E6 protein from human papillomavirus type 16 （HPV16）, the eukaryotic expression vector pcDNA3.1 （-）/E6 was used for the study on the effect of E6 protein to influence the secretory activity of LPS-induced 3MP-1-macrophages, and the reconstructed plasmid pcDNA3.1 （-）/E6 was transfected into THP-1-macrophages. The expression of E6 gene was assayed in macrophage lysates by using Western blot analysis and the level of TNF-α or IL-1β was examined by ELISA. All of data were analyzed by SPSS12.0. As demonstrated by Western blot analysis, the expression of E6 protein with a molecular weight of about 18 kDa by plasmid pcDNA3.1 （-）/E6 in THP-1-macrophages could be detected. However, as demonstrated by ELISA assay, the level of TNF-α or IL-1β in lysates of THP-1-macrophages showed an obvious difference between the pcDNA3.1 （-）/E6 group and the LPS control group or the pcDNA3.1 （-） control group （P 〈 0.01）, but no significant difference existed between pcDNA3.1 （-） control group and LPS control group （ P 〉 0.05）. All these results illustrate that the transient over-expression of HPV6 E6 protein reduces the production of TNF-α and IL-1β induced by LPS in THP-1-macrophages.

Human papillomavirus in upper digestive tract tumors from three countries

AIM: To clarify human papillomavirus (HPV) involvement in carcinogenesis of the upper digestive tract of virological and pathological analyses. METHODS: The present study examined the presence of HPV in squamous cell carcinomas of the oral cavity (n = 71), and esophagus (n = 166) collected from Japan, Pakistan and Colombia, with different HPV exposure risk and genetic backgrounds. The viral load and physical status of HPV16 and HPV16-E6 variants were examined. Comparison of p53 and p16INK4a expression in HPV-positive and HPV-negative cases was also made. RESULTS: HPV16 was found in 39 (55%) oral carcinomas (OCs) and 24 (14%) esophageal carcinomas (ECs). This site-specific difference in HPV detection between OCs and ECs was statistically significant (P < 0.001). There was a significant difference in the geographical distribution of HPV16-E6 variants. Multiple infections of different HPV types were found in 13 ECs, but multiple infections were not found in OCs. This difference was statistically significant (P = 0.001). The geometric means (95% confidence interval) of HPV16 viral load in OCs and ECs were 0.06 (0.02-0.18) and 0.12 (0.05-0.27) copies per cell, respectively. The expression of p16INK4a proteins was increased by the presence of HPV in ECs (53% and 33% in HPV-positive and-negative ECs, respectively; P = 0.036), and the high-risk type of the HPV genome was not detected in surrounding normal esophageal mucosa of HPV-positive ECs. CONCLUSION: Based on our results, we cannot deny the possibility of HPV16 involvement in the carcinogenesis of the esophagus.

Expression of Messenger RNA of Oncoproteins E6 and E7 of Human Papilomavirus in Women with Negative Oncotic Cytologies, Epithelial Squamous Atypias of Undefined Significance and Low-Grade Intraepithelial Lesions

Objectives: To verify the prevalence of E6/E7 RNAm expression of HPV in patients with negative cervicovaginal cytology, ASC-US and LSIL;to correlate with negative anatomopathological exams and/or squamous intraepithelial neoplasy grade I (SIN 1) of the lower genital tract (LGT);to relate the RNAm expression with viral infection types;to assess the genotyping in single infections. Methods: Findings from 825 women submitted to E6/E7 RNAm survey and 422 women submitted to LGT biopsies were analyzed. Results: A larger percentage of E6/E7 in ASC-US and LSIL cytologies occurred. Negative results of RNAm expression were in accordance with negative cytologies and negative anatomopathological exam. In positive cases, the infection by a single HPV type was most prevalent, with the type 16 being the most common. Conclusions: the expression of mRNA was most prevalent in ASC-US and LSIL cytologies, comparing with the negative ones. The findings of SIN 1 biopsies were related to the positive expression of mRNA and negative cytologies;the negative expression was in agreement with negative anatomopathological exam. The infection by a HPV type was more frequent in cases of positive expression, the HPV type 16 being most frequently found. Patients with low grade intraepithelial lesion cytologies had a higher percentage of multiple infections.

