Effect Of Cystic Fibrosis Transmembrane Transduction Regulator (CFTR) On The Characteristics Of Lung Cancer Stem Cells In Response To Nicotine

Objective:To investigate the effects of CFTR on the characteristics of tumor stem cells in lung adenocarcinoma A549 cells treated with nicotine by regulating the expression of CFTR gene in lung adenocarcinoma A549 cells and the expression of CFTR gene in clinical smoking and non-smoker lung cancer tissues.It reveals the mechanism of action of CFTR gene in lung cancer metastasis,exacerbation and drug resistance,and provides a new target for the treatment of clinical lung cancer.Methods:A549 cells were inoculated into 96-well plates at 10~5/well,cultured at 37°C,5%CO2,and cultured for 24 h.A549 cells were stimulated with different concentrations of nicotine for 48 h to select the optimal nicotine concentration by MTT assay.The lung adenocarcinoma cell line A549 was infected by constructing the CFTR adenovirus overexpression vector and the CFTR interfering adenovirus vector,and the A549 cells of different treatment groups were stimulated by the above selected nicotine concentration,and the whole protein and cell of the different treatment groups were respectively extracted.Using Western Blot analysis of the expression of CFTR gene in lung adenocarcinoma cells,revealing the effect of nicotine on CFTR gene expression in lung adenocarcinoma cells.The thoracic surgery in the operating room for lobectomy or one-sided pneumonectomy in lung cancer patients and non-smoking lung cancer patients with adjacent normal lung tissue specimens and cancer tissue specimens in 30 cases,using RT-PCR analysis of CFTR in these tumor tissues Expression to elucidate the clinical relevance of the preliminary determination of nicotine and CFTR genes in the pathogenesis of lung adenocarcinoma.The effects of CFTR expression on the malignant characteristics of proliferation,migration and invasion of non-small cell lung cancer A549 cells were detected by CCK8 cell proliferation assay,cell scratch assay,Transwell cell invasion assay and colony formation assay.At the same time,the effect of CFTR gene expression on the expression of tumor stem cell-associated transcription factors was analyzed by Western blotting.Results:Nicotine inhibits the expression of the CFTR gene and enhances the characteristics of lung cancer stem cells.Western blot results showed that CFTR protein was significantly reduced in A549 cells infected with nicotine-adsorbed Ad/CFTR compared to controls(p<0.01).Immunofluorescence staining further revealed that nicotine significantly reduced the expression of CFTR in A549 cell membrane and cytoplasm.These results indicate that nicotine can inhibit the expression and function of CFTR protein in A549 cells.The effects of CFTR on proliferation,migration and invasion and colony formation of A549cells were examined by CCK8 assay,cell scratch assay,Transwell assay and colony formation assay.Cells exposed to nicotine showed lower proliferative capacity compared to control cells,indicating that nicotine at a concentration of 200 nm/L exhibited marginal toxicity in A549cells.The concentration of nicotine at a concentration of 200 nm/L showed enhanced cell migration and colonization of A549 cells.It is worth noting that overexpression of CFTR significantly inhibited the migration and colonization of A549 cells,regardless of the presence or absence of nicotine(p<0.01).In contrast,knockdown of CFTR by shRNA showed a significant increase in the efficiency of migration invasion and colony formation in A549 cells(p<0.01).These results indicate that CFTR can inhibit the metastatic characteristics of A549tumor cells.Expression by Westernblot of stem cell associated transcription-related factor,compared to control infected with Ad/CFTR and Ad/BgLII cells,although many putative stem cell associated protein was not detected significant change,but Ad/CFTRi infected A549 cells A significant increase in CD133,OCT3/4 protein abundance was observed.The A549 cells were treated with Ad/BgLII(BgLII),Ad/CFTR(CFTR)and Ad/CFTRi(CFTRi)24 hours for infection,then exposing them to nicotine or control an additional 24 hours,cells were then harvested and stained for flow ALDH Cytological analysis.Quantitative analysis of flow cytometry data compared to Ad/BgLII-infected cells compared to cells treated with no nicotine showed that CFTR did not significantly alter the proportion of ALDH-positive cells in A549 cells(p>0.05).To further investigate the clinical relevance of CFTR and lung adenocarcinoma expression and function,RT-PCR analysis was used to assess the relative CFTR transcripts in human lung tissue and its matched tumor adjacent lung tissue and normal lung tissue of lung adenocarcinoma patients.Abundance.Surprisingly,unexpectedly richer CFTR transcripts were detected in tumor tissues compared to normal lung tissue(p=0.0107)and adjacent tumor tissues(p=0.0053)in lung adenocarcinoma patients,but no statistical difference was observed between normal lung tissue and adjacent tumor tissues.Conclusion:1.Nicotine inhibits the expression of CFTR protein in A549 cells.2.CFTR decreases the characteristics and motility of A549 cells.3.CFTR inhibits the clonogenic capacity of A549 cells.4.CFTR reduces the expression of stem cell-related transcriptional factorsand CD133 in A549 cells.