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.

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.

HPV DNA、E6/E7 mRNA在各子宫颈病变及子宫颈鳞状细胞癌中的表达

目的探讨HPV DNA和E6/E7 mRNA在各子宫颈病变及子宫颈鳞状细胞癌(cervical squamous cell carcinoma,CSCC)中的表达,并分析其与CSCC发生、发展的相关性。方法应用PCR-反向杂交法和支链DNA技术对210例低级别鳞状上皮内病变(low-grade squamous intraepithelial lesion,LSIL)、200例高级别鳞状上皮内病变(high-grade squamous intraepithelial lesion,HSIL)及240例CSCC进行HPV DNA及E6/E7 mRNA的检测。结果 HSIL、CSCC中,HPV DNA百分率分别为100.00%、89.58%,呈递减趋势(P<0.001);E6/E7 mRNA百分率分别为52.50%、95.83%,呈递增趋势(P<0.001);HPV DNA+/mRNA+的百分率分别为52.50%、87.50%,呈递增趋势(P<0.001);HPV DNA-/mRNA-的百分率分别为0、6.25%,呈递增趋势(P<0.001)。LSIL、HSIL中,HPV DNA百分率均大于E6/E7 mRNA百分率,差异有统计学意义(P<0.001);而CSCC中HPV DNA百分率均小于E6/E7 mRNA百分率,差异有统计学意义(P=0.008)。结论 HPV DNA在LSIL、HSIL中的表达较E6/E7 mRNA高;CSCC中HPV DNA表达较E6/E7 mRNA低,因此HPV DNA标志物可能与CSCC的发生过程关系较为紧密;E6/E7 mRNA标志物可能与CSCC的发展相关性更高。

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。

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。

不同地区HPV16 E7基因的克隆及序列差异分析

采用PCR扩增、pGEM T载体克隆和核苷酸序列分析的方法对一例武汉地区及两例五峰县高发区宫颈癌患者体内HPV16型的E7基因编码区进行序列分析并与野生型 (德国标准株 )及已发表的HPV16湖北株 (HPVHB)进行了比较。结果发现武汉地区HPV16型E7基因仅第 5 4位出现一个同义突变 ,而高发区HPV16型E7基因存在差异 ,第 77位氨基酸由精氨酸 (Arg)变为半胱氨酸 (Cys) ,第 96位由谷氨酰氨酸 (Gln)变为精氨酸 (Arg) ,E7蛋白的二级结构及亲、疏水性也相应改变 。

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.

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.

HPV16/18E_6和P_(53)基因蛋白在喉鳞癌中的表达及其相关性研究

目的 探讨人乳头瘤病毒16 / 18型早期蛋白E6 (HPV16 / 18E6 )、P53基因蛋白在喉鳞癌(L SCC)中的表达及其相关性。方法 免疫组化SABC法检测HPV16 / 18E6 和P53基因蛋白在L SCC及声带息肉中的表达。结果 HPV16 / 18E6 在声带息肉中未见表达,在L SCC中的表达率为4 0 .0 % ,差异有显著性(P <0 .0 5 )。P53基因蛋白在声带息肉与L SCC中的表达率分别为6 .6 6 %和6 6 .2 % ,差异有显著性(P <0 .0 5 )。HPV16 / 18E6 及P53基因蛋白的表达与L SCC临床分期、淋巴结转移有关。二者的表达无明显相关性。结论 HPV 16 / 18型感染和P53基因异常与L SCC发生、发展有关。HPV16 / 18E6 与P53基因功能异常无明显相关性。

Induction of SiHa Cells Apoptosis by Nanometer Realgar Suspension and Its Mechanism

The effects of nanometer realgar uterine cervix cancer cell line SiHa cells and suspension on proliferation and apoptosis of human oncogenic genes HPV16E6/E7 were investigated. A ＂micro-jet effiux＂ strategy was used for the preparation of nanometer realgar suspension. SiHa cells were treated with nanometer Realgar suspension in various concentrations （6.25, 12.5, 25 and 50 mg/L） for different durations （12, 24, 48 and 72 h）. The inhibitive effect of nanometer realgar suspension on growth of SiHa cells was detected by MTT method. Special morphological changes of apoptosis were observed by transmission electron microscopy （TEM） and DNA fragments electrophoresis. The apoptotic rate was quantified by flow cytometry （FCM）. The expression of HPV 16E6/E7 mRNA and protein was assayed by RT-PCR and Western blot respectively. The results showed after being treated with 25--50 mg/L nanometer realgar suspension for 48 h, the survival rate of SiHa cells was decreased, and apoptotic rate markedly increased in a time- and concentration-dependent manner. TEM and DNA electrophoresis revealed the special morphological changes of apoptosis. The apoptotic rate of SiHa cells treated with nanometer realgar suspension was significantly higher than in the control group （P〈0.01）, and G0/G1 phase arrest appeared following treatment with nanometer realgar suspension in 25 and 50 mg/L for 48 h. RT-PCR and Western blot assay indicated that nanometer realgar suspension reduced the HPV16E6/E7 gene expression. Nanometer realgar suspension could inhibit the proliferation and induce apoptosis of SiHa cells. The mechanism may be related to the down-regulation of the HPV16E6/E7 gene expression.

常见HR-HPV各型在子宫颈组织中致癌能力的评估

目的探讨高危型人乳头状瘤病毒(high-risk human papilloma viruses,HR-HPV)各型与子宫颈病变进展的关系。方法对135例诊断结果为45例非子宫颈上皮内瘤变(non-squamous intraepithelial lesions,NSIL)、42例低级别鳞状上皮内病变(low-grade squamous intraepithelial lesions,LSIL)、40例高级别鳞状上皮内病变(high-grade squamous intraepithelial lesions,HSIL)、8例子宫颈癌(squamous cervical cancer,SCC)的子宫颈活检组织蜡块进行HPV及E6/E7 mRNA的检测,分别应用PCR-反向杂交法、支链DNA技术(Quantivirus冷光仪)检测。结果 HPV 16在NSIL、LSIL、HSIL、SCC中感染率依次22.22%、21.43%、47.50%、62.50%,呈逐渐递增趋势(P=0.007);单一感染时,HPV 16的E6/E7 mRNA拷贝数依次是1 425.76、3 747.25、1 325.98、86 015.36,与子宫颈病变严重程度具有相关性(P=0.028);多重感染时,HPV 16伴其它HPV感染组在NSIL、LSIL、HSIL、SCC中的E6/E7 mRNA拷贝数呈递增趋势,差异有统计学意义(P=0.040),而其它HPV(不包括HPV 16)感染组表达的mRNA拷贝数与病变严重程度呈负相关性(P=0.044)。结论 HPV 16与子宫颈病变进展的关系最为紧密;在所有HR-HPV中HPV 16的致癌能力最强。