高级别鳞状上皮内病变中E6/E7 mRNA检测诊断价值的Meta分析

目的:探讨HPV E6/E7 mRNA检测对高级别鳞状上皮内病变(HSIL)的诊断价值。方法:通过检索PubMed、Cochrane、中国知网等数据库以及文献追溯的途径,收集HPV E6/E7 mRNA检测用于诊断HSIL的研究数据进行Meta分析。结果:检索到8篇文献,共4417例。HPV E6/E7 mRNA检测方法诊断出HSIL的合并敏感度、合并特异度、阳性似然比和阴性似然比分别为0.88(95%CI为0.82~0.92)、0.63(95%CI为0.55~0.71)、2.40(95%CI为2.00~2.80)和0.19(95%CI为0.14~0.26);SROC曲线下面积(AUC)为0.82(95%CI为0.79~0.86),诊断比值比为13.00(95%CI为10.00~16.00)。结论:HPV E6/E7 mRNA检测诊断HSIL的敏感度高、诊断效果好、应用价值大等,值得推广应用。

口腔鳞状细胞癌中人乳头瘤病毒16型转化基因E_7的检测

【目的】准确检测口腔鳞状细胞癌及正常口腔粘膜中人乳头瘤病毒 16型 (HPV16)的感染状况 ,探讨人乳头瘤病毒 16型与人口腔鳞状细胞癌之间的关系。【方法】从 30例未经治疗的口腔鳞状细胞癌患者肿瘤组织及 30例健康志愿者正常口腔粘膜切取标本 ,液氮冷冻保存 ;常规提取组织DNA ,设计HPV16E7转化基因特异性引物 ,进行聚合酶链反应 ,对PCR结果进行检测 ,记录结果。【结果】 30例口腔鳞状细胞癌患者中检测到 11例HPV16阳性 ,感染率 36 7% ;30例正常对照组中检测到 4例HPV16阳性 ,感染率 11 1%。经 χ2 检验 ,两者差别有显著性。【结论】人乳头瘤病毒 16型与人口腔鳞状细胞癌有一定关系 ,但HPV16在人口腔鳞状细胞癌发生发展过程中所起的作用还有待进一步研究。

HPV E6/E7 mRNA阳性而阴道镜检查阴性患者中漏诊HSIL及以上病变的危险因素分析

目的探讨HPV E6/E7 mRNA阳性而阴道镜检查阴性患者中漏诊HSIL及以上病变的危险因素。方法对HPV E6/E7 mRNA初筛阳性患者,进一步进行液基薄层细胞学(TCT)检查、HPV分型检测、阴道镜检查及病理活检。对其中466例阴道镜检查阴性患者经病理确诊为HSIL及以上的病例进行分析,探讨这部分患者漏诊HSIL及以上病变的危险因素。结果以病理为金标准,466例HPV E6/E7 mRNA阳性而阴道镜检查阴性的患者中检出42例组织学HSIL,检出率为9.01%(42/466);腺癌1例,检出率为0.21%(1/466)。466例的TCT结果NILM、ASC-US、LSIL、ASC-H、HSIL分别为80.47%、13.95%、4.29%、0.43%、0.86%。随着TCT异常级别的升高(因ASC-H例数少,将ASC-H并入HSIL中进行统计分析),组织病理为HSIL病例的检出率呈上升趋势,差异有统计学意义(χ^(2)=14.440, P=0.002)。HSIL及以上病例检出率在HPV16中最高,但与HPV18/45、其他型别间比较,差异无统计学意义(P>0.05)。随着年龄的增加,组织学HSIL及以上病例的检出率呈上升趋势,且差异有统计学意义(χ^(2)=13.328, P=0.020)。Logistic回归分析也显示年龄是HPV E6/E7m RNA阳性而阴道镜检查阴性患者漏诊HSIL及以上病例的危险因素,其O^R值为1.070,95%CI:1.027-1.114 (P<0.05)。结论对于HPV E6/E7 m RNA阳性的女性,如果阴道镜检查阴性,需要参考TCT结果并结合年龄因素的影响,必要时行宫颈活检和/或ECC,以免遗漏HSIL及以上病例。

HPV16E7-HSP70 Hybrid DNA Vaccine Induces E7-Specific Cytotoxic T Cells and Antitumor Immunity

