| Background and Specific AimsLung cancer is one of the most common malignancies. National Cancer Institute in 2015 released data show that China's male lung cancer incidence rate in the No.2, female cancer incidence rate in the No.4. Among non-small cell lung cancer (non-small cell lung cancer, NSCLC) is the most common, accounting for about 80%-87% of the total lung cancer. With NSCLC pathogenesis and genetic research driven deep, epidermal growth factor (Epidermal Growth Factor Receptor, EGFR, also known as ErbBl or HER1) pathway signal role in the development process takes place in the NSCLC constantly being taken seriously and understanding. Development of the important role of EGFR overexpression and mutations in lung cancer, making it one of the target molecule targeted therapy. Targeted therapy has become an important treatment for NSCLC. Although the treatment of NSCLC targeted drug has a good clinical efficacy, but with the extension of treatment time, the resistance comes, continue to impede the application of targeted drugs. Therefore, a method of treating resistance to reverse current target is an important issue in targeted therapeutic areas.Astragalus is commonly used in traditional Chinese medicine, contains many chemical ingredients, recent studies have reported that Astragalus in vivo and in vitro have anti-tumor effect, astragaloside (Astragaloside IV, AS-IV) is a major component of astragalus. Studies have shown that, AS-IV can inhibit migration and invasion of lung cancer cells by regulatory T cells and cytotoxic T lymphocytes and signaling pathway.SIRT6 is ten NAD-dependent Class ? histone acetyl enzyme family (Sirtuin family) an important part of it. Epigenetic regulation is referred to a series of biological processes, modification, chromosomal DNA and tissue changes related proteins involved by methylation, acetylation of chromatin remodeling and other processes to regulate gene expression. Now researchers found, SIRT6 expression and tumor metastasis in tumor tissue, postoperative survival and recurrence rates are closely related, is now considered a new tumor suppressor gene, inhibit or even reverse tumor resistance to chemotherapy is a new target.The purpose of this study was to (1) explore the expression of abundance and localization SIRT6 expression in NSCLC, as well as in human lung cancer NCI-H1299, PC9/GR, A549 cells expressing SIRT6; (2) different concentrations Astragaloside effect on people lung cancer NCI-H1299, PC9/GR, A549 cells were observed for different mutation status of lung cancer cells proliferation; (3) different concentrations Astragaloside joint gefitinib acts on the human lung cancer NCI-H1299, PC9/GR, A549,observed its different mutation status of lung cancer cell proliferation; (4) After the AS-IV alone intervention cells and AS-IV combined with gefitinib combined intervention of cell changes in the expression levels of SIRT6; (5) Lentivirus transfection PC9/GR cells detected AS-IV combined with gefitinib on PC9/GR horizontal cell proliferation after transfection; (6) To investigate the interaction between SIRT6 and NF-?B signaling pathway in lung cancer cells resistant..Research methods:1, by immunohistochemistry assay NSCLC tumor tissues, adjacent tissues and benign lesions expression SIRT6 and to collect clinical data, analyze the relationship between SIRT6 and clinical parameters;2, qRT-PCR and Western blot test methods detection BEAS-2,NCI-H1299, PC9/GR, A549 cells SIRT6 expression;3, the effect of different concentrations of gefitinib imatinib on human lung cancer NCI-H1299, PC9/GR, A549 cells, CCK-8 assay Cell proliferation was read OD value, calculated according to the following formula cell growth inhibition rate and cell growth curve depicted;4 different concentrations AES-? acts on human lung cancer NCI-H1299, PC9/ GR, A549 cells, CCK-8 assay Cell proliferation was Read OD in a microplate reader, according to the following formula cell growth inhibition rate and cell growth curve depicted;5. AS-IV combined with different concentrations of gefitinib imatinib role in human lung cancer NCI-H1299, PC9/GR, A549 cells, CCK-8 assay Cell proliferation, cell growth inhibition rate calculated depicts the growth curve of cells;6, combined with different concentrations of AS-IV gefitinib gefitinib Nepalese effect of different concentrations in the human lung cancer alone PC9/GR cells using qRT-PCR detected SIRT6 mRNA expression; protein expression using Western-blot technique to detect changes SIRT6:7. choose Lentivirus transfection transfection. SIRT6 eukaryotic expression vector was transfected PC9/GR cells, inverted fluorescence microscope to observe the expression of green fluorescence 48 hours after transfection, determination of viral titer. using Western-blot technique to detect changes SIRT6 protein. Detected AS-IV Joint gefitinib on PC9/GR cell proliferation levels after transfection:8, Western-blot detection I?B and NF-?Bp65, study of the interaction between SIRT6 and NF-?B signaling pathway in lung cancer cells resistant.Results:1, The result SIRT6 lower than in NSCLC tissue and benign tissue adjacent to cancer; the higher the degree of differentiation IOD higher than average degree of differentiation, in patients with