An Experiment Study For The Interactions Between GRIM-19and E6,E6AP And Their Sites Of Interaction

[Background] Cervical cancer is the most common malignant tumor in the threemalignant tumors of the female reproductive system and seriously threat to the healthand lives of women. Every year, Approximately500,000new cases of cervical cancerare diagnosed and about230,000women died of cervical cancer worldwide, of whichthe majority (80%) occur in developing countries. The region of the highest incidencerate is South Africa, next is Asia. China’s new cases a year is about130,000cases,accounting for nearly1/4of the world’s. The cause of cervical cancer has not been sofar entirely clear. Molecular biology studies have shown that more than90%ofcervical cancer associated with high-risk human papillomavirus (HR-HPV) infection,mainly16and18subtypes. High-risk HPV persistent infection plays a vital role in theoccurrence and development of cervical cancer, the integration of HR-HPV genomeexists in more than70%of cervical cancer.The current study confirmed that early genes E6, E7of high-risk HPV16and18encode early proteins E6and E7, which play a key role in the process of deteriorationof cervical epithelial cells. E6, E7protein combine with P53and Rb respectively, spurP53and Rb degradation, eventually leading to the occurrence of cervical cancer. Thecarcinogenic effects of E6protein is mainly depended on the recruitment of E6AP(E6-associated protein) to form a complex with the P53core area to promote P53ubiquitin proteasome degradation.E6AP is a ubiquitin ligase (the ubiquitin-ligase byenzyme, E3) HECT domain family member. E6hijacks E6AP, this complex bindswith p53, E6AP transfers the activated ubiquitin to p53protein, resulting indegradation of p53by the26S proteasome.interaction of E6and P53plays a key role in the occurrence and development of cervical cancer.GRIMs (genes associated with retinoid-IFN-induced mortality) are a group ofinterferon and retinoic acid merger-induced apoptosis genes recent years, screeningand identified used antisense RNA knockout technology in breast cancer cells andfirst reported the selection of apoptosis proteins by Kalvakolanu. GRIM-19is themember of GRIMs family, located in19p13.2, the area have multiple genes forprostate cancer suppressor gene. GRIM-19is involved in mitochondrial respirationand expression in most human tissues, like Hurthle cell tumors of the thyroid, mouthcarcinoma, squamous cell carcinoma of the head and neck, human keratinocytes andhuman colorectal carcinoma. GRIM-19is low expression in cervical carcinoma.GRIM-19was mainly localized in the mitochondria, less in nucleus. The geneinvolved in cell proliferation, apoptosis regulation and control process, the reductionof its expression or mutation sites can cause abnormal cell proliferation and malignanttransformation. expression of GRIM-19was found to be reduced in many tumors,such as thyroid cancer, oral cancer, kidney cancer, colorectal cancer. However, thefunction of GRIM-19in cervical carcinoma is still unclear.Our early study have confirmed that GRIM-19can up-regulate the expression ofp53proten and it’s downstream genes p21and PUMA in cervical carcinoma cell linesHeLa, SiHa and CaSki, otherwise this phenomenon not occurred in ovrian carcinomacell lines HO8910, which is HPV negative.We envisage that GRIM-19could inducep53protein accumulation in cervical carcinoma cell lines, GRIM-19induces theaccumulation of p53protein through prolonging the half-life of p53protein. It’sclearly that p53degradation depends on the E6and E6AP protein in cervicalcarcinoma. further study the interaction of GRIM-19and18E6get results as follow:GRIM-19and E6have protein interactions, but not with p53; GRIM-19induced theaccumulation of p53protein through interacting with E6, preventing the E6-mediateddegradation of p53; E6AP can competitive inhibition the interaction between GRIM-19and18E6. This experiment will further explore the interactions betweenGRIM-19and HPV16E6, HPV18E6and their sites of interaction in cervical cancercells.[Objective] To explore the interactions between GRIM-19and HPV16E6, HPV18E6and their sites of interaction in cervical cancer cells, and will provide a theoreticalbasis and treatment of clues.[Methods](1) Construction of the GRIM-19-HPV16E6prokaryotic expression vector:pGST-G19①[pGEX-4T-3-GRIM-19(1-35)], pGST-G19②[pGEX-4T-3-GRIM-19(36-70)], pGST-G19③[pGEX-4T-3-GRIM-19(71-144)], pET-flag-16E6(pET-22b-3×flag-16E6),16E6eukaryotic expression plasmid:p3×Flag-16E6(p3×Flag-CMV-Myc-24-16E6);(2) co-immunoprecipitation method(CO-IP) analysis of GRIM-19and16E6protein interactions in vivo;(3) Induced expression and purification of fusionprotein in E. coli;(4) GST pull down analysis of GRIM-19and16E6,18E6proteininteractions in vitro;(5) GST pull down analysis of GRIM-19and E6AP proteininteractions in vitro.[Results](1) result of CO-IP shows that GRIM-19and16E6has interaction in vivo;(2)result of GST pull down result shows that GRIM-19①and HPV16E6has interactionin vitro;(3) result of GST pull down result shows that GRIM-19①and HPV18E6has interaction in vitro;(4) result of GST pull down result shows that GRIM-19andE6AP①d ose not have interaction in vitro;(5) result of GST pulldown result showsthat GRIM-19and E6AP②dose not have interaction in vitro;(6) result of GST pulldown result shows that GRIM-19and E6AP③has interaction in vitro;(7)result ofGST pull down result shows that GRIM-19①and E6AP③has interaction in vitro.[Conclusion] GRIM-19and E6have protein interactions and the binding site ofGRIM-19is the first1-35amino acids; GRIM-19and E6AP have protein interactions and the binding site of GRIM-19is the first1-35amino acids and the binding site ofE6AP is its HECT domain.