Using human papillomavirus type 16 （HPV16） E7 as an antigen and Heat Shock Protein 70 as adjuvant, we constructed a DNA vaccine by linking HSP70 gene to E7^C91G; gene. Mice, after being immunized with E7^C91G;-HSP70, E7^C91G/HSP70, E7^C91G, and wild E7 DNA vaccines respectively, produced E7 specific CD8^＋ T-cell precursor frequencies of 280.33±2.52, 144.34±4.04, 164.34±5.13 and 82.33±3.51 respectively within every 1 × 10^5 mouse splenocytes. This proves that E7^C91G-HSP70 fusion vaccine can significantly enhance the E7 specific cellular immunity within the mice body（p〈0. 01）. After being immunized with E7^C91G-HSP70 fusion vaccine, tumor-bearing mice of the group being treated have significantly longer latency and survival periods, comparing with other three categories of E7 vaccines. Experiment shows that this vaccine has a significant effect on enhancing E7 positive tumor-treatment within mice body. After being immunized with E7^C91G-HSP70 vaccine, there were no pathological changes found in livers, kidneys and spleens of the mice, which proves that the vaccine is quite safe. After all, E7^C91G-HSP70 fusion vaccine has a much stronger tumor- treatment effect than thai of wild type E7 DNA vaccine.

Transforming Activity of a Novel Mutant of HPV16 E6E7 Fusion Gene

An optimized recombinant HPV16 E6E7 fusion gene (HPV16 ofE6E7) was constructed according to codon usage for mammalian cell expression, and a mutant of HPV16 ofE6E7 fusion gene (HPV16 omfE6E7) was generated by site-directed mutagenesis at L57G, C113R for the E6 protein and C24G, E26G for the E7 protein for HPV16 ofE6E7 [patent pending (CN 101100672)]. The HPV16 omfE6E7 gene constructed in this work not only lost the transformation capability to NIH 3T3 cells and tumorigenicity in SCID mice, but also maintained very good stability and antigenicity. These results suggests that the HPV16 omfE6E7 gene should undergo further study for application as a safe antigen-specific therapeutic vaccine for HPV16-associated tumors.

HPV18 E6 and E7 Intratumour Heterogeneity in Esophageal Cancer

The development of esophageal cancer accompanied by the presence of human papillomavirus (HPV) DNA into the host genome. By evaluating the expression of this virus for tumor cell origin and also their cell grows and migrations, we examined esophageal cancer clonality in the context of intra-tumor heterogeneity. In this research, we have checked the expression of HPV18 E6 and E7 in different single cell clones by the manual cell picking method in the HPV positive esophageal cancer (EC109), EC109 cell line used as a negative control, and Hela cell line used as the positive control. Quantitative real-time PCR (QRT-PCR) was run to detect the expression levels of HPV E6 and E7, Cell Counting Kit-8 (CCK-8) assay was used to examine cell proliferation, invasion assays performed using Costar chambers and wounding assay to study cell migrations in vitro. We investigated the intra-tumor heterogeneity of HPV E6 and E7 in esophageal cancer and the evaluation of the growth and migrations at the clonal level, using 10 single cell clones. In particular clones, C7 & C10 displayed a highly variable expression in both HPV E6 and E7 and weak in four clones (C1, C3, C4, and C9) consequently, the cell invasion, proliferation, and migration increase with increasing the level of HPV expression and inverse. In conclusion, the resulting based on single cell cloning showed the relationship between HPV and cell growth and migration in esophageal cancer. Future study in HPV DNA integration needed to explore the mains specific integration site of HPV DNA in esophageal cancer and molecular monitoring of the HPV for future prevention researches and also effective therapeutic strategies.

Two less common human microRNAs miR-875 and miR-3144 target a conserved site of E6 oncogene in most high-risk human papillomavirus subtypes

Human papillomaviruses （HPVs） including high.risk （HR） and low-risk （LR） subtypes have distinguishable variation on both genotypes and phenotypes. The co- infection of multiple HR-HPVs, headed by HPV16, is common in cervical cancer in female. Recently accu- mulating reports have focused on the interaction be- tween virus and host, particularly the role of human microRNAs （miRNAs） in anti-viral defense by targeting viral genome. Here, we found a well-conserved target site of miRNAs in the genomes of most HR-HPVs, not LR-HPVs, by scanning all potential target sites of human miRNAs on 24 HPVs of unambiguous subtypes of risk. The site is targeted by two less common human miR- NAs, miR-875 and miR-3144, and is located in E6 onco- gene open reading frame （ORF） and overlap with the first alternative splice exon of viral early transcripts. In validation tests, miR-875 and miR-3144 were identified to suppress the target reporter activity markedly and inhibit the expression of both synthetically exogenous E6 and endogenous E6 oncogene. High level of two miRNAs can inhibit cell growth and promote apoptosisin HPV16-positive cervical cancer cells. This study pro- vides a promising common target of miRNAs for most HR-HPVs and highlights the effects of two low ex- pressed human miRNAs on tumour suppression.

