An Experiment Study For Induction Of P53 Protein Accumulation By GRIM-19 In Cervical Carcinoma Cells

[Background] Cervical carcinoma is one of the three malignant tumors in female reproductive system and serious threat to women's health. The mortality is always in high level and the age of onset trends to become younger. HR-HPV continuous infection is the most dangerous factor of cervical cancer occurrence, so looking for the methods to against HPV become urgent. The gene therapy which represents the biotherapy has become the fourth treatment modalities after surgery, radiotherapy, chemotherapy. Gene therapy is an experiment technique that uses genes to suppress or kill tumor cells by inserting a gene into a patient's cells. Although it has become the new trend of cervical carcinoma treatment, effective gene therapy depends on the stable expression of target gene.p53, a tumor suppression gene,is located in 17p13.1,and the full length is between 16 and 20 kb. Its protein is low in cervical cancer tissues with the main wild-type. In cervical cancer cells, the half-life of wtp53 protein is shorter, and the function is inactivated. The functions of wtp53 are the regulation of cell cycle, promotion of tumor cells apoptosis, and inhibition of tumor cell growth. The concrete mechanism is to regulate cell cycle by p21 expression adjustment, to suppress tumor cell growth by NFκB-Bcl-2 pathway, to induce cell apoptosis by increasing downstream gene expression like PUMA, NOXA, and Perp gene. E6 protein encoded by HR-HPV inhibits p53 function, induces cells malignant transformation. Therefore, low expression or inactivation of p53 gene is the major factor in development and progression of cervical carcinoma,and increasing expression or reactivating has become the significant treatment strategy. GRIMs (genes associated with retinoid-IFN-induced mortality) are recently discovered a group of interferon and retinoic acid merger-induced apoptosis gene, and Kalvakolanu are screening and identified used antisense RNA knockout technology in breast cancer cells and first reported the selection of apoptosis proteins. Grim-19 is the member of Grims family, located in 19p13.2, a novel 16-kD protein, the area have multiple genes for prostate cancer suppressor gene. GRIM-19 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. For example, Hurthle cell tumors of the thyroid, squamous cell carcinoma of the head and neck, human colorectal carcinoma, human keratinocytes and oral carcinoma. STAT3 is a major oncogenic transcription factor involved in the development and progression of a huge of human tumors. STAT3 is also constitutively activities in a number of human cancers by autocrine growth factors or activated oncogenes. It is reported than GRIM-19 binds to STAT3 and inhibits its transcription-activating function. However, the function of GRIM-19 in cervical carcinoma is still unclear.Our early study firstly showed a significant down-regulation of GRIM-19 protein levels in several primary human cervical cancers, and we established a stable expression GRIM-19 cell lines which is named HeLa/GRIM-19. In vivo and in vitro experiment both demonstrated that restoration of GRIM-19 levels was able to inhibit STAT3 transcription-activating function, suppress cell growth and promote cell apoptosis. In the light of former results has proved GRIM-19 is low in cervical carcinoma,GRIM-19 induce cell growth arrest and apoptosis, and p53 induce cell apoptosis to against tumor, we discussed that GRIM-19 have relationship with p53 even regulate it,and found that GRIM-19 induces p53 protein accumulation in HR-HPV infected cervical tumor cells but not in HR-HPV uninfected cells,and this accumulated p53 protein have the function to suppress cell growth;.[Objective] To research on the regulation p53 activity and mechanism by GRIM-19, provide a theoretical basis and treatment of clues.[Methods]⑴Western blot detection of p53 and its downstream gene p21, PUMA in HeLa cells transient transfected with pGRIM-19 or control vector;⑵Western blot detection of p53 and its downstream gene p21, PUMA in HeLa cells transient transfected with GRIM-19 siRNA or control siRNA;⑶Western blot detection of p53 and its downstream gene PUMA in SiHa, Caski cells transient transfected with pGRIM-19 or control vector;⑷Western blot detection of p53 and its downstream gene PUMA in SiHa, Caski cells transient transfected with GRIM-19 siRNA or control siRNA;⑸Western blot detection of p53 in HO8910 cells transient transfected with pGRIM-19 or GRIM-19 siRNA respectively;⑹FACS detection the cell-cycle profiles of HeLa/GRIM-19 and HeLa/Con cells which are treated with double thymidine;⑺FQ-PCR detection of p53 in HeLa cells transient transfected with pGRIM-19 or control vector;⑻Luciferase reporter gene detection of p53 promoter in HeLa cells transient transfected with pGRIM-19 or control vector;⑼Western blot detection of p53 in HeLa, SiHa cells transient transfected with STAT3 siRNA or control siRNA;⑽Western blot detection of p53 in HeLa, SiHa cells transient transfected with STAT3C or STAT3DN vector;⑾Western blot detection of p53 half-life in HeLa/GRIM-19 and HeLa/Con cells at various intervals after treatment with CHX;[Results]⑴Western blot detection of p53 and its downstream gene p21, PUMA expression levels in HeLa cells transient transfected with pGRIM-19 are increased significantly. There is a significantly difference between two groups (p<0.05);⑵Western blot detection of p53 and its downstream gene p21, PUMA expression levels in HeLa cells transient transfected with GRIM-19 siRNA are decreased significantly. There is a significantly difference between two groups (p<0.05);⑶Western blot detection of p53 and its downstream gene PUMA expression levels in SiHa, CaSki cells transient transfected with pGRIM-19 are increased significantly;⑷Western blot detection of p53 and its downstream gene PUMA expression levels in SiHa, CaSki cells transient transfected with GRIM-19 siRNA are decreased significantly;⑸Western blot detection of p53 expression levels in HO8910 cells transient transfected with pGRIM-19 or GRIM-19 siRNA is not varied;⑹FACS detection of cell-cycle profiles of HeLa/GRIM-19 treated with double thymidine are G1 phase arrest signalificantly;⑺FQ-PCR detection of p53 gene expression levels in HeLa cells transient transfected with pGRIM-19 or control vector are not varied. There is no significantly difference between two groups (p>0.05);⑻Luciferase reporter gene detection of p53 promoter activity in HeLa cells transient transfected with pGRIM-19 or control vector are not varied. There is no significantly difference between two groups (p>0.05);⑼Western blot detection of p53 expression levels in HeLa, SiHa cells transient transfected with STAT3 siRNA or control siRNA are not varied;⑽Western blot detection of p53 expression levels in HeLa, SiHa cells transient transfected with STAT3C or STAT3DN are not varied;⑾Western blot detection of p53 protein half-life in HeLa/GRIM-19 after treatment with CHX are prolonged. There is a significantly difference between two groups (p<0.05).[Conclusion] GRIM-19 induces p53 protein accumulation in HR-HPV infected cervical tumor cells but not in HR-HPV uninfected cells,and this accumulated p53 protein is able to suppress cell growth; GRIM-19 induced the accumulation of p53 protein through prolonging the half-life of p53 protein neither by transcriptional regulation nor by inhibiting of STAT3 activity.