| Laryngeal cancer is a common malignant tumors of the head and neck, and its incidence rate is rising year by year. At present, traditional therapies of the laryngeal and other malignant tumors are operation, radiotherapy and chemotherapy. However, these traditional methods for the treatment of advanced laryngeal cancer patients don't have great significance, so in recent years the comprehensive treatment was concerned very much, a-nd the interferon is one of that. Some reasch find, as one of the HIN-200protein family member which is called the interferon inducible family, IFI16protein in hunman and p202protein in mouse were associated with systemic lupus erythematosus. Multiple studies demonstrate that HIN-200protein family can inhibit cell proliferation. For this reason, some researchers began to study on the antitumor effect of IFI16gene. In a previous study, it was demonstrated that IFI16gene had a role in anti breast cancer and prostate cancer, and exhibited cell growth inhibitory effect in head and neck squamous cell carcinoma HNO136cells.In this study, IFI16gene amplified from RT-PCR was constructed into eukaryotic expression plasmid pVR1012to obtain pVR1012-IFI16recombinant plasmid, and then this recombinant plasmid was transfected into Hep-2cells with Lipofecter transfection. After transfection, the ectopic expression of IFI16gene in Hep-2cells was analyzed using semi-quantitative RT-PCR method, Hep-2cells proliferation influenced by the ectopic expression of IFI16gene was determined using cell growth curve drawing method and MTT method, and Hep-2cell cycle and apoptosis influenced by the ectopic expression of IFI16gene was analyzed using flow cytometry, Synthetic shRNA for IFI16gene was constructed into lentiviral expression vector pGreen Puro to obtain pGreen Puro-IFI16recombinant plasmid. And then this recombinant plasmid was transfected into Hep-2cells with Calcium phosphate to construct Hep-2cell line for stable expression of this recombinant plasmid. Semi-quantitative RT-PCR result showed that the mRNA level of IFI16gene was noticeably increased in pVR1012-IFI16recombinant plasmid transfected Hep-2cells. It was found from cell growth curve assay that Hep-2cells grew slower than control cells after one day of transfection with pVR1012-IFI16recombinant plasmid, and the growth rate of Hep-2cells was obviously slower than control cells when transfected with pVR1012-IFI16recombinant 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 transfection with pVR1012-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 apoptotic cells in Hep-2cells transfected with pVR1012-IFI16recombinant plasmid for48h were obviously increased comparing to that of control cells. The above results illustrated that the pVR1012-IFI16recombinant plasmid could ectopicly express IFI16gene in Hep-2cells and the ectopicly expressed IFI16gene could inhibit Hep-2cells proliferation, retard Hep-2cells cycle in sub GO and induce Hep-2cells apoptosis. we constructed the pGreen Puro-IFI16-shRNA and a Hep-2cell lines for stable expression of pGreen Puro-control-shRNA of successfully, and a Hep-2cell lines for stable expression of pGreen Puro-IFI16-shRNA is in constructing.In this 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 IFI16inhibits the Hep-2cell proliferation. |
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