| Purposes:cancer is one of the most serious diseases which threatening people's health, in recent years, the incidence rate and mortality are increased sharply. the mechanism of tumor are most likely to be the gene disease which happened when the gene got mutation ,deletion abnormalities. Therefore, we should be clear from the perspective of lung cancer genes, and to find effective mechanisms for the development of therapeutic targets. The development of modern molecular biology techniques and applications gene therapy has opened a new way for cancer. Recently thyroid transcription factor -1 (thyroid transcription factor-1, TTF-1) as a target for cancer gene therapy is paid attention for its role, in the thyroid tumor and lung tumor development process. Its belongs to the NKX-2 gene family, and is a DNA binding protein, molecular weight of about 38KD, but by the autosomal 14q13 gene encodes one point, involved in human lung, thyroid and brain tissue of embryonic development among a variety of transcription Therefore, differentiation and morphogenesis plays an important role in these cell. In lung tissue, TTF-1 can control the morphology of lung development and composition of surface proteins play a key role in A, B, C and Clara cell secretory protein gene; in thyroid tissue may activate some gene promoters, such as thyroglobulin ( TG), thyroid peroxidase (TPO) and thyroid hormone receptor (TSHR) and other transcriptional activity. The current results show that the TTF-1 only specific highly expressed in lung cancer and thyroid cancer, as a specific transcription factor and DNA binding proteins, TTF-1 may be the target gene transcription through the protein binding site combination, or change the reactivity of DNA binding proteins to positive or negative effects on gene transcription. This study aimed to investigate the effect of down regulation of TTF-1 on the apoptosis of A549. Methods:Firstly, transfect human lung adenocarcinoma cell line A549 with the synthetic siRNA of TTF-1 gene fragment, then observe the morphological changes of these cells by Inverted microscope; flow cytometry apoptotic index in each group is used to detect the apoptosis of these cells.; reverse transcription - polymerase chain reaction (PCR) is used to detect the level of TTF-1 expression at the mRNA; immunofluorescence is used to detect the TTF-1 expression in these transcriptional cell.TTF-1 antibody expression.Results:Morphological changes showed by inverted microscope before and after transfection : before transfection, the cell epithelial grew, good condition, most of cells are spindle-shaped, medium size, clear nucleolus. 48 hours after transfection, irregular shape, cell shrinkage, poor epithelial grew,cell processes disappear. Apoptosis was detected by flow cytometry: the apoptotic rate, TTF-1 siRNA transfection group (32.38±2.03)%, control group (0.38±0.25)%, the negative control group (0.75±0.35)%. This indicates that TTF-1 siRNA could inhibit TTF-1, and induce apoptosis. RT-PCR detection of TTF-1 mRNA expression showed that after TTF-1 siRNA was transfected into A549 cells 48h ,the TTF-1 mRNA expression were significantly reduced, while in the control group and negative control group, TTF-1 mRNA showed no significant change. TTF-1 siRNA could induce TTF-1 mRNA-specific degradation. By immunofluorescence,the expression of TTF-1 protein level,was significantly reduce after transfection.Conclusion:RNAi silencing of the TTF-1 gene can promote apoptosis of A549 cells,which may offer a new targets for the gene theyapy of lung cancer. |
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