Effect And Mechanism Of CFTR Chloride Channel On Proliferation And Migration Of Lung Cancer Cells

CFTR（Cystic Fibrosis Transmembrane Regulator）is a c AMP-dependent chloride channel.CFTR is widely expressed in a variety of epithelial tissues,heart,smooth muscle,neurons,endothelial cells,sperm,and eggs.CFTR plays an important role in the secretion and absorption of airways,gastrointestinal,reproductive,sweat and salivary glands.Therefore,defects in CFTR function are related to diseases,including secretory diarrhea,cystic fibrosis（CF）,and chronic obstructive pulmonary disease（COPD）.Recent research findings,in addition to being an ion channel,CFTR is also an important regulator of multiple signaling pathways related to cancer progression.Its mutation can increase the risk of cancer in the gastrointestinal tract,pancreas,etc.Studies have shown that the expression of CFTR in non-small cell lung cancer is lower than that in normal lung tissues,and the low expression of CFTR is significantly related to the progression of non-small cell lung cancer and tumor invasion ability.Therefore,CFTR may be a biomarker for the prognosis and prediction of lung cancer patients.In this study,qPCR,Western Blot,and immunocytochemistry were used to compare CFTR in human normal bronchial epithelial cells HBE,human bronchial epithelial cells BEAS-2B,human bronchial alveolar adenocarcinoma cells NCI-H1650,human alveolar type Ⅱ gland Differences in the expression levels of cancer cell A549,human lung squamous carcinoma cell NCI-H226,human KRAS mutation grade Ⅲ epidermoid lung cancer cell Calu-1.Using immunofluorescence experimental methods to study the location of CFTR in lung cancer cells.Using three CFTR inhibitors(CFTR_inh-172,GlyH-101,BPO-27)and activators（Genistein,Forskolin,IBMX）as tool drugs,through cell proliferation,apoptosis,cell scratches,cell invasion,Western Blot Experiments to study the effect and mechanism of CFTR chloride channel function on the proliferation,apoptosis,migration and invasion of the above-mentioned different types of lung cancer.The main findings are as follows:1.The results of qPCR,Western Blot and immunocytochemistry experiments showed that the expression levels of CFTR in the following cells from high to low were HBE,H1650,A549,Calu-1,H226,BEAS-2B.Among them,the expression of CFTR in HBE is much higher than that of human bronchial epithelial cells BEAS-2B.The expression of CFTR in lung cancer cells is higher than that of human bronchial epithelial cells BEAS-2B.The expression of CFTR in lung adenocarcinoma cells H1650 and A549 was higher than that in lung squamous cell carcinoma H226.The expression of CFTR in bronchoalveolar adenocarcinoma cell H1650 was higher than that of alveolar type Ⅱ adenocarcinoma cell A549.The expression level of CFTR in KRAS-mutated lung cancer cell Calu-1 was lower than that of non-mutated lung adenocarcinoma cells H1650 and A549.2.The results of immunofluorescence experiments show that CFTR is localized in the cell membrane and cytoplasm of lung cancer cells,and is more localized in the cytoplasm.3.Cytotoxicity test results showed that when the concentration of CFTR_inh-172 is less than 100 μM,it is not toxic to H1650 and H226;when it is less than 50 μM,it is not toxic to A549 and Calu-1.When the concentration of GlyH-101 is less than 50 μM,it is not toxic to Calu-1;when it is less than 25 μM,it is not toxic to H1650,A549 and H226.When the concentration of BPO-27 is less than 25 n M,it is not toxic to H1650,Calu-1 and H226;when it is less than 20 n M,it is not toxic to A549.When the concentration of Genistein is less than 100 μM,it is not toxic to H1650 and Calu-1;when it is less than 50 μM,it is not toxic to A549 and H226.When the concentration of IBMX is less than 100 μM,it is not toxic to Calu-1;when it is less than 50 μM,it is not toxic to H1650,A549 and H226.When the concentration of Forskolin is less than 100 μM,it is not toxic to H1650,A549,Calu-1 and H226.4.Cell proliferation experiment results show that CFTR inhibitors CFTR_inh-172,GlyH-101 and BPO-27 