Research On The Cellular Malignant Transformation Induced By HPV18 E5 Protein And Its Regulation Mechanism

Human papillomaviruses(HPVs) is a kind of DNA viruses, which can infect mucosal and cutaneous epithelial cells and cause cellular proliferation. High-risk HPV have been shown to be responsible for many cancers, i.e., uterine cervix carcinoma, oesophageal carcinoma, laryngocarcinoma, nasopharyngeal carcinoma and breast carcinoma. HPV encodes eight proteins, among which E6、E7、E5 are oncoproteins, they are known to contribute to cellular proliferation and malignant transformation by targeting several oncogenes, tumor suppressor genes and cellular receptors within the cell. Recent studies have showed the key roles of E6 and E7 viral oncoproteins in HPV induced malignancy, while the research on E5 function and its carcinogenic mechanism is still limited, and mainly focused on the HPV16 E5 protein. HPV18 belongs to high-risk HPV, while the function of HPV18 E5 in the carcinogenic process is still unknown, thus, it is necessary to analyze the cellular malignant transformation ability and regulation mechanism of HPV18 E5 protein. If the role and the regulation mechanism of HPV18 E5 are known, corresponding vaccines and targeted drugs can be designed.In the present research, we studied the relationship between phylogenesis and carcinogenicity of E5 proteins from the aspects of phyletic evolution, and detected the presence of HPV18 E5 gene in esophageal carcinoma tissue specimens. The results showed that the same “genus” or same “species” of E5 proteins belong to the same branch in the phylogenetic tree, and there is a correlation between the phylogenetic and carcinogenicity of E5 protein. High risk HPV E5 proteins have the closely phylogenetic relationship, and low risk HPV E5 proteins also present a closely phylogenetic relationship. We studied the presence of HPV18 E5、E6、E7、L1 genes in esophageal cancer specimens from the Tangshan area of China, the results showed that the presence of the HPV18 E5 gene was detected in 34/94(36%) samples, HPV18 E5, E6 and L1 genes were detected in 28/94(30%) samples, HPV18 E5, E6 and E7 genes were detected in 25/94(27%) samples. In HPV18 E5-positive samples, 4 cases showed sequence mutations, among which 3 were in the initiation codon sites.A pSecTag-HPV18E5 eukaryotic expression vector was constructed, and positive cell lines were screened by culture medium containing bleomycin. The influence of HPV18 E5 on cell proliferation and cell cycle were detected by CCK-8 assay and flow cytometry respectively. The results showed that compared with Balb/c 3T3 group, the ability of cell proliferation in HPV18 E5 stable expressed cells was obviously increased, and the ratios of S and G2/M phases in cell cycle were also increased, while the ratio of G1 phase was decreased.Recombinant adenovirus expressed HPV18 E5 gene was constructed and two stage cell transformation assay was used to analyze the malignant transformation ability of HPV18 E5 protein on Balb/c 3T3 cells. Soft agar assay was used to detect the clone formation ability of transformed cells. Wound healing and cell invasion assays were used to explore the migration and invasion abilities of transformed cells. SCID mice were used to detect the tumor formation ability of transformed cells. The results showed that the cells which were infected with Ad-E5 commonly exhibited altered cellular morphology and function, including multilayer growth, basophilic hyperchromatic, lost cell contact inhibition and anchorage dependence. Transformed cells could form 20.00±0.58 transformed foci and had 26.2±0.63% colony formation rate in soft agar, while normal cells could not grow in soft agar. Transformed cells had the ability of migration and invasion when compared with control group and formed tumors in SCID mice(1.5±0.3 cm). The findings indicated that HPV18 E5 protein displayed transformation activity on Balb/c 3T3 cells.Real-time PCR and western blot techniques were used to analyze the changes of p21, p27 and EGFR genes from transcription and protein expression levels. The results showed that HPV18 E5 transformed cells presented down-regulation of p21 and p27 genes, and presented up-regulation of EGFR gene in both transcription and protein expression levels. The findings indicated that HPV18 E5 protein influenced the expression of p21, p27 through EGFR signaling pathways which finally caused cellular malignant proliferation and transformation.In summary, this thesis proved that HPV18 E5 protein had the capability of inducing cellular transformation. The mechanism may involve the activation of EGFR signaling pathway, and influencing the expression of p21 and p27 tumor suppressor genes which finally caused cellular malignant transformation. This study provides a basis for elucidating the carcinogenic mechanism of HPV18, and gives a guide for the development of HPV vaccine.