The Correlation Research Of GRIM-19 And Its Target Gene Product STAT3 In Human Cervical Carcinoma

[Background]Cervical cancer is one of the most common malignant tumors,and its incidence in women's cancer is at the second place.All over the world around two hundred thousand women have died from this disease per year.Mapping the human genome and studing human genome have provided a new way for prevention and treatment of malignant tumors from the source of the tumor.Recent study shows that in the human genome activation of oncogene and inactivation of tumor suppressor gene related to cell proliferation and differentiation cause the occurrence and development of the malignant tumor.On this basis the theoretical and practical exploration of gene therapy for malignant tumors origin.Looking for and determining the target gene associated with therapy is to implement a prerequisite for tumor gene.Gene transfection is to transfer or delivery a nucleic acid to cells and to maintain its biological functions in cells.Nucleic acid is including DNA(plasmid and linear double-stranded DNA), antisense oligonucleotide and RNAi(RNA interference).Gene transfer technology has been widely used in genomic functional research and gene therapy research.The former includes gene expression regulation,gene function,signal transduction and drug screening research.Liposome plays an important role on vector introducted into cell in all scientific research and clinical trials,and in recent years are the most promising non-viral vector for transfering DNA into culture cells,and are currently the most convenient of transfection methods.Compared with the viral vector,liposome-mediated method is more simple,safe and has low immunogenicity,can be mass-produced,and not subject to the restrictions on the size of portable devices.IFN/RA combination induced cell apoptosis-related gene(gene associated with retinoid-interferon-induced mortality-19,GRIM-19) is a new apoptosis gene.GRIM-19 involves in cell apoptosis by a variety of mechanisms.Normal expression of GRIM-19 can maintain a normal cell cycle so that cells enter apoptosis.On the contrary,low expression of certain cells should not enter apoptosis,and thus trigger the occurrence of tumor.GRIM-19 is one of the basic components of mitochondrial NADH-dehydrogenase 1 complex and plays an important role on the mitochondrial respiratory processⅠ.It also involves in energy and oxygen supply,activation of apoptosis in cells.Signal transducersand activators of transcription 3(STAT3) is a member of the Src tyrosine kinases family.STAT3 and its mediated signal transduction pathway involve in the regulation of cell proliferation,apoptosis,differentiation and mediate cellular malignant transformation.GRIM-19 is STAT3-specific inhibitory protein.A variety of tumor tissues have lower or none expression of GRIM-19.[Objectives]To investigate the expression of GRIM-19 and its target gene STAT3 in human cervical cancer tissues.To establish stable expression of GRIM-19 in the HeLa cell lines.To analyze GRIM-19 and its inhibition to STAT3 and its effect on the biological behavior of the human HeLa cervical cancer cell line and explore their roles in the occurrence of the cervical cancer.To provide a theoretical and experimental basis for for clinical treatment.[Methods]①Immunohistochemical detection of the expression of GRIM-19 in normal cervical tissue and cervical cancer tissues;②Western Blot and RT-PCR detection of the expression of GRIM-19 and STAT3 in cervical cancer tissues;③GRIM-19 sequences transfected into HeLa cervical cancer cell lines to establish an stable transfection of GRIM-19;④Identify the expression of GRIM-19 in Hela/GRIM-19 and Hela/control cell line and its inhibitory to STAT3 by RT-PCR and Western-Blot method;⑤Assay cell proliferation ability of Hela/GRIM-19 and Hela/control cells after different treatments by MTT and draw growth curve;⑥Set a tumor growth experiment of Hela/GRIM-19 and Hela/control cell line in nude mice and draw a growth curve mapping. [Results]①Immunohistochemistry result indicates that the expression of GRIM-19 protein in normal cervical tissues is significantly higher than that in cervical cancer organizations;②Western blot detection of protein expression levels in squamous epithelial carcinoma that GRIM-19 is lower either STAT3 is increase;③RT-PCR detection of mRNA expression level of GRIM-19 significantly decreased yet STAT3 expression enhanced in squamous epithelial carcinoma;④Hela/GRIM-19 and Hela/control cell lines which have a stable expression of GRIM-19 are established;⑤MTT test indicates that cell proliferation of Hela/GRIM-19 group is much lower than that of Hela/control group(p＜0.05);⑥Tumor cell's growth rate and the size of tumor in Hela/GRIM-19 group by subcutaneous inoculation in nude mice was significantly lower than that in Hela/GRIM-19 group.(p＜0.01)...