low. There was a significant difference between the average IOD lymph node metastasis and lymph node metastasis did not occur in patients SIRT6.2, By CCK-8 experiments, different concentrations of gefitinib intervention A549, after PC9/GR and H1299 after 48h, inhibition of cell proliferation as the concentration increased and increased, reflecting the dose-dependent inhibition. Compared with the control, gefitinib cell inhibition significantly different. Gefitinib half inhibitory concentrations were 21.29?M,45.98±1.62?M,26.29±2.25?M;3,Using CCK-8 method, choose different concentrations of AS-? acts on the A549 and PC9/GR cell lines, the researchers found that when intervention time is at 48h,3ng/ml and 6ng/ml concentration of AS-? of the A549 and PC9/GR cells can not produce significant inhibition, while greater than 12ng/ml of AS-? significantly inhibited the growth of cells. Select different concentrations of AS-? acts on H1299 cell line, when intervention time is 48h when,3ng/ml and 6ng/ml can produce significant inhibition;4, With 4 concentrations (3ng/ml,6ng/ml,12ng/ml,24ng/ml) of the AS-? and 10?M gefitinib medium combined effect on A549, H11299, PC9/GR cells after 48 hours, CCK-8 reagent microplate reader, the same grfitinib concentrations, different concentrations of AS-? acts on the same cell lines, inhibition of cell proliferation was significantly different, inhibition of cell proliferation with AS-? concentrations increases.3 types of cells are visible gefitinib combined with AS-? at a concentration of 24ng/highest rate of cell proliferation inhibition. At the same concentration of gefitinib, with the drug concentration increased proliferation of different cells, which PC9/GR cell proliferation worst;5, By qRT-PCR and Western blotting techniques, compared PC9/GR, A549, H1299 cells with normal lung tissue glandular epithelial BEAS-2B cells, SIRT6 mRNA and protein expression level decreased significantly;6, qRT-PCR analysis showed that, compared with the control group, AS-? 12ng/ ml,24ng/ml combined with gefitinib 10?M intervention PC9/GR, the increased expression of SITR6mRNA. Separate AS-? 24ng/ml alone intervention PC9/GR, compared with the control group, a statistically significant, but separate AS-? 12ng/ ml alone intervention PC9/GR, there was no statistically significant;Western blotting detection results, compared with the control group, AS-? 12ng/ ml,24ng/ml combined with gefitinib 10?M intervention PC9/GR, increased protein expression SITR6 separate AS-? 24ng/ml alone intervention PC9/GR compared with the control group, while a separate AS-? 12ng/ml alone intervention PC9/GR, compared with the control group, no statistical significance;7, Gefitinib concentration gradient 0,5,10,20,40?M alone intervention PC9/GR, compared with the control group, SIRT6mRNA cells and protein levels were higher, but not statistically significant;8,Constructed stably transfected SIRT6 overexpression of PC/GR, inverted fluorescence microscope observed, cell transfection efficiency is estimated at about 80%-85%. By Western Blot testing, compared with the control group, the protein expression of SIRT6 has greatly improved;9, With the 10?M gefitinib and concentration gradients AS-? intervention pLVTHM-SIRT6 and pLVTHM-scramble cells,with the other three groups, SIRT6, gefitinib and AS-? when the combined effects of cell growth inhibition rate increased significantly;pLVTHM-SIRT6 group pLVTHM-scramble+gefitinib group showed no significant statistical significance, compared with pLVTHM-scramble group, these two groups of cell growth inhibition rate were statistically significant;By AS-? and concentration 12ng/ml gradient gefitinib intervention,compare with pLVTHM-SIRT6 and pLVTHM-scramble cells, SIRT6, AS-IV and gefitinib when the combined effects of cell growth inhibition rate was significantly enhanced, pLVTHM-SIRT6 group pLVTHM-scramble+AS-IV group showed no significant statistical significance, compared with pLVTHM-scramble group, these two groups of cell growth inhibition rate were statistically significant;10, Western blot detection pLVTHM-scramble and Plvthm-SIRT6 cells, I?B expression level in pLVTHM-scramble cells lower than the expression level in Plvthm-SIRT6 cells, by AS-IV24ng/ml intervention pLVTHM-scramble cells and Plvthm-SIRT6 cells, I?B in pLVTHM-scramble expression levels belower than Plvthm-SIRT6 cells, as compared to cells without treatment, both I?B levels were raised;AS-IV24ng/m interventions pLVTHM-scramble and Plvthm-SIRT6 cells and cells after 48hs, extract nuclear proteins in each group, western blot to detect the expression of NF-KBp65 subunit,NF-?Bp65 subunit Plvthm-SIRT6+AS-IV Intervention cells was the lowest, compared with pLVTHM-scramble cells, SIRT6 overexpression reduced NF-?Bp65 subunit Plvthm-SIRT6 accumulate in the nucleus.Conclution:1,Levels of SIRT6 falled in NSCLC tissues and lung cancer cells; 2, the AS-IV has inhibitory effect to the lung cancer cells, showed a trend of concentration;3, AS-? combined with gefitinib increase in tumor inhibition rate;4, Overexpression of SIRT6 can inhibit the activation of NF-?B;5, AS-? can rais SITR6 level, inhibit NF-?B activation, promoting gefitinib sensitivity. |
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