An Experimental Study For GRIM-19 In The Apoptosis Induced By IFN/RA Combined On Ovarian Carcinoma Cell Line SK-OV-3

[Background]Ovarian malignant tumor is one of the three malignant tumors on female reproductive system;it is a serious threat to women's health and life on account of difficulty to early detection and high mortality.Conventional surgery and radiotherapy and chemotherapy are difficult to significantly improve the long-term survival rate of patients,especially survival rate of advanced stage patients,it is hightime to looking for new methods of treatment of ovarian cancer.The modern medicine has gained great achievement,but the morbidity and mortality of the ovarian carcinoma is still very high. The accomplishment of the human genome map and the deep investigation of the human geonme provide a new way to prevent and cure the carcinoma.Lots of recent research has demonstrated that the activation of the oncogene and the inactivation of the anti-oncogene correlated with the cell proliferation and differentiation gene in the genome,cause the occurrence and development of the carcinoma.Based on this theory, some theoretical and clinical researches of the gene therapy have been implemented to the carcinoma.Targeting gene therapy is that the normal gene or therapeutic gene target specificly to tumor cells by molecular biology,and inhibit tumorigenic gene or repair defective genes,so that tumor cell growth was inhibited without affecting other normal cells.Targeting gene therapy has laid a foundation in the field of successthl application of gene therapy for ovarian cancer,targeted gene therapy for ovarian cancer has become the new trend of treatment.GRIMs(genes associated with retinoid-IFN-inducedmortality) 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,the area have multiple genes for prostate cancer suppressor gene.Grim-19 is involved in mitochondrial respiration and new genes of control apoptosis and IFN/RA induced tumor cell apoptosis,its abnormal expression may be involved in a variety of tumor formation.For example,Hurthle cell tumours of the thyroid,squamous cell carcinoma of the head and neck,human colorectal carcinoma, human keratinocytes and mouth 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.In the pathogenesis of Ovarian cancer,the signal transduction pathway of STAT3 is essential to activate the continuity of its activation and participated with matrix metalloproteinases,Bcl-2 family members,vascular endothelial growth factor,Her-2 gene in anti-tumor cells apoptosis,vascular remodeling,invasion,metastasis,drug resistance.We detected GRIM-19,STAT3 in epithelial ovarian cancer clinical specimens so that we observed what happened on apoptosis gene GRIM-19 and the proto-oncogene STAT3 in epithelial ovarian cancer.In clinical ATRA and IFN united applicated on leukemia,liver,breast,head and neck cancer,cervical cancer,vulvar cancer,renal cell carcinoma,ovarian cancer,neuroblastoma and other malignant tumors. Other studies have shown that,over-expression of GRIM-19 leads to increase the rate of apoptotic breast cancer cells induced by IFN / RA.Zhang and other people transfected with pCMV-Tag-2B and pCMV-GRIM-19 in HeLa cells,and found that transfected with GRIM-19 cells,have about 82.5±3.6%of apoptosis compared with the control group only 42.4±2.6%of apoptosis after treatment with IFN-β/RA.This shows that the over-expression of GRIM-19 has been effective in promoting apoptosis induced by IFN-β/RA.Therefore,we envisaged to transfected GRIM-19 gene to SK-OV-3 cells comparedwith SK-OV-3/Control and observe the impact of the biological characteristics and thus it Provides theoretical and experimental basis on clinical application in the future.[Objective]To research on all-trans retinoic acid combined with interferon-αon SK-OV-3 cells(human ovarian cancer cell line) growth inhibition,and to observe their apoptosis-related gene GRIM-19,proto-oncogene STAT3 expression for epithelial ovarian cancer treatment and provide a theoretical basis and treatment of clues.[Methods]①Western blot,RT-PCR and Fluorescence Quantitative PCR(FQ-PCR) detection of GRIM-19,STAT3 mRNA,protein expression lever in epithelial ovarian cancer clinical specimens;②MTT colorimetric method observation of ATRA/IFN-αcombined application or a separate application of SK-OV-3 cell proliferation rate at different times;③FQ-PCR detection of GRIM-19 mRNA expression lever after ATRA/IFN-αunited or combined;④High expression of GRIM-19 recombinant plasmid by lipofection into SK-OV-3 medium,via puromycin selection of stable transfected cell lines SK-OV-3/GRIM-19,and establish the control group SK-OV-3 / Control,RT-PCR and Western blot identification of GRIM-19,STAT3 expression;⑤MTT assay detection of SK-OV-3/GRIM-19,SK-OV-3/Control cell proliferation,as well as to sensitivity of cisplatin.[Results]①FQ-PCR and RT-PCR detection of mRNA expression level of GRIM-19 significantly decreased yet STAT3 expression enhanced in serous ovarian cancer;②Western blot detection of protein expression levels in serous ovarian cancer that GRIM-19 is lower either STAT3 is increase;③It is significantly growth inhibition that ATRAluM united IFN-α1b500u/ml acting on SK-OV-3 cells,2d,4d,6d,8d and ATRA or IFN-αcombined or separated to the cells(P＜0.05);④FQ-PCR showed that GRIM-19 expression increased significantly at mRNA expression level in ATRA/IFN-α, compaired with the control group and the ATRA group,IFN-αgroup(P＜0.05);⑤successfully set up a high expression of the stability of GRIM-19 transfected cell lines SK-OV-3/GRIM-19,and established control Group SK-OV-3/Control,RT-PCR and Western blot results showed that at mRNA and protein expression levels of GRIM-19 is upgrade either STAT3 is decreased,the difference has statistical significance(p＜0.05);⑥MTT results that SK-OV-3/GRIM-19 group is significantly lower than SK-OV-3/Control in cell proliferation(p＜0.05);⑦Cisplatin resistance research:the cell inhibition ratio of SK-OV-3/GRIM-19 is accrete on the role of DDP,up to 48.76% at 6d,compared with the control group there was a significant difference(p＜0.05);after combined with IFN/RA,the cell inhibition ratio of SK-OV-3/GRIM-19 is notably upgrade,up to 60.76%at 6d,compared with the control group there was a significant difference(p＜0.05).[Conclusion]ATRA combined with IFN-αwould inhibited cell proliferation and induction of apoptosis of SK-OV-3,and its one of the mechanisms for the increase in GRIM-19 expression,so down-oncogene STAT3 expression.