Effects Of Transfections With MT-3 And MT-1E Genes On Cell Cycle In Cell Lines Of Squamous Cell Carcinoma Of The Esophagus

Objective:Esophageal cancer pose a serious threat to human health and to life as one of the malignant tumors. The mortality of esophageal cancer in the world ranks the seventh of malignant tumors .China is a country of high incidence of esophageal cancer which the incidence ranks the fourth place, especially in the south of Hebei, the north of Henan and parts of Shanxi as the high-risk area.Therefore,in-depth study of the pathogenesis of esophageal cancer in order to effectively prevent and treat them have very important significance.Studies have shown that the incidence and development of esophageal cancer is non-single molecular events,but a multi-factor,multi- stage,long multi-step process,involving multiple oncogenes and tumor suppressor genes and cell regulatory mechanism to change the exception.There are many reports about the MT-1 and MT-2 genes,which exists in esophageal carcinoma with overexpression condition.But,there are little reports about the MT-3 gene,It is well-known that MT-3 gene encodes metallothionein in cells,which not only has many important physiological functions, but also inhibits cells proliferation.The hypermethyla- tion of the MT-3 gene extensively exists in esophageal carcinoma in our early day,s study,moreover this hypermethylation results in the expression down regulation of the MT-3 gene.Based on the above studies,this study was designed to research effects of transfections with MT-3 and MT-1E genes on cell cycle in cell lines of squamous cell carcinoma of the esophagus.Methods: 1 The Eca-109 and TE13 cells of squamous cell carcinoma of the esophagus were cultured in six-well plate with RPMI1640 medium,in cell confluence of 70%,preparation for cell transfection.2 Four kinds of plasmid DNA of E.coli strains which contain EX-T3737-M03-MT-3, EX-T3737-M03, EX-T2598-M56-MT-1E and EX- T2598-M56 were obtained,through one night,extracted plasmid DNA and determined its concentration and purity; then prepared plasmid DNA/liposome complexes, and established a blank control, carried out cell transfection.3 The above plasmid DNA/liposome complexes and the two kinds of cell lines were cultured, and fluorescent protein expression of plasmid DNA were abserved in the cells with the fluorescence microscope in 24h, 48h and 72h ,respectively ,and calculated cells transfection efficiency in order to determine the optimal time of transfection of plasmid DNA in the cells.4 After the Eca-109 and TE13 cells which were transfected by plasmid DNA for 48h, the cells of the transfection groups and control groups were collected in six-well plate and observed the change of cell cycle by flow cytometry.5 All measurement data were demonstrated with the mean±standard deviation,and two samples mean were selected t test or t, test ,P<0.05 as statistically significant difference, and statistical process was completed by the SPSS13.0 statistical software.Results:1 After transfected the Eca-109 and TE13 cells of squamous cell carcinoma of the esophagus by four kinds of plasmid DNA,fluorescent marker were observed in all groups by fluorescence microscope in 24h,48h and 72h.The transfection groups of EX-T3737-M03-MT-3 and EX-T3737-M03 were green fluorescent.The transfection groups of EX-T2598-M56-MT-1E and EX-T2598-M56 were red fluorescent. the fluorescence intensity was the strongest after transfection cells for 48h,accounting for 80% of the total number of cells, suggesting that transfection was successful and efficient.2 After transfected the Eca-109 and TE13 cells by EX-T3737-M03-MT-3 and EX-T3737-M03 for 48h,its proportion of G0/G1 phase cells ratios were 70.97±1.58% vs 53.20±2.13 %(P<0.05),S phase cell ratios were 14.32±1.23% vs 31.50±1.59%(P<0.05), G2/M phase cells ratios were 14.72±1.08% vs 15.30±1.38%(P>0.05).3 After transfected the Eca-109 and TE13 cells by EX-T2598-M56-MT- 1E and EX-T2598-M56 for 48h,its proportion of G0/G1 phase cells ratios were 54.58±2.38% vs 53.02±2.16%(P>0.05),S phase cell ratios were 30.15±1.77% vs 31.90±1.71%(P>0.05),G2/M phase cells ratios were 15.27±1.06% vs 15.08±0.78 %(P>0.05).4 After transfected the Eca-109 and TE13 cells by EX-T3737-M03-MT-3 and EX-T2598-M56-MT-1E for 48h,its proportion of G0/G1 phase cells ratios were 70.97±1.58% vs 54.58±2.38%(P<0.05),S phase cell ratios were 14.32±1.23% vs 30.15±1.77%(P<0.05),G2/M phase cells ratios were 14.72±1.08% vs 15.27±1.06 %(P>0.05).Conclusion:1 After transfection with the MT-3 gene in Eca-109 and TE13 cells of squamous cell carcinoma of the esophagus, the proportion of G0/G1 phase cell ratios were significantly increased,and the S phase cell ratios were significantly reduced,so the gene of MT-3 was speculated to inhibit the cell growing of esophageal carcinoma through changing the cell cycle.2 After transfection with the MT-1E gene in Eca-109 and TE13 cell of squamous cell carcinoma of the esophagus, the proportion of the cell cycle ratios were no difference, so the gene of MT-1E was speculated that it may be no effect on the cell growing of esophageal carcinoma.