Research On E2, E6and E7Genes Of HPV16in The Samples Of Different Clinical Stages Of Cervical Cancer

Cervical cancer samples of HPV16infection (including some paracarcinoma tissues)were collected to analyze the sequence of E6, E7gene, subsequently, phylogenetic trees for E6and E7gene were constructed. Real-time quantitative PCR was used to detect E2/E6values on DNA level, by which status of integration of HPV16gonomic DNA could be identified, and to detect the reletive level of E2,E6,E7mRNA comparing with β-actin. The alternative splicing of E6mRNA was also analysed by the semi-quantitative RT-PCR.The main results are listed as follow.1. Both of E6and E7Phylogenetic trees show that only Asian and European variants, not any African type1, African type2, Asian-American and North-American variants were found. And the T178G-A647G-T846comutation has no correlation with the clinical stage of cervical cancer (χ2=0.046, P<0.05).2. E2/E6gene ratio was used to detect the physical status of HPV-16DNA in73patients. The results show45cases (61.64%) of episomal form, and18cases (24.66%)of mixed form,10cases (13.70%) of integrated form. The integration of HPV does not show correlation with clinical data, which include the clinical stage (χ2=0.006, P<0.05) and histologic type of cervical carcinoma. The T178G-A647G-T846comutation is not associated with the status of virus (χ2=3.696, P<0.05).3. Splicing isoform E6*I is dominant in E6mRNA comparing with E6full length (P <0.0001), with some samples showing nagetive results for very low E6mRNA level. E6and E6*I have a positive correlation (r=0.6335, P<0.001)。 The change of the alternative splicing of E6has no correlation with the integration of HPV (F=0.0667, P>0.05), the T178G-A647G-T846comutation (P>0.05) and the clinical stage (F=0.6399, P>0.05)4. There is a significant difference among the transcripts of E2,E6,E7(P<0.001).The amount of E6transcript and E7transcript are significantly higher than that of E2(P <0.001). But the result does not show significant difference between the transcripts of E6and E7(P=0.9907). The E2transcripts of episomal form are obviously higher than those of mixed form (P<0.01) and integration form (P<0.05). The levels of E6and E7transcripts in the status of integrated form are both higher than those of the mixed form (P<0.01, P<0.001) and those of the episomal form (P<0.05, P<0.05). There is not any difference between the levels of the E2, E6and E7transcripts and the clinical stages. The T178G-A647G-T846C mutation does not influence the amount of E2, E6, E7transcripts (P>0.05).Conclusion:Within these detections and analyses of the E2, E6and E7of HPV16, three oncogenic genes, some significant differences and relevances were found, but any factor relevant to the clinical stages of cervical cancer was not found.