A Study On Type-specific Difference In Human Papillomavirus Induced Cervical Carcinogenesis

In the worldwide, cervical cancer is the second most common malignancy, with 527,624 new cases and 265,653 deaths each year (www.hpvcentre.net in Oct 31th, 2014), of which 85% of cervical cancer is occurred in the developing country. The cause of cervical cancer is clear, a lot of studies show that the persistent infection of high-risk human papillomavirus (HPV) is the main pathogenic factor of cervical cancer and precancerous lesions. HR-HPV include HPV16, HPV18, HPV31, HPV33, HPV35, HPV45, HPV51, HPV52, HPV56, HPV58 and HPV61. Another study showed that the oncogene E6 and E7 expressed by HR-HPV virus can degradate P53 and Rb, respectively, cause the changes on cell cycle, proliferation, invasion, metastasis and other biological behaviors, promote the malignant transformation of cells, and ultimately lead to the occurrence and progression of cervical cancer. But it is still unclear about the differences between viral oncoproteins in different type HPVs. Epidemiological evidence display that the frequnencies of different types of HPV in patients with normal cytology and cervical lesions are not the same, it shows that different types of HPV have different infective ability and pathogenicity, but whether is associated with host susceptibility to type specific HPV, is still unclear.In this study, we focus on the susceptibility of different types of HPV to host and the different pathogenicity of different types of HPV. First, we designed two HPV58 E6 specific peptide fragments, then cross-linked to three different carrier proteins, immunizated rabbits, got the immuned serum, checked the serum titer by ELISA, isolated splenocytes of the rabbits and cell fusion, detected the cell surpernant, screened the positive clones, got the recombinant antibody, sequenced and analyzed, so as to obtain specific HPV58 E6 rabbit monoclonal antibody. Secondly, constructed pFLAG-CMV5.1-HPV58E6/E7 and pFLAG-CMV5.1-HPV16E6/E7 eukaryotic expression plasmid and transfected into 293 T and U2OS cells, observed the function of HPV58E6/E7 and HPV16 E6/E7 gene on cell cycle, apoptosis, invasion ability and the downstream molecules, compared their carcinogenic potential. Thirdly, collected 3299 cases of cervical exfoliated cell samples, got 875 cases with single HPV16,18,52,58 positive and HPV negative, extracted DNA, genotypes 96 SNPs of HPV receptors (EGFR, PPIB, HSPG2, FGFR2, FURIN, ITGA6, TSPAN1 and SDC2) by Illumina BeadXpress VeraCode platform, analyzed the relationship between the target SNPs and type-specific HPV infection and cervical lesions progression.The study successfully obtained HPV58E6 monoclonal antibody for the first time; in vitro experiments, compared to HPV58E6/E7, HPV16E6/E7 have more ability to promote cell cycle progression, inhibit cell apoptosis and promote cell invasion, E6-P53 and E7-pRB may play a key role in the process of cervical carcinogenesis caused by HPV; and found out the significant different SNPs, genotypes and haplotypes related to type-specific HPV infection and cervical lesions progression. We further showed the distribution characteristics of HPV distribution in nature and different degree of cervical lesions, for a better understanding of the carcinogenic process, benefit to the treatment and prevent of the cervical cancer, and also provide scientific evidence to explore the blocking of cervical cancer, enrich the theory of etiology of cervical cancer.This study preliminarily explain the mechanism of HPV in the distribution characteristics in the nature and different lesions, for a better understanding of the differences carcinogenicity relate to HPV type specific, provide scientific evidence for the cervical cancer prevention and control strategy for the development.Part I:Invent and create HPV58E6 specific rabbit monoclonal antibodyObjectiveinvent and create HPV58E6 specific rabbit monoclonal antibody for further study of HPV58 in the vitro.MethodsWe compared the HPV58E6 sequence with the homology sequences of HPV16E6, HPV31E6, HPV33E6, HPV52E6 and HPV67E6, and found out the specific sequences of HPV58E6, designed two HPV58E6 polypeptide fragments, then crosslinked to three different carrier proteins, immunized New Zealand white rabbits, got immune serum, screened the positive antibody rabbits and tested the titer by ELISA, isolated the rabbit spleen cells and fused with 240E-W2 cells, then plated into 96 well culture plates, incubated in 1640AB medium for cloning, screening the positive cell clones by ELISA, and found out HPV58E6 specific antibody positive clones, extracted the RNA, did RT-PCR amplification used primers Fl/Rl, got multiple heavy chain cDNA and light chain cDNA, cDNA were cloned into pTT5 plasmid expression vector, paired heavy chain cDNA plasmid pTT5 with light chain cDNA pTT5 plasmids and co-transfected into HEK-293-6E cells; screened