can promote the proliferation of H1650,A549,Calu-1 and H226 cells.In H1650,the optimal concentration of CFTR_inh-172 is 12.5 μM,and the optimal concentration of BPO-27 is 12.5 n M.In A549,the optimal concentration of CFTR_inh-172 is 3.125 μM,the optimal concentration of GlyH-101 is 1.5625 μM,and the optimal concentration of BPO-27 is 6.25 n M.In Calu-1,the optimal concentration of GlyH-101 is 3.125 μM,and the optimal concentration of BPO-27 is 3.125 n M.In H226,the optimal concentration of CFTR_inh-172 is 12.5 μM,the optimal concentration of GlyH-101 is 1.5625 μM,and the optimal concentration of BPO-27 is 12.5 n M.5.Cell proliferation experiment results show that CFTR activators Genistein,IBMX and Forskolin can inhibit the proliferation of H1650,A549,Calu-1,H226 cells.In H1650,the optimal concentration of IBMX is 25 μM,and the optimal concentration of Forskolin is 25 μM.In A549,the optimal concentration of Genistein is 50 μM,the optimal concentration of IBMX is 50 μM,and the optimal concentration of Forskolin is 25 μM.In Calu-1,the optimal concentration of Genistein is 50 μM,the optimal concentration of IBMX is 50 μM,and the optimal concentration of Forskolin is 6.25 μM.In H226,the optimal concentration of Genistein is 50 μM,and the optimal concentration of Forskolin is 6.25 μM.6.The results of apoptosis experiments show that CFTR inhibitors can inhibit the apoptosis of A549 cells,CFTR_inh-172 has the most obvious effect,and only Genistein among CFTR activators promotes the apoptosis of A549.7.The results of cell scratch experiments show that CFTR inhibitors CFTR_inh-172,GlyH-101 and BPO-27 can promote the migration of H1650,A549,Calu-1 and H226 cells.In H1650,the optimal concentration of CFTR_inh-172 is 10 μM,the optimal concentration of GlyH-101 is 5 μM,and the optimal concentration of BPO-27 is 16 n M.In A549 cells,the optimal concentration of BPO-27 is 16 n M.In Calu-1,the optimal concentration of CFTR_inh-172 is 10 μM,and the optimal concentration of GlyH-101 is 5 μM.In H226,the optimal concentration of CFTR_inh-172 is 10 μM,the optimal concentration of GlyH-101 is 5 μM,and the optimal concentration of BPO-27 is 16 n M.8.The results of cell scratch experiments show that CFTR activators Genistein,IBMX and Forskolin can inhibit the migration of lung cancer cells H1650,A549,Calu-1 and H226.In H1650,A549,Calu-1 and H226,the optimal concentration of Genistein and IBMX are both 50 μM,and the optimal concentration of Forskolin is 20 μM.9.The results of cell invasion experiments show that both CFTR_inh-72 and GlyH-101 can promote the invasion of A549 and H226.Forskolin,Genistein and IBMX can inhibit the invasion of A549 and H226.10.Western Blot experiments showed that CFTR_inh-172 significantly promoted the expression of cyclin D1,Cyclin E1 and vimentin;Forskolin significantly inhibited the expression of cyclin D1,Cyclin E1 and vimentin.11.Western Blot experiment results showed that Forskolin significantly inhibited the EMT process of A549 cells,and the addition of BPO-27 reduced the inhibition of Forskolin.CFTR_inh-172 promoted the EMT process of A549 cells,and the addition of Genistein reduced the promotion effect of CFTR_inh-172.12.Western Blot experiments showed that CFTR inhibitors CFTR_inh-172 and BPO-27 promoted the expression of p65,a major NF-κB family member protein,and CFTR activators Genistein and Forskolin suppressed p65 expression.In summary,this study found that the expression of CFTR in different types of lung cancer cells may be related to the grade of bronchi to alveoli,the classification of lung cancer cells,and the source of the cells.CFTR chloride channel can affect the proliferation of lung cancer cells by regulating the expression of cyclin,and affect the migration of lung cancer cells by regulating the protein expression of EMT process and NF-κB signaling pathway.Therefore,the study of the mechanism of lung cancer progression in this paper has very important theoretical significance and can provide a theoretical basis for the treatment of lung cancer with specific targets.