HPV16-E6蛋白在梅州地区客家人食管鳞癌组织及非癌组织中的表达

目的通过分析HPV16-E6蛋白在食管正常组织、不典型性增生组织和癌组织中的表达,探讨HPV16-E6蛋白在梅州地区客家人食管鳞癌组发生、发展中的生物学意义。方法应用免疫组织化学方法检测对68例食管正常黏膜组织、20例上皮不典型增生组织及68例癌组织中HPV16-E6蛋白的表达情况进行研究。结果 HPV16-E6蛋白阳性率在正常食管黏膜组织中为14.7%,在上皮不典型性增生组织中为45.0%,在癌组织中为70.6%,各组间差异有统计学意义(P<0.05)。结论 HPV16-E6蛋白与梅州地区客家人食管鳞癌的发生、发展关系密切。

人乳头瘤病毒16型E_6基因的T-A克隆及在真核细胞中的表达

由于ＨＰＶ１６Ｅ６蛋白能诱导机体保护性免疫反应，可作为基因治疗的靶抗原。用杆状病毒昆虫细胞表达系统制备了ＨＰＶ１６Ｅ６基因工程蛋白，拟用于宫颈癌细胞系小鼠模型抗癌的免疫治疗。用ＰＣＲ技术从ＨＰＶ１６基因组中扩增获得转化基因Ｅ６的完整ＯＲＦ，按ＴＡ策略将其克隆到自行制备的杆状病毒转移载体ｐＶＬ１３９３Ｔ尾载体中，置于杆状病毒ＡｃＭＮＰＶＰｏｌｈ晚期启动子控制之下，用此重组转移质粒ｐＶＬ１３９３Ｅ６与杆状病毒ＤＮＡ共转染昆虫细胞Ｓｆ９，经噬斑筛选获得带有编码Ｅ６蛋白基因的重组杆状病毒株，并在昆虫细胞Ｓｆ９中表达为非融合性Ｅ６蛋白。ＳＤＳＰＡＧＥ电泳分析其分子量约为１８ｋＤ，免疫印迹实验表明，此重组蛋白能被兔抗ＨＰＶ１６Ｅ６抗体所识别。

Anal human papilloma viral infection and squamous cell carcinoma:Need objective biomarkers for risk assessment and surveillance guidelines

High grade anal intraepithelial neoplasia due to human papilloma viral(HPV)infections is a precursor lesion for squamous cell carcinoma especially in high risk populations.Frequent examination and anal biopsies remain unpopular with patients;moreover they are also risk factors for chronic pain,scarring and sphincter injury.There is lack of uniform,surveillance methods and guidelines for anal HPV specifically the intervals between exam and biopsies.The aim of this editorial is to discuss the intervals for surveillance exam and biopsy,based on specific HPV related biomarkers?Currently there are no published randomized controlled trials documenting the effectiveness of anal screening and surveillance programs to reduce the incidence,morbidity and mortality of anal cancers.In contrast,the currently approved screening and surveillance methods available for HPV related cervical cancer includes cytology,HPV DNA test,P16 or combined P16/Ki-67 index and HPV E/6 and E/7 mRNA test.There are very few studies performed to determine the efficacy of these tests in HPV related anal precancerous lesions.The relevance of these biomarkers is discussed in this editorial.Longitudinal prospective research is needed to confirm the effectiveness of these molecular biomarkers that include high risk HPV serotyping,P16 immuno-histiochemistry and E6/E7 mRNA profiling on biopsies to elucidate and establish surveillance guidelines.

