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Objective To explore the clinical value of HPV E6/E7 mRNA in screening lesions at grade CIN2 and above.Methods A total of 120 cases with CIN and suspected cervical cancer treated in our hospital from January 2014 to September 2016 were selected.According to the results of pathological examination,60 patients with CIN2,CIN3 and invasive cancer were selected as the research group.60 patients with normal CIN1 and CIN1 were selected as control group.The results of TCT,HPV DNA and HPV E6/E7 mRNA were detected and analyzed.Results In 120 patients,the total positive rate of TCT was 55%,the total positive rate of HPV DNA was 87.5%,the total positive rate of HPV E6/E7 mRNA 60.8% HPV E6/E7;control group mRNA positive rate of 78.3% was significantly higher than the positive rate of HPV DNA 36.7%(P<0.05),HPV in the study group were E6/E7 mRNA positive rate of 85.0% was significantly lower than the positive rate of HPV DNA(96.7%);HPV E6/E7 mRNA CIN1,quantitative CIN2,CIN3 and invasive carci-noma were(4 867.31 ± 694.84),(8 943.51 ± 986.23),(28 243.10 ± 10 963.21),(3 610.84 ± 412.64)copies/mL;HPV E6/E7 mRNA quantitative CIN3 values were significantly higher than that of CIN 2,CIN1 and infil-tration of cancer and the control group(P<0.05),E6/E7 mRNA CIN2 quantitative HPV the value of CIN1 was significantly higher than that of control group(P< 0.05).Two methods of detecting HPV DNA and HPV E6/E7 mRNA in the screening of CIN2 and above lesions,the sensitivity of 84.5% DNA 93.8% HPV sensitivity was higher than that of HPV E6/E7 mRNA(P<0.05)HPV E6/E7 21.6%;specificity the speci-ficity of mRNA was 53.6% higher than that of HPV DNA(P<0.05),HPV DNA.HPV ROC curve E6/E7 mRNA were 0.581 and 0.681,the difference was statistically significant(P<0.05).Conclusion The specific-ity and accuracy of HPV,E6/E7 and mRNA in detecting cervical lesions over CIN2 and above are higher than those of HPV and DNA.It is of certain diagnostic value for screening cervical lesions over CIN 2 and above.
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Objective To evaluate the effect of combined detection of cervical E 6/E7 and liquid based cytology(TCT)in the screening of cervical cancer.Methods A total of 206 cases of high-risk patients with cervical cancer from March 2015 to March 2017 in Yongding Hospital of Suzhou were taken the cervical E6/E7 detection and TCT two screening methods respectively,and the pathological diagnosis was taken as the gold standard,the efficiency difference among TCT,cervical E6/E7 detection,and the com-bined detection of cervical cancer was compared.Results The sensitivity and negative predictive value of E6/E7 were significantly higher than those of TCT(P<0.05).The specificity of TCT was significantly higher than that of cervical E 6/E7(P<0.05).The accuracy of combined diagnosis was significantly higher than that of cervical E6/E7 and TCT alone.The difference was statistically significant(P<0.05).Compared with the cervical E6/E7,the specificity and positive predictive value of the combined diagnosis were higher,and the difference was statistically significant(P<0.05).The sensitivity and negative predictive value of the combined diagnosis were significantly higher than those of TCT,and the difference was statistically significant(P<0.05).The receiver oper-ating characteristic curve(ROC curve)showed that the area under the combined diagnostic curve(AUC)was significantly higher than that of the cervical E6/E7 and TCT alone,and the difference was statistically significant(P<0.05).Conclusion Cervical E6/E7 detection has a low specificity,and sensitivity of TCT is low;the combined detection of cervical E6/E7 detection and TCT has a high screening accuracy,and can improve the sensitivity and specificity of individual diagnosis.
