An Experiment Study For The Mechanism Of P53 Protein Accumulation By The Interaction Between GRIM-19 And HPV18 E6 In Cervical Carcinoma Cells

[Background] Cervical carcinoma is the most common malignant tumors on female reproductive system. Every year, approximately 470,000 new cases of cervical carcinoma are diagnosed and approximately 230,000 women worldwide die of the disease, with the majority (~80%) of these cases and deaths occurring in developing countries, seriously threat to women's health.Nobel laureate Harald zur Hausen first described a possible link between the human papillomavirus (HPV) and cervical carcinoma. It's confirmed that, the E6 and E7 proteins, encoding by the high-risk HPV(HR-HPV), specifically HPV16 and HPV18, can inactivate the p53 and retinoblastoma (pRb) tumor suppressors, leading to the cervical carcinoma. The E6 protein, that mainly located in nucleus is small basic protein containing approximately 160 amino acids, which plays an important role in vrius life cycle and tumor progression to malignancy. HPV E6 binds with p53 tumor suppressor, resulting in the p53 of the ubiquitylation and degradation. E6AP (E6-associated protein), a cellular ubiquitin ligase encoded by the UBE3A gene, plays an important role in this process. E6 hijacks E6AP, this complex binds with p53, E6AP transfers the activated ubiquitin to p53 protein, resulting in degradation of p53 by the 26S proteasome. Not as many other tumors like ovarian carcinoma and lung cancer, p53 is wild type with short half-life and low expression in cervical carcinoma. The role of E6 degradation of p53 is a key element in HPV infection and proliferation, the cells with dysfunction genes could perstetur proliferation, leading to the infected cells aggravation and becoming tumor cells.GRIMs (genes associated with retinoid-IFN-induced mortality) are a group of interferon and retinoic acid merger-induced apoptosis genes, screening and identified used antisense RNA knockout technology in breast cancer cells and first reported the selection of apoptosis proteins by Kalvakolanu. GRIM-19 is the member of GRIMs family, located in 19p13.2, the area have multiple genes for prostate cancer suppressor gene. GRIM-19 is involved in mitochondrial respiration and expression in most human tissues, like Hurthle cell tumors of the thyroid, mouth carcinoma, squamous cell carcinoma of the head and neck, human keratinocytes and human colorectal carcinoma. GRIM-19 is low expression in cervical carcinoma. The gene involved in cell proliferation, apoptosis regulation and control process, the reduction of its expression or mutation sites can cause abnormal cell proliferation and malignant transformation. binds to STAT3, which is a major oncogenic transcription factor involved in the development and progression of a huge of human tumors, inhibits its transcription-activating function. However, the function of GRIM-19 in cervical carcinoma is still unclear.Our early study have confirmed that GRIM-19 can up-regulate the expression of p53 proten and it's downstream genes p21 and PUMA in cervical carcinoma cell lines HeLa, SiHa and CaSki, otherwise this phenomenon not occurred in ovrian carcinoma cell lines HO8910, which is HPV negative.We envisage that GRIM-19 could induce p53 protein accumulation in cervical carcinoma cell lines, GRIM-19 induces the accumulation of p53 protein through prolonging the half-life of p53 protein. It's clearly that p53 degradation depends on the E6 protein in cervical carcinoma. Our study will research on this specific regulation,further study on the molecular mechanism of p53 protein accumulation by the interaction between GRIM-19 and HPV18 E6 in cervical carcinoma cells.[Objective] To research on the mechanism of p53 protein accumulation by the interaction between GRIM-19 and HPV18 E6 in Cervical Carcinoma Cells, and will provide a theoretical basis and treatment of clues.[Methods]⑴Construction of GRIM-19, HPV18 E6 protocaryon expression plasmid: pGEX-4T-3-GRIM-19 (pGST-G19), pET-22b-18E6-Flag (pET-E6) and 18E6 eukaryon expression: p3×Flag-CMV-Myc-24-18E6 (p3×Flag-E6);⑵RT-PCR analysis of the mRNA expression level of E6 in HeLa cells transient transfected with pGRIM-19 or control vector;⑶U biquitin analysis of p53 protein ubiquitylation in HeLa cells transient transfected with pGRIM-19 in vivo;⑷co-immunoprecipitation (CO-IP) analysis of GRIM-19 and 18E6/p53 protein interactions in vivo;⑸GST pull down analysis of GRIM-19 and 18E6 protein interactions in vitro;⑹GST pull down competitive inhibition analysis of the E6AP competitive inhibition the interaction between GRIM-19 and 18E6 in vitro.[Results]⑴RT-PCR analysis of HPV E6 mRNA levels in HeLa cells transient transfected with pGRIM-19 or control vector. There is no significantly difference between two groups (p>0.05);⑵Ubiquitin analysis of p53 protein ubiquitylation in HeLa cells transient transfected with pGRIM-19 is decreased significantly, There is a significantly difference between two groups (p<0.05);⑶CO-IP analysis of GRIM-19 with 18E6 has protein interactions, but not with p53;⑷GST pull down analysis of GRIM-19 with 18E6 has protein interactions;⑸GST pull down competitive inhibition analysis of the E6AP can competitive inhibition the interaction between GRIM-19 and18E6.[Conclusion] GRIM-19 and E6 have protein interactions, but not with p53; GRIM-19 induced the accumulation of p53 protein through interacting with E6, preventing the E6-mediated degradation of p53; E6AP can competitive inhibition the interaction between GRIM-19 and 18E6.