Human papillomavirus(HPV) E6/E7 mRNA detection in cervical exfoliated cells:a potential triage for HPV-positive women

Cytology triage has been generally recommended for human papillomavirus （HPV）-positive women, but is highly dependent on well-trained cytologists. The present study was designed to explore whether HPV E6/E7 mRNA detection in cervical exfoliated cells can be a potential triage for HPV-positive women from a clinic-based population. Both the primary HPV testing and Papanicolaou （Pap） test were performed on all eligible HPV-positive women. HPV E6/E7 mRNA was detected by QuantiVirus HPV E6/E7 mRNA assay in cervical exfoliated cells. All HPV-positive women underwent colposcopy and further biopsy if indicated. The data were assessed by Pearson＇s Chi-squared test and the receiver operating characteristic curve. A total of 404 eligible HPV-positive women were enrolled. Positive rate of E6/E7 mRNA in high-grade squamous intraepithelial lesion （HSIL） cases was higher than that in low-grade squamous intraepithelial lesion （LSIL） or normal cases. There was no statistical difference found between mRNA and cytological testing with sensitivity （89.52% vs. 86.67%, P=0.671）, specificity （48.96% vs. 48.96%, P=1.000）, positive predictive value （39.00% vs. 38.24%, P=1.000）, and negative predictive value （92.76% vs. 90.97%, P=-0.678） for detecting ≥HSIL. HPV E6/E7 mRNA detection in cervical exfoliated cells shows the same performance as Pap triage for HSIL identification for HPV-positive women. Detection of HPV E6/E7 mRNA may be used as a new triage option for HPV-positive women.

Clinical Value of Human Papillomavirus E6/E7 mRNA Testing in Patients with Atypical Squamous Cells of Undetermined Significance and Low-Grade Squamous Intraepithelial Lesion

Objective: To explore the clinical significance of the quantitative detection of human papillomavirus(HPV) E6/E7 mRN A in triage of patients with atypical squamous cells of undetermined significance(ASC-US) and low-grade squamous intraepithelial lesion(LSIL).Methods: A cross-sectional screening study was conducted among women who underwent outpatient gynecological screening at the Obstetrics and Gynecology Hospital of Fudan University from September 2015 to July 2016. A total of 500 patients from our hospital with ASC-US or LSIL based on cytology testing were subjected to HPV DNA and HPV E6/E7 mRNA quantitative analysis.Results: The specificity of the HPV E6/E7 mRNA test for detecting ≥high-grade squamous intraepithelial lesion(HSIL+) was statistically higher than that of the HPV DNA test(61.3% vs. 40.0%, P< 0.05), whereas there was no significant difference in the sensitivity of HPV E6/E7 mRNA test and HPV DNA test(90.0% vs. 95.0%, P > 0.05). The positive rates of HPV in the participants tested by HPV E6/E7 mRNA and HPV DNA were, respectively, 42.8%(214/500) and 62.8%(314/500), with statistical significance(P < 0.05).Conclusions: The HPV E6/E7 mRNA test was slightly less sensitivity than that of the HPV DNA test for diagnosing HSIL+ in patients with ASC-US and LSIL, but the difference was not significant, although the specificity of the former was significantly higher. HPV E6/E7 mRNA detection can effectively reduce overdiagnosis and overtreatment of patients with ASC-US and LSIL and has important clinical value in triage of patients with ASC-US and LSIL.

Increased expression of 70 kD heat shock protein in cultured primary human keratinocytes induced by human papillomavirus 16 E6/E7 gene

Background Heat shock protein 70 （HSP70） is expressed highly in epithelial tumours associated closely with human papillomavirus 16 （HPV16） infections. However, evidence about the direct relationship between HSP70 expression and HPVs infections are still lacking. In the present study, we examined the expression of HSP70 in keratinocytes introduced with HPV16 E6/E7 oncogenes. Methods Stable transfected cells were established by transfection of the plasmids pLXSN16E6/E7 into cultured primary keratinocytes and subsequently selected by plasmid specific selection antibiotic （G418） at the required concentration. The expression of HSP70 in pLXSN16E6/E7 transfected keratinocytes was determined by Western blot. The correlation of HSP70 expression and E6/E7 transfeetion was further confirmed by doubly labelled immunofluorescent staining. Results Compared to non-transfected keratinocytes, there was a significant trend for higher levels of HSP70 in pLXSN16E6/E7 transfected keratinocytes. Doubly labelled immunofluorescent staining experiment showed that the co-localization of HPV16 E6/E7 and HSP70 in transfeeted keratinoeytes was observed and increased expression of HSP70 was strongly associated with the transfection of HPV16 E6/E7. Conclusions Our studies demonstrated increased levels of HSP70 proteins in keratinocytes stably transfected by HPV16 E6/E7 oncogenes. It suggests that the expression of HSP70 is modulated by HPV16 E6/E7 proteins, which may be involved in HPV16 E6/E7 induced immortalization.