the pair with the highest IgG concentrations and OD value larger than 0.3, which is the hpv58e6 specific rabbit monoclonal antibody, preparing recombinant antibody and sequencing analysis, to obtain the specificity of hpv58e6 rabbit monoclonal antibody.Results1. The serum from the New Zealand white rabbits which were immuned by the specific HPV58E6 polypeptide fragments could combine with the protein extracted from pEGFP-C1-HPV58E6 plasmid transfected 293T cells.2. the supernatant of the hybridoma cell could combine with the protein extracted from pEGFP-C1-HPV58E6 plasmid transfected 293T cells, while not from the pEGFP-C1-HPV 16E6 plasmid transfected 293T cells.3. the recombinant antibody ZJM1B-9 could combine with the protein extracted from pEGFP-C1-HPV58E6 plasmid transfected 293T cells, while not from the pEGFP-C1-HPV16E6 plasmid 293T cells.Conclusion 1. HPV58E6 rabbit monoclonal antibody was successfully obtained usinghybridoma technique.2. HPV58E6 can combine with HPV58E6 protein, but can not with HPV16E6 protein.Part II:The differences between the carcinogenic ability of HPV16 and HPV58 oncogenic gene E6 and E7ObjectiveTo confirm the differences of cell phenotype between HPV16 and HPV58 viral oncogenes E6 andE7, and elaborate the differences the carcinogenic ability of HPV16 and HPV58 in the phenotype and molecular level.Methodsconstructed the pFLAG-CMV5.1-16E6, pFLAG-CMV5.1-16E7, pFLAG-CMV 5.1-58E6 and pFLAG-CMV5.1-58E7 eukaryotic expression plasmids and transfected into 293T and U2OS cells, detected the cell cycle and apoptosis by flow cytometry, and invasion by transwell, detected E6-P53 and E7-pRB co-expression and co-location by confocal immunofluorescence.Results1. In the 293T and U2OS cells, compared with negative control, over-expression of HPV58E6/E7 promotes cell proliferation, while compared with HPV58E6/E7, over-expression of HPV16E6/E7 promotes cell promote cell proliferation more obviously.2. In 293T cells, in HPV58E6/E7group the S phase is 42.68% and 45.39%, while 52.66% and 49.18% in HPV16E6/E7 group. In U2OS cells, in HPV58E6/E7group the S phase is 32.73% and 27.03%, while 37.32% and 36.24% in HPV16E6/E7 group.3. In 293T cells, the proportion of early apoptosis in HPV58E6/E7 group is 9.66% and 12.1%, while 6.6% and 14.0% in HPV16E6/E7 group, respectively. In U2OS cells, the proportion of early apoptosis in HPV58E6/E7 group is 5.63% and 5.22%, while 3.84 and 3.44% in HPV16E6/E7 group, respectively.4. In the 293T and U2OS cells, compared with negative control, in HPV58E6/E7 group the invasive ability is enhanced, while compared with HPV58E6/E7 group, more in HPV16E6/E7 group.5.In the 293T and U2OS cells, compared with HPV58E6 group the expression of P53 is lower in HPV16E6 group, especially in the nuclear; compared with HPV58E7 group, the expression of pRB is higher in HPV16E7 group, especially in the nuclear.ConclusionCompared with HPV58E6/E7, HPV16E6/E7 showed more powerful in promote cell proliferation and invasion, inhibit cell apoptosis, promote G1 to S transition and degradate P53 and pRB, it hint that HPV16 has more carcinogenic ability than HPV58.Key words:HPV58; HPV16; P53; pRB; cervical cancer.Partâ…¢the study on the association of gene variants in HPV receptors with type specific cervical infection and disease progressionObjectiveto confirm the Single nucleotide polymorphisms (SNPs) in human papillomavirus (HPV) receptors associated with type-specific HPV infection and cervical lesion progressionMethodsWe examined 96 SNPs in 8 genes which may participate in the HPV infection process in 875 samples with HPV negative or single HPV16,18,52,58 positive from 3299 cervical exfoliated cell samples, by Illumina BeadXpress VeraCode platform, and analyzed the correlation between the target gene SNPs and type-specific HPV infection and cervical lesions progression.ResultsWe found two SNPs (rs28384376, rs12034979) significantly and three haplotypes critically correlated to HPV16 infection; four SNPs (rs2575738, rs6697265, rs2575712, rs2575735) and three haplotypes significantly correlated to HPV18 infection, two SNPs (rs10510097, rs12718946) and two haplotypes significantly correlated to HPV52 infection, three SNPs (rs4947972, rs2981451, rs2575735) and two haplotypes significantly correlated to HPV58 infection; and five SNPs (rs3135772, rs2556537, rs12034979, rs1047057, rs16894821)and one haplotype significantly correlated to cervical lesion progression induced by HPV 16 infection, three SNPs (rs6697265, rs6680566, rs16860426) and one haplotype by HPV18 infection, three SNPs (rs878949, rsl2718946, rsl2668175) and one haplotype by HPV52 infection, and no SNP or haplotype by HPV58 infection.ConclusionOur findings suggest genetic variants of HPV receptors may influence the susceptibilities to HPV type-specific infection and cervical lesion progression, which might have a potential application value in cervical cancer screening and therapy.