A Study Of The Effects And Mechanisms Of HPV16 E6 And E7 Mediated Pathways By GSK3β On Cervical Squamous Carcinoma Cells

Cervical cancer(CC) has the highest morbidity and is the most common malignant tumor in the clinical of female reproductive system. China is high-risk areas of cervical cancer in the world. Although the prevention and treatment of cervical cancer has been carried for several decades, the incidence rate and morbidity rate of cervical cancer remains high levels. Recent twenty years, the incidence rate and morbidity rate of cervical cancer increased markedly. The prevention and treatment of cervical cancer, specially the prevention and screening in early stage of cervical cancer, has become priority work.The human Papilloma virus human(HPV) has more than 200 kinds of subtypes. According to its biological behavior, HPV is divided into two categories: high risk type and low risk type. It has been confirmed that persistent infection of high risk type HPV is-8-the leading cause of cervical cancer. It has been identified that the E6, E7 are mainly oncology proteins of HPV. E6, E7 play important roles in malignant transformation of cervical cells by combining and degrading the tumor suppressor gene P53, p Rb. While HPV infection is not enough to cause the malignant transformation of cervical cells. The mechanism of HPV in the cervical cancer development is still a puzzle. To explore the mechanism of HPV in cervical cancer development and to find new effective target gene in in cervical cancer development mediated by HPV would be helpful to screen the susceptible population of HPV, and would be useful to provide a new diagnostic and therapeutic target in cervical cancer.Synthase kinase 3β(glycogen synthase kinase 3β, GSK3β) is a common serine/ threonine protein kinase(glycogen) in eukaryotic cells. GSK3β is not only the key limited enzyme in the glycogen Metabolism, but also play important role of embryonic development, apoptosis, differentiation and the regulation of several transcription factors in tumor cells. There are a lot of researches on GSK3β in tumor area, but the role of GSK3β in tumor development and progression is still controversial. It was speculated that GSK3β would play different roles in different human tissue type.However, the clinical value of GSK3β activity and the relationship between HPV and GSK3β in cervical cancer have not been described. What role does the GSK3β activity play in the cervical cancer development mediated by HPV? In this study, we detected the expression of HPV, GSK3β and phosphorylation levels of GSK3β(p-GSK3βser9 and p-GSK3βtyr216) protein in 80 cases cervical squamous cell cancer patients and analyzed the clinical value of GSK3β and phosphorylation levels of GSK3β(p-GSK3βser9 and p-GSK3βtyr216) in cervical cancer tissues and the relationship between HPV and GSK3β and phosphorylation levels of GSK3β(p-GSK3βser9 and p-GSK3βtyr216) in cervical cancer tissues. According to characteristics of HPV structure, we constructed HPV E6, E7, E6/E7 recombinant plasmids, which contains E6, E7, E6 and E7 structure, respectively. We choose the cervical cancer cell line C33 A which has not HPV infection as research object. The infection efficiency of HPV E6, E7, E6/E7 recombinant plasmids was confirmed by q RT-PCR and western blot were performed to evaluate the expression of E6 and E7 m RNA and protein respectively. Flow cytometry analysis clone formation assays, wound healing assays transwell assays were performed to evaluate the effects of HPV E6, E7, E6/E7 recombinant plasmids on cell cycle, proliferation, migration and invasion of C33 A cell line. After transfected with HPV E6, E7, E6/E7 recombinant plasmids, q RT-PCR was performed to evaluate the GSK3β m RNA levels. Meanwhile, western blot were performed to evaluate the expression of GSK3β hosphorylation levels of GSK3β(p-GSK3βser9 and p-GSK3βtyr216), GSK3β target genes(Cyclin D1 and β-catenin) respectively after transfected with HPV E6, E7, E6/E7 recombinant plasmids. This study would clarify the relationship between HPV and GSK3β activity in cervical squamous cell cancer, and explore the GSK3β activity regulation mechanism mediated by HPV.Some results have been obtained:1. In cervical squamous cell cancer, the positive expression of GSK3β, p-GSK3βtyr216 in early stage group were significantly higher than those in advanced stage group(25.5% vs. 4.0%, χ2=5.193, p=0.029; 76.4% vs. 52.0%, χ2=4.749, p=0.039). While the positive expression rate of p-GSK3βser9 in the patients with early stage is obviously lower than the patients with advanced stage(5.5% vs. 28.0%, χ2=7.988, p=0.009). The positive expression rate of p-GSK3βser9 in well-moderately differentiated tumors were lower compared with poorly differentiated tumors( 7.4%,7.9% and 33.3%, χ2=7.329, p=0.031).The expressions rate of GSK3β and p-GSK3βtyr216 were significant correlation with lymph node status(5.9% vs. 28.3%, χ2=6.427, p=0.018; 52.9% vs. 80.4%, χ2=6.878, p=0.014), while the expression rate of p-GSK3βser9 were increased in lymph node metastases compared with non- lymph node metastases(23.5% vs. 4.3%, χ2=6.577,p=0.015).2. The percentage of S stage of C33 A cell were obviously increased after HPV E7 plasmid infection. It was supposed that HPV E7 would accelerate the DNA duplication in cervical cancer cells, and promote the malignant transformation of cervical cancer cells. Meanwhile, both the HPV E6 and E7 overexpression obviously promoted the proliferation ability, the migration ability and the metastatic ability in cervical cancer cells, and there were synergistic effect between HPV E6 and E7 in promoting the proliferation ability,the migration ability and the metastatic ability in cervical cancer cells.3. HPV E6 and E7 invoked the GSK3β activity by regulating phosphorylation levels of GSK3β(p-GSK3βser9 and p-GSK3βtyr216) in 24 hours. There were synergistic effect between HPV E6 and E7 in promoting the GSK3β activity by regulating phosphorylation levels of GSK3β(p-GSK3βser9 and p-GSK3βtyr216) in cervical cancer cells. Cyclin D1 and β-catenin protein levels were regulated by HPV E6 and E7 through invoking the GSK3β activity in cervical cancer cells.