Stably Expressed IFI16Gene Inhibited The Colony Formation Of Hep-2Cells

The tumor is a serious threat to human health and a worldwide problem. The treatment of tumors also gradually from a single therapeutic method to comprehensive treatment age. The comprehensive treatment of tumor is based on patients’ body and mind status, the location of the tumor, the pathology type of the tumor, clinical analysis and development trend and combined the changes of molecular biology of the cell. It has scientificly, planfully and reasonablely to use the existing multidisciplinary effective treatment method for tumor therapy. Then increase the cure rate of tumorous patients in large, degree the pain of the patients for tumor in large, at the same time, enhance the patients’ quality of life, to achieve the best cure purpose. For the moment, The comprehensive treatment of tumor is getting more and more concerns.In addition, the level expression of IFI16genes is very low in cancer cells, but One major antiapoptotic mediator that is overexpressed in cancers is transcription factor NF-κB. NF-κB is well known as a mediator of immune and inflammatory responses, Studies have proved NF kappa-B nuclear factor can promote cancer cell proliferation, invasion and metastasis, and it also activates the transcription of proteins that inhibit apoptosis. Suppressed activation of NF-κB treatment with drugs to explore the clinical theraphy of cancer. Inhibition of NF-kappaB may be of potential therapeutic benefit in the treatment of Laryngeal squamous cell carcinoma. The antioxidant pyrrolidine dithiocarbamate (PDTC) has been shown in previous work to selectively inhibit NF-kappaB activation. In recent years, studies have reported the curcumin inhibits NF-kappaB activation and it could induce cell apoptosis in cancers.we will use PDTC and Curcumin to inhibit NF-κB and to test the synergistic antiproliferative effect of the curcumin and PDTC on Hep-2cell. The results show that decrease proliferation ability and induce apoptosis in Hep-2cells. The results is play a key role for our subsequent research and it is a very important to clinical treatment with the laryngeal squamous carcinoma.Laryngeal squamous cell carcinomas is a common malignant tumors of the head and neck, its incidence rate is rising year by year, and the increasing tendency is more and more to be younger, But it is not clear with the mechanism of development of Laryngeal squamous cell carcinomas is not clear. In addition, the prognosis of traditional treatment methods for treatment of advanced laryngeal cancer are often not ideal and have a great side effects. In recent years, the comprehensive treatment for cancers are more and more attention from people, and the Gene therapy is one of that. Therefore, looking for related gene of the occurrence of laryngeal cancer is play a very important role in the reveal molecular mechanism of laryngeal cancer and early diagnosis and prevention. In the past, the research data shows that P53protein (tumor suppressor gene) was associated with the occurrence of laryngeal cancer in hunman. Besides, at present, the IFI16gene that is interferon inducible IFI-200family members is also about with the occurrence of laryngeal cancer. In a previous study, it was demonstrate that IFI16gene had a role in anti-breast cancer and prostate cancer, and exhibited cell growth inhibititory effect in head and neck sqamous cell carcinoma HNO136cells.This research will RT-PCR amplification get IFI16gene cloning to pEGFP-C1carrier to construct recombinant plasmid pEGFP-IFI16The amplified coding region of IFI16gene by the RT-PCR technique was cloned into the green fluorescent protein eukaryotic expression vector-pEGFP-C1To construct pEGFP-IFI16eukaryotic expression vector and analyze the influence of its expression on Hep-2cell proliferation.In the study, IFI16gene amplified from RT-PCR was constructed into eukaryotic expression plasmid pEGFP-C1to obtain pEGFP-IFI16recombinant plasmid, and this recombinant plasmid was transfected into Hep-2cells with Lipofecter transfection. After transfection, the ectopic stable expression of IFI16gene in Hep-2cells was analyzed using semi-quantitative RT-PCR method, Hep-2cells proliferation influenced by the ectopic stable expression of IFI16gene determined using cell growth curve drawing method and MTT method, and Hep-2cell cycle and apoptosis influenced by the ectopic stable expression of IFI16gene was analyzed using flow cytometry, Synthetic shRNA for IFI16gene was constructed into lentiviral expression vector pGreenPuro to obtain pGreenPuro-IFI16recombinant plasmid. And then this recombinant plasmid was transfected into into Hep-2cells with Calcium phosphate to construct Hep-2/pEGFP-IFI16cell line for stable expression of this recombinant plasmid. Semi-quantitative RT-PCR result showed that the mRNA level of IFI16gene was noticeably increased in pEGFP-IFI16recombinant plasmid transfected Hep-2cells. It was found from cell growth curve assay that Hep-2cells greow slower than control cells after one day of transfection with pEGFP-IFI16recombinant plasmid. and the growth rate of Hep-2cells was obviously slower than control cells when transfected with pEGFP-1FI16recombinant plasmid for two days and statistically different to that of control cells(p<0.05). MTT result showed that the relative viable cell number of Hep-2cells was remarkably decreased after48h of tranfection with pEGFP-IFI16recombinant plasmid and significantly different to that of control cells(p<0.05and p<0.01). flow cytometry analysis disclosed that the ratios of sub GO cells and apoptosis cells in Hep-2cells transfected with pEGFP-IFI16recombinant plasmid for48h were obviously increased comparing to that of control cells. The above results illustrated that the pEGFP-IF116recombinant plasmid could inhibit Hep-2cells proliferation, retard Hep-2cells cycle in sub GO and induce Hep-2cells apoptosis. We constructed the pGreenPuro-control-shRNA of successfully, and a Hep-2cell lines for stable expression of pGreenPuro-control-shRNA is in constructing.In the study, we evaluated the effect of IFI16gene for Hep-2cells by over-expression technology and as an auxiliary, we demonstrated the effect by silencing technology It was showed that IFI16inhibits the Hep-2cell proliferation from the above results. And using the liposomal transfection, flow cytometry and other experiments to verify this speculation. All of these will provide basis for further clarify the molecular mechanisms of IF116inhibits the Hep-2cell proliferation.