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Objective To investigate the value of HPV E6/E7 mRNA test combining liquid-based cytology test in cervical cancer screening. Methods A total of 377 samples from Wenzhou People's Hospital from June 2014 to September 2015 were collected and screened by HPV E6/E7 mRNA test combining with liquid-based cytology test , and the results was compared with the findings from the gold criteria of histology and pathology. Results The combination of HPV E6/E7 mRNA test and liquid-based cytology test can enhance the testing sensitivity and specificity. The sensitivity of the combination of HPV E6/E7 mRNA test and liquid-based cytology test for the diagnosis of LSIL was 94.41%, and that for the diagnosis of HSIL was 96.36%. Based on the gold criteria of histology and pathology , the sensitivity , specificity , positive-predictive value and negative predictive value of HPV E6/E7 mRNA test for the diagnosis of HSIL was 90%, 60.67%, 48.53% and 92.49%respectively. The sensitivity, specificity, positive-predictive value and negative- predictive value of liquid-based cytology test for the diagnosis of HSIL was 72.73%, 75.28%, 54.79% and 87.01% respectively. Conclusions The sensitivity of HPV E6/E7 mRNA test is superior to that of liquid-based cytology test , while the specificity of HPV E6/E7 mRNA test is inferior to that of liquid-based cytology test. The negative predictive value of HPV E6/E7 mRNA test is more meaningful than that of liquid-based cytology test. The combination of HPV E6/E7 mRNA test and liquid-based cytology test can enhance the testing sensitivity , but it does not increase the specificity.
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Objective To analyze the differences of positive detection rate and copy number of human papillomavirus (HPV) DNA and E6/E7 mRNA between different grades of cervical lesions, and evaluate their clinical values in early screen?ing of cervical cancer. Methods The cervical exfoliated cell samples from 154 women undergoing biopsy examination and 32 objects undergoing hysterectomy (control group) were collected in Tianjin Central Hospital of Gynecology Obstetrics in 2014. According to the pathological results of cervical biopsy, 154 samples were divided into low-grade squamous intraepi?thelial lesion group (LSIL, n=51), high-grade squamous intraepithelial lesion group (HSIL, n=71), and squamous cell carci?noma group (SCC, n=32). HPV DNA was tested with hybrid capture technology, and E6/E7 mRNA was detected with fluores?cence quantitative hybridization. Immunohistochemistry was performed by detecting E6/E7 protein in all patients after sur?gery or cervical biopsy. Results Combined results of HPV DNA and E6/E7 mRNA demonstrated that the positive detection rate was significantly lower in control group than that of all levels of lesion groups (P 10 000 E6/E7 were significantly increased in high-grade squamous intraepithelial lesion group. Immunohistochemical results showed that the positive detection rate of E6/E7 was significantly lower in control group than that of all levels of lesion groups (P<0.05). The positive rate of E6/E7 was significantly higher in the high-grade squa?mous intraepithelial lesion group than that of low-grade group (P<0.05). Conclusion HPV infection is closely related to cervical abnormalities, which is one of effective measures for early screening of cervical cancer. The negative result of HPV DNA is very helpful to exclude the cervical abnormality, whereas the positive detection of mRNA has great value in predict?ing the disease. Combined results of positive detection and copy number make a comprehensive evaluation for the risk of cer?vical lesions.
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Objective To explore the clinical value of examining HPV E6/E7 mRNA level in assessing cervical le?sions infected with high-risk human papillomavirus (HR-HPV). Methods The cervical epithelial cells were collected from 265 patients with HR-HPV infection, including 100 cases of neoplasia free/inflammation group (control group), 88 cas?es of cervical intraepithelial neoplasia (CIN)Ⅰ, 33 cases of CINⅡ, 28 cases of CINⅢand 16 cases of cervical carcinoma and the transcription of HPV E6/E7 mRNA level was examined using branched DNA (b-DNA) technology. Results The positive rate HPV E6/E7 mRNA were higher in CIN Ⅱ(81.82%), CINⅢ(89.29%) and cervical cancer group (100.00%) than tthat in control group (20.00%) and CINⅠ(35.23%) with significant difference, and there were no significant differences between other groups;The positive rate and transcription level of HPV E6/E7 mRNA in HSIL (high grade squamous intraepi?thelial lesion)and cancer group were significantly higher than normal, ASC(atypical squamous cell carcinoma) and LSIL(low grade squamous intraepithelial lesion) group (P<0.05). Conclusion The transcription level of HPV E6/E7 mRNA may re?flect the activity of the virus and the progression of disease, and could be use as an effective indicator to screen high grade cervical pathological changes and a complementary method of cervical lesion screening.
