| Objective To explore the relationship between copy number of HPV-16 E6 and E7 oncogene and cervical carcinoma, and the potential clinical uses of real-time PCR and RT-PCR methods for evaluating HPV-16 E6 and E7 oncogene expression.Methods Plasmids containing HPV-16 E6 or E7 oncogene, which produced by TA cloning method using the PCR products, were used to generate absolute sdandard curves. Three cell lines, 98 cases of TCT samples and 55 cases of cervical tissues were tested for expression of oncogenes of HPV-16 by real-time PCR and RT-PCR analysis and were compared with detection of expression by conventional PCR and RT-PCR analysis. All data were analyzed by SPSS software.Results 1. The correlation coefficients of sdandard curves were larger than 0.99, and the PCR efficiencies were larger than 90%.2. The relative level of HPV-16 E6 and E7 DNA and RNA were CaSki > SiHa > HeLa cells. In SiHa cells,the copy number of E6 DNA and RNA were 9.42 X 103 copy/ng DNA and 2.01 X 104 copy/ng RNA respectively,and the copy number of E7 DNA and RNA were 1.49X 104copy/ng DNA and 4.43 X 104copy/ng RNA respectively. The DNA copy number per ng DNA in CaSki cells was 292 times greater (E6) and 297 times greater (E7) than the copy number in SiHa cells. For RNA copy number, CaSki cells was 41 (E6) and 9.5 (E7) times greater than the copy number in SiHa cells respectively.3. ThinPrep samples were capable of being amplified by real-time PCR and RT-PCR.4. The rate of agreement of real-time and conventional PCR analysis was 88.8%. Between real-time and conventional RT-PCR analysis, The rate of agreement was 87.8%.5.The percentage positive for HPV-16 E6 or E7 DNA in a series of Thinprep cervical cytologic samples was 0% for negative samples, 13.6% for ASCUS, 16.7% for LSIL, 60.0% for HSIL. The percentage positive for HPV-16 E6 or E7 RNA was 0% for negative samples, 4.5% for ASCUS, 10.0% for LSIL, 56.7% for HSIL. There was significant difference between HSIL and negative samples, ASCUS, LSIL (P < 0.05) . The copy number per nanogram for both DNA and RNA E6 and E7 were increased as severity of the lesions progressed. In HSIL, the average copy number per nanogram was 3. 56 X 106 for E7 DNA and 2. 02 X 106 for E7 RNA, which were higher significantly than the number in negative samples, ASCUS and LSIL (P < 0.05) .6. In SCC samples, the percentage positive for HPV-16 E6 or E7 DNAwas 61. 5% and 56. 4% for RNA. There was significant difference between SCC and negative samples, ASCUS, LSIL (P < 0.05) . The average copy number per nanogram was 3. 60 X 10s for E7 DNA and 1. 79 X 107 for E7 RNA, 8. 39X106 for E6 RNA, which were higher significantly than the number in negative samples, ASCUS and LSIL (P<0.05) .7. There was no relationship between the quantity of HPV-16 E6 or E7 DNA or RNA and clinical stage, pathological grade, pelvic metastasis, vaginal involvement and lymph nodes involvement (P > 0.05) . Conclusions The expression of HPV-16 E6 and E7 may associate with the carcinogenesis of cervix. The quantitation of HPV-16 E6 and E7 oncogene by real-time PCR or RT-PCR analysis in ThinPrep cervical cytologic samples may serve as a quick, reliable, and sensitive tool. It can provide important information to incorporate in the evaluation and treatment of women with a diagnosis of SIL.
|
PDF Full Text Request