| The incidence of hepatocarcinoma in the world ranked sixth in malignant tumors and the mortality rate was third.Surgical resection is the preferred way for hepatocarcinoma and the comprehensive antitumor treatment should be done also.The fast pace of development and the late diagnosis of hepatocarcinoma make more patients get no chance to get surgical resection.So many scholars pay more energy to research more effective way for the treatment.In these years more development have been made in antitumor targeted therapy. Scholars make the DNA, metabolic pathways and micro environment be the target. The scholars get a lot of achievements, they commit to cure cancer but no destroy to normal cells.There are some targets to cure hepatocarcinoma, such as tumor suppressor gene or oncogene, angiogenic gene and cell adhesion factor. Scholars wants to inhibit tumor cell growth, inhibit the produce tumor blood vessels. Endocan gene shows close relationship to tumor cells and tumor angiogenesis in literature reports.This study through the following measures to research wether Endocan can be a target gene for treatment to hepatocarcinoma: 1. RNAi technology to silence Endocan gene of SH-HEP-1 cells.In this experiment three sequences of RNA interference and one negative control sequence are designed, by plasmid as the carrier transfected into hepatocarcinoma cells, we select the best silencing effect through Endocan protein detection to do the next step experiment.2. Research the proliferation and invasion ability of hepatocaicinoma cells by MTT test and Transwell little room experiment.In this section through no transfection of plasmid group(Control group), RNA interference group(si RNA group), negative control group(NC group) were compared, to observe wether the cell proliferation and invasion ability of si RNA group will be suppressed. 3. Research the hepatocacinoma cells apoptosis after Endocan gene silencing.In this section using the apoptosis detection kit, flow cytometry instrument to observe apoptosis between the Control group, the si RNA group and the NC group after the Endocan gene silencing.4. Research the hepatocaicinoma cells autophagy after Endocan gene silencing.Autophagy death is also a kind of programmed cell death like apoptosis.In this section by detecting the autophagy related proteins, cells of the Control group, si RNA group, NC group are observated after Endocan gene silencing. 5. Endocan gene silencing affect cells survive through the NF-κB pathway.To study Endocan gene pathway how to affect the cells survive, this study examined the NF-κB pathway, hope to further understand Endocan gene and protein from the gene pathway for cell survival.Experiment and research scheme based on the above, we got the following results through a series of research work in the process of the experiment: 1. Endocan silenced hepatocarcinoma cells SH-HEP-1 as the research object,they are tested by MTT and Transwell Chambers experimental to observe the ability of the proliferation and to attack.The experimental results shows that when Endocan gene be silenced, si RNA group multiplication rate decreased significantly, and cell number count showed which can through the Transwell little room is less than that Control group and NC group. 2. Cells’ apoptosis rate is 15% after Endocan gene silencing, while the control group werenot more than 5%. The other ways by Western Blot and m RFP-GFP-LC3 double fluorescence system detection from autophsgy related proteins found that when Endocan gene is silenced, autophagy flow obviously happened. 3. By Western Blot and EMSA methods to detect the NF-κB in the nuclei, we found that the NF-κB quantity was obviously reduced in the nuclei of si RNA group.Comprehensive the above study get the following conclusions: Endocan gene silencing inhibits hepatocarcinoma cells proliferation and invasion; Endocan gene silencing induces apoptisis and autophagy;Endocan gene silencing affects cell survival through the NF-κB pathway. |
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