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To study the apoptosis induced by cisplatin in cervical cancer cell line HeLa and its mechanism, cell growth inhibition of cisplatin on HeLa cells was analyzed by MTT assay. Cell apoptosis was examined by cytometry and Hoechst33258 staining after treatment with cisplatin. The ef- fects of cisplatin on transcription of E6 were analyzed by RT-PCR. The protein expressions of E6, p53, p21, Bax and Bcl-2 were studied by Western blotting. Cisplatin inhibited proliferation in a time- and dose-dependant manner. Cytometically, sub-G1 peak showed higher apoptosis rates in the ex- perimental group than those in the control. Hoechst33258staining exhibited apoptosis induced by cis- platin. RT-PCR revealed that cisplatin decreased transcription of E6. Western blotting showed that cisplatin decreased protein expression of E6 and increased protein expression of p53, p21and Bax. It had no effect on protein expression of Bcl-2. It is concluded that cisplatin can induce apoptosis in HeLa cells by suppressing HPV E6 and thereby restoring the function of p53.
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OBJECTIVE: Telomerase is a ribonucleoprotein enzyme which stabilizes chromosomal structure, thereby inducing cellular immortality. We investigated telomerase activity and human telomerase reverse transcriptase (hTERT) mRNA expression in relation to high-risk human papillomavirus (HPV) DNA presence in cervical carcinomas. METHODS: From December 1995 to December 1999, at the department of obstetrics and Gynecology of Kyung-Hee University Hospital, 32 cervical carcinomas and 5 corresponding nontumor cervical tissues were obtained and the samples were immediately frozen and stored at -70 degree C. Telomerase activity was measured by using telomerase PCR ELISA, a modified version of the TRAP. Analysis of the expression of hTERT mRNA was performed by quantitative RT-PCR and the analysis of the HPV E6 gene was performed by DNA-PCR. RESULT: All of the carcinomas examined exhibited strongly positive for telomerase activity (OD>0.24), whereas telomerase activity was week or not found in the 5 corresponding nontumor cervical tissues. Quantitative RT-PCR analysis demonstrated significantly increased hTERT mRNA expression levels (>0.024) in most carcinomas comparing to control groups. There was no obvious relationship between telomerase activity levels and the clinical parameters examined including age, clinical stage, pathology, differentiation, tumour size, LN involvement and invasion depth except lymphovascular space invasion (p=0.03). In the correlation between the levels of hTERT mRNA expression and telomerase activity, correlation index which was 0.916, shows high correlation (p=0.01). According to the analysis of HPV E6 gene, 29 of 32 (90.6%) carcinomas showed HPV E6 positivity. CONCLUSION: There is a strong association between telomerase activity and hTERT mRNA expression, and up-regulation of hTERT probably plays a role in the progression of cervical carcinomas. Telomerase is at least partially activated by viral oncogenes of high-risk types. There is no obvious relationship between telomerase activity levels and the clinical parameters except LSVI (p=0.03). These findings provides that telomerase may play an important role in the early stage of carcinogenesis.
