| OBJECTIVETo examine the role of human papillomavirus (HPV) type 16 viral load, HLA class I antigen (HLA-I) expression during cervical carcinogenesis and try to approach the feasible mechanism of carcinogenesis of HPV 16 E7.METHODS1. Real-Time Quantitative HPV DNA PCR Genomic DNA was extracted from from 795 cervical paraffin-embedded and frozen tissue from Shandong province. The primers and probe were designed to specifically amplifying an 132-bp fragment for HPV 16 E7 in this study. Purified genomic DNA was amplified by Real-Time Quantitative PCR for the HPV-16 E7 gene as well as for an internal reference gene, β-actin. Standard curves for these two genes were developed and run in parallel with each analysis. To adjust for differences in the extraction and output of genomic DNA among samples, we calculated HPV 16E7 load through dividing E7 copy number by β-actin copy number (CH/Cβ), thus the final quantity could be considered as the number of viral copies/genome/cell.2. Immunohistochemical S-P staining 3-μm sections were cut from paraffin blocks and mounted on polylysine-coated slides. After deparaffinization in xylene and rehydration in graded ethanols, heat induced epitope retrieval (HIER) was performed using a microwaveable vessel where the slides were immersed in Trisodium citrate Retrieval Solution and boiled for 15 minutes at 98°C in microwave oven. 5-βm sections were also cut from frozen tissue on the cryostat and placed on polylysine-coated slides. Sections were air-dried and then slides were fixed for 10 minutes in acetone at -20℃. After pretreatment above, all sections were stained with anti- HLA Class I protein using Histostain-Plus kit. The specificity of the reaction was confirmed by use of substitution control in which PBS was used instead of the primary antibody. Positive control slides were included in each run and examined for appropriate staining.RESULTS1. Among samples positive for β-actin, quantitative PCR identified the highest HPV16E7 viral load (copy number of HPV /genome /cell) in patients with cervical squamous carcinomas (median, 2453), followed by a level of 44.96 copies in cervical intra-epithelial neoplasia (CIN), 18.6 copies in adenocarcinoma and 0 copies in the cervicitis. There was a significant difference in viral load among groups (cervical cancer versus cervicitis, P< 0.001; cervical cancer versus CIN, P< 0.001; CIN versus cervicitis, P< 0.001). HPV16E7 load was highly and positively associated with the development of cervical lesion (Spearman's correlation coefficient r = 0.848, P < 0.001).2. Positive rate for HPV 16 E7 were 98.3% in squamous carcinomas of the cervix (SCC), 22.6% in adenocarcinoma of the cervix (AUC), 78.8% in CIN and 10.5% in cervicitis. Differences of positive rate was significantly distinguished among groups (CIN versus cervicitis, P< 0.001; SCC versus cervicitis, P< 0.001; AUC versus cervicitis, P< 0.001; SCC versus CIN, P< 0.001; AUC versus CIN, P< 0.001; SCC versus AUC, P< 0.001).3. HLA-I expression decreased progressively with the grades of cervical lesion (Pearson's correlation coefficient r= 0.434, P<0.001) and significant differences could be observed between CIN and cervicitis (P< 0.001), between SCC and cervicitis (P< 0.001), between AUC and cervicitis (P< 0.001), between AUC and CIN and between AUC and SCC (P < 0.001).4. HPV16 infection and down-regulation of HLA -1 antigen was highly correlated in cervical lesions (Pearson's correlation coefficient r= -0.475, P<0.001). CONCLUSIONOur study demonstrated that HPV16E7 load was highly and positively associated with the development of cervical lesion and HLA-I expression decreased progressively with the grades of cervical lesion. In addition, HPV 16 E7 plays an important role in down-regulation of HLA -1 antigen in cervical lesions which may lead to the immune escape of virus. Furthermore, quantitative PCR for HPV 16E7 can be reliably applied to cervical samples collected for risk assessment. |
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