Effect Of Transfection Of TFPI-2 Plasmid On Lung Cancer NCI-H460 Cells

Background: In a worldwide, the morbidity and mortality of Lung cancer run the first place and impact seriously on human's life and health. Non-small cell lung cancer (non small cell lung cancer ,NSCLC) accounts for about 80%. Beacause of the high malignant and high frequency of metastasis, 5-year survival rate of NSCLC patients is poor, just 25% to 30%,and 3/4 of newly diagnosed NSCLC patients are found to have the existence of metastasis,which greatly increases the difficulty of clinical treatment. Molecular biology studies have shown that lung cancer is a complexity of multi-stage biological process with multi-factors being involved, including activation of oncogene and inactivation of tumor suppressor gene. Because the clinical treatment of NSCLC is limited to chemotherapy, radiotherapy and surgery, gene therapy is still in research stage.Therefore, to explore the molecular mechanisms of pathogenesis of NSCLC and the new target for intervention is very important for clinical treatment.Studies show that the human tissue factor pathway inhibitor-2 (Tissue Factor Pathway Inhibitor-2, TFPI-2) is a tumor suppressor gene.TFPI-2 express in a low status in the higher degree malignant tumor cells,and in a relatively higher status in the relatively low degree of malignant tumor cells. Sajani S et al carry out a large number of studies on the effection of TFPI-2 in human lung tumor invasion and metastasis.The results confirm that in lung cancer cells, TFPI-2's low expression can greatly enhance the viability and growth rate of tumor cell A549, and have a certain relationship with invasion and metastasis of lung tumors. So,the purpose of our study is to detect the expression condition of TFPI-2 in NSCLC cells NCI-H460, and to us gene interference methods to improve the TFPI-2 expression status in NCI-H460, thereby to reduce the growth rate of tumor cells and the risk of tumor metastasis.Objective: To investigate the morphology changes of lung tumor cell and detect the expression status of TFPI-2 after gene interference in NCI-H460 cells , so as to give a comprehensive analysis of the role of TFPI-2 in lung cancer occurrence, and offer a new theoretical basis for the clinical treatment of lung cancer.Methods: Liposomes (LipofectamineTM 2000) was used to transfect human TFPI-2 plasmid into human lung cancer cell NCI-H460 in order to improve the expression of TFPI-2 gene. Fluorescence microscopy was used to detect transfection efficiency and investigate the morphology changes of lung tumor cells. RT-Real Time PCR was used to detect the level of RNA expression. Immunofluorescence and Western blot techniques was used to detect the level of TFPI-2 protein expression of transfected NCI-H460 cells. Flow cytometry techniques was used to detect the apoptosis and the cell cycle of transfected NCI-H460 cells.Result:1. Transfection efficiencyThe transfer rate was more than 98% under fluorescence microscope.2. The result of RT-Real Time PCRCompared with the control group and negative group, the expression of TFPI-2 mRNA levels of the experimental group was improved. And with the increase of the number of PCR cycles, the concentration of TFPI-2 CDNA was significantly increased. Using repeated measures analysis of variance data, the differences between the experimental group and blank control group and negative control group were significant. But there was no significant difference between blank control group and negative control group ( P >0.05).3. Result of detecting protein expression level by Immuno-fluorescence and Western blot TechnologyUnder fluorescence microscope, compared with the blank control group and negative control group, the levels of TFPI-2 protein in experimental group was greatly improved. Western blot showed that the levels of TFPI-2 protein in transfected NCI-H460 cells was about double of that in the blank control group and negative control group. The software analysis system named labworks was used for the identification of the results of absorbance ratio of TFPI-2/β-actin between the experimental group and blank control group and negative control group. The differences were both significant (p<0.05) . The difference between blank control group and negative control group was not significant(P >0.05). 4. The results of flow cytometry4.1 The results showed that cell cycle was clearly arrest in the cells 48- 72 hours after transfection.Cells in the experimental group was arrested at G1 / S phase, while the blank control group and negative control showed normal growth trend of the composition distribution.4.2 Detection of apoptosis rate showed that 48-72 hours after transfection, apoptosis rate in experimental group was (24.28±0.33)%, while the blank control group was (4.83±0.17)% and the negative control group was(4.85±0.21)%. Using repeated measures analysis of variance data, the differences between the experimental group and blank control group and negative control group are significant (p<0.05). The apoptosis rate between blank control group and negative control group was not significantly different(P >0.05).Conclusion:Increasing TFPI-2 expression levels in NCI-H460 cells results in reduction of proliferation of NCI-H460 cells and promotion of apoptosis.