Subject(s)
Humans , Carcinogenesis , DNA , Enzyme-Linked Immunosorbent Assay , Gynecology , Obstetrics , Oncogenes , Pathology , Polymerase Chain Reaction , Ribonucleoproteins , RNA, Messenger , Telomerase , Up-Regulation , Uterine Cervical NeoplasmsABSTRACT
OBJECT: In this study, to evaluate the putative role of telomerase in gynecologic malignancies (cervical ca, ovarian ca, endometrial ca), we measured telomerase activity in malignant gynecologic tumor tissues and normal tussues, and compared it with prognostic factors in cervical cancer. To evaluate the correlation of telomerase activity and human papillomavirus (HPV) infection in cervical cancer, the analysis of HPV E6 gene was performed. METHOD: Specimens were obtained from 51 women who underwent gynecologic radical operation and 13 normal tissues (from December 1995 to December 1996) in the Department of Obstetrics and Gynecology, Kyung-Hee Univ. Medical Center. With Telomerase PCR ELISA (Boehring Mannheim), modified TRAP (Telomere Repeat Amplication Protocol), we examined telomerase activity of 32 cervical carcinomas, 11 ovarian carcinomas, 8 endometrial carcinomas, 5 normal cervical tissues, 4 normal ovarian tissues and 4 normal endometrial tissues. The analysis of HPV E6 gene was performed by PCR amplication. We compared the abnormally high telomerase activity with prognostic factors, also compared the telomerase activity with the expression of HPV E6 gene in cervical cancer tissues. RESULT: We detected the abnormally high telomerase activity in all cervical carcinomas, 10 of 11 (90.9%) ovarian carcinomas, 6 of 8 (75.0%) endometrial carcinomas, but couldn't detect in each normal tissues. There was statistically no significant difference of telomerase activity levels according to age, clinical stage, pathology, differentiation, LN involvement, depth of invasion and tumor size except lymphovascular space invasion in cervical carcinomas (p<0.05). According to the analysis of HPV E6 gene, 29 of 32 (90.6%) in 32 cervical cancer tissues showed HPV E6 positivity. So it was considered that telomerase activation was closely related with the expression of HPV E6 gene. CONCLUSION: Telomerase activation is associated with immortalization or malignant transformation of gynecologic cancers. The expression of HPV E6 gene is considered to activate telomerase in cervical cancer.
Subject(s)
Female , Humans , Endometrial Neoplasms , Enzyme-Linked Immunosorbent Assay , Gene Expression , Gynecology , Obstetrics , Ovarian Neoplasms , Pathology , Polymerase Chain Reaction , Telomerase , Uterine Cervical NeoplasmsABSTRACT
OBJECTIVE: Human papillomavirus (HPV) is strongly implicated as a causative agent in the etiology of cervical cancer. Of its gene products, E6 and E7 oncoproteins play major roles by inactivation of cellular p53 and pRb tumor suppressor proteins, respectively. However, it has been recently suggested that p53 and/or pRb-independent functions of E6 and E7 are involved in cervical carcinogenesis. The purpose of this study is to identify novel a cellular target, p73, of E6 and to determine how E6 inactivates p73 function, METHODS: The interaction between E6 and p73 were identified by the yeast two-hybrid assay in vivo and the GST pull-down assay in vitro. The function of the interaction was determined by transient transfections using p21 promoter-CAT reporter plasmid. The molecular mechanism underlying the functional significance of the interaction was further assessed by in vivo and in vitro protein degradation assays, and gel mobility shift assays. RESULTS: Yeast two-hybrid and GST pull-down assays indicate a physical interaction between p73 and either HPV-16 or HPV-11 E6 proteins in vivo and in vitro, respectively. Transactivation domain (amino acid residues 1-49) is found to be absolutely required for this interaction. Transient co-expression of E6 significantly inhibits the p73-mediated activation of p21WAF1 promoter in a p53-defective C33A cell line. Using Ga14-p73 fusion protein, we demonstrate that E6 inhibition of p73 transactivation function is independent of sequence-specific DNA binding, which is confirmed by direct electrophoretic mobility shift assay. Moreover, E6 inhibits p73 function by interfering with the activity of the amino-terminal activation domain. The protein degradation assays in vivo and in vitro indicate that p73, unlike p53, is not susceptible to E6-dependent proteolysis. CONCLUSION: Throughout this study, we identified p73 as a novel cellular target of HPV-E6 protein and found that E6 binds p73 through the amino-terminal transactivation domain, and inhibits its transactivation function independent of the protein degradation and DNA binding. These overall results, consequently, suggest that in addition to the inactivation of p53, the functional interference of p73 by HPV-E6 may, at least in part, contribute to E6-mediated cellular transformation.