| BackgroundHepatocellular carcinoma(HCC)has become one of the most lethal kind of tumors up to now,and situations are worse in China,with the highest incidence and mortality,threatening the health of Chinese people severely.The mainstream therapies against HCC include hepatectomy,ablation therapy,interventional therapy,molecular targeting treatment,radiotherapy and chemotherapy,et al.Though integrated treatments are employed,the prognosis of HCC patients remains grim.To date,there's much evidence suggesting that the accumulated activation of oncogenes,inactivation of tumor suppressor genes and aberration in some genes such as cycle-regulation in hepatocytes,which normally last for a long period,gradually lead to tumorigenesis in liver.For instance,the protein encoded by Ras takes part in the signaling pathway regulating transcription so as to influence the growth and differentiation of cells.This gene would be activated as oncogene once point mutation took place.The Ras/MAPK pathway was found activated in at least 50% of human HCC.On the other hand,PTEN is a tumor suppressor gene,whose function is to prohibit the proliferation and growth of cells and induce apoptosis in some cases through hydrolysis to decrease the activity of downstream molecules.Missense mutation or deletion would weaken the activity of PTEN protein,and eventually its tumor suppressing function.Theoretically,all genes mentioned above possess the potent to be the therapeutic targets to HCC.Therefore,as the researches of gene therapy go further,there exists great possibility to shed light on the new and effective treatments against HCC.Tg737 was first found in algae,encoding the homolog of intraflagellar transport 88(IFT88),both of which are essential to cilia and flagella.Tg737 was found to take part in polycystic kidney disease,and recent studies suggested that it was also a tumor suppressor gene which could play a role in hepatocellular carcinoma cells.As reported,the expression of Tg737 was downregulated in 59% tissues in HCC,and this gene was found to be able to exert influence on cell cycle,proliferation and apoptosis in HCC cells,as well as the migration and invasion in hypoxia state.PurposeThis study intends to achieve the overexpression of Tg737 in HepG2 cells to detect its effects in the following aspects: cell cycle,apoptosis,proliferation,migration and invasion,hoping to provide HCC therapy with a new treating target.Method1.To construct the Tg737 overexpression model of HepG2 cells.The overexpression was achieved by gene transfection using pcDNA3.1-Tg737 plasmid,and all cells involved were sorted into 4 groups: the group of cells transfected with pcDNA3.1-Tg737(experimental group),the group incubated with liposome(liposome group),the group transfected with pcDNA3.1(-)(vector group)and the group without transfection(blank control group).2.To detect the possible influence of Tg737 overexpression on cell cycle and apoptosis in HepG2 cells.48 hours after transfection,flow cytometry was used to detect the distribution of cell cycle in 4 groups of cells,Annexin V/ propidium iodide(PI)double staining and morphological methods were employed to detect apoptosis status in different groups of cells.The expression of proteins associated with apoptosis and cell cycle was assessed by Western blot.3.To detect the possible influence of Tg737 overexpression on migration and invasion in HepG2 cells.24 hours after transfection,the abilities of migration and invasion of different groups of cells were tested by transwell migration and invasion assay.Phalloidin dye was carried out to determine the morphological changes of cells.The expression of proteins of EMT was assessed by Western blot.Result1.Western blot was employed to assess the expression of Tg737 at both 24 hours and 48 hours after transfection in different groups of HepG2 cells,which manifested that the expression was increased significantly in experimental group(all P < 0.05),while the expression levels among the rest groups showed no significant difference.The possible interference on overexpression by plasmid vector or liposome was excluded.2.48 hours after transfection,compared with 3 control groups,the proportion of cells staying in S phase increased significantly in experimental group,correspondently,cells in G0/G1 and G2/M phase decreased remarkably(all P < 0.05).The result of Annexin V/PI staining showed that there existed a few apoptotic or necrotic cells normally,however,48 hours after transfection,the quantity of normal cells decreased and apoptotic cells increased significantly(all P < 0.05),while that of necrotic cells remained close(all P > 0.05).Morphological assays found that typical changes of apoptosis took place in experimental cells,and cells in control groups showed scarcely any.Western blot revealed the elevated expression level of cyclin A and Bax,and down-regulated expression of Bcl-2 in experimental group(all P < 0.05).3.24 hours after transfection,both transwell migration and invasion assay came to the same conclusion that the abilities of migration and invasion of cells in experimental groups were inferior to those in control groups(all P < 0.05).Through phalloidin dye,apparent pseudopodium was observed in control group cells but not in experimental group.Result of Western blot showed the expression of E-cadherin was elevated,expression of Vimentin and Fibronectin spontaneously were found to decrease significantly in experimental group(all P < 0.05).Conclusion1.The overexpression of Tg737 could induce cycle arrest and promote apoptosis to prohibit proliferation of HepG2 cells,the mechanism of which may partly lies in the cyclin A,Bax,Bcl-2 signal pathway mediated by Tg737.2.The overexpression of Tg737 could weaken the abilities of migration and invasion of HepG2 cells,may partly owing to the inversion of EMT influenced by Tg737. |
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