Role Of The TMEM16A Calcium-activated Chloride Channel In The Proliferation And Migration Of Colorectal Cancer

TMEM16A(also known as ANO1)is a calcium-activated chloride channel that belongs to the TMEM16 membrane protein family and has 10 transmembrane domains.TMEM16 A is widely expressed in a variety of epithelial cells,smooth muscle cells,cardiomyocytes,neurons,etc,and has a variety of important functions,such as regulating secretion of epithelial cells to secrete fluid,smooth muscle contraction,nerve and myocardial excitation,etc.Its dysfunction is associated with many diseases such as cancer,gastrointestinal diseases,hypertension,and cystic fibrosis.In recent years,studies have found that in a variety of cancers,such as breast cancer,squamous cell carcinoma of the head and neck,prostate cancer,etc.,TMEM16 A expression is up-regulated and is involved in regulating the proliferation and migration of cancer cells.Colorectal cancer is a malignant tumor that seriously endangers human health.The study that TMEM16 A is expressed in colorectal cancer cells and its expression may be related to the occurrence and progression of colorectal cancer.Therefore,TMEM16 A can be used as a target for studying the progress of colorectal cancer.In this study,the expression of TMEM16 A in human normal intestinal epithelial cells NCM460,human colorectal adenocarcinoma cells SW480,SW620 is detected by the methods of qPCR,Western blot,and immunocytochemistry.Using TMEM16 A inhibitors T16Ainh-A01,Ca CCinh-A01,Luteolin and TMEM16 A activators Eact,Ionomycin,the effect of TMEM16 A on colorectal cancer cells proliferation and migration has been investigated by CCK-8 method to detect cell proliferation,flowing cytometry to detect cell cycle Distribution,Hoechst 33258 staining to observe nuclear shrinkage and chromatin fragmentation,cell scratches and Transwell detection of cell migration.The experimental results are drawn as follows:1.The experiment results of qPCR,Western blot and immunocytochemistry: TMEM16 A is expressed in colorectal cancer cell lines SW620,SW480 and normal intestinal epithelial cell line NCM460.The expression levels of the m RNA and protein of TMEM16 A gene are SW620,SW480,NCM460 from higher to lower,respectively.Compared with NCM460 cells,the expression of TMEM16 A is significantly up-regulated in SW620 and SW480 cells.Compared with SW480 cells,SW620 cells expressed higher TMEM16 A.2.The method of CCK-8 is used to detect the proliferation of SW620,SW480 and NCM460 cells.By statistically analyzing the cell proliferation rate,the order of the proliferation from large to small is that: SW620,SW480,and NCM460.Similarly,the cellmigration rates are in the same order.It is suggested that the cell proliferation rate and migration rate are positively correlated with the expression of TMEM16 A in the three cell lines.3.The T16Ainh-A01,Ca CCinh-A01 and Luteolin inhibitors can inhibit the proliferation and migration of NCM460,SW620 and SW480 cells.The optimal concentrations of three inhibitors are 25,12.5 and 50 ?M,respectively.4.TMEM16 A activator Eact promotes the proliferation of NCM460,SW620 and SW480 cells.The optimal concentrations of NCM460,SW620 and SW480 cells are 6.25,25,and 25?M respectively.The activator Ionomycin can significantly promote the proliferation of NCM460 cells,and its optimal concentration is 0.63 ?M.However,it has no effect on the proliferation of SW620 and SW480 cells.Eact and Ionomycin can promote the migration of NCM460,SW480 and SW620 cells,and the optimal concentrations are 0.5,25 ?M,respectively.5.Cell cycle test results: Inhibition of TMEM16 A channel activity(T16Ainh-A01,Ca CCinh-A01,Luteolin)reduces the proportion of S-phase cells in colorectal cancer cells and inhibits the replication of DNA molecules in cells.The optimal concentration is 25,12.5 and50 ?M.6.Eact can increase the proportion of SW620 S-phase cells,indicating that it accelerates the replication of DNA molecules in SW620 cells,accelerates the cell cycle,and promotes the proliferation of SW620 cells.Its optimal concentration is 25 ?M;while for SW480 cells,S-phase cells the number ratio increased slightly,but there are no significant effect,and Eact has no effect on the cycle of SW480 cells.Ionomycin can slightly increase the proportion of SW620 and SW480 S-phase cells without a significant effect.It can be seen that Ionomycin has no effect on the cycle of SW620 and SW480 cells.7.Hoechst 33258 staining results show that T16Ainh-A01,Ca CCinh-A01 and Luteolin inhibited channel TMEM16 A activity,which cause nuclear shrinkage and chromosome fragmentation of NCM460,SW620,and SW480 cells,producing apoptotic bodies.The optimal concentrations of T16Ainh-A01,Ca CCinh-A01 and Luteolin are 25 ?M,12.5 ?M and 50 ?M,respectively.8.Transwell detectes the migration of SW620 cells,and the results show that inhibition of TMEM16 A activity could inhibit the migration of SW620 cells.9.Western blot is used to detect the expression of EMT(Epithelial-Mesenchymal Transition)marker protein—Vimentin.The expression levels of Vimentin are SW620,SW480,NCM460 from higher to lower,respectively.Conclusion: The expression of TMEM16 A is up-regulated in colorectal cancer cells,and it is relatively high in colorectal cancer cells with high metastatic potential.Inhibiting theactivity of TMEM16 A channel can inhibit the proliferation of cells by blocking the replication of S-phase DNA molecules,and can induce nuclear shrinkage and chromosome fragmentation,and produce apoptotic bodies;activating the activity of TMEM16 A channel can promote the migration of colorectal cancer cells,but has little effect on cell proliferation.In addition,EMT progression may be a key factor in the conversion of primary colorectal cancer to metastatic colorectal cancer.Therefore,TMEM16 A may be the target of affecting the proliferation and migration of colorectal cancer cells,which provides a new strategy for the treatment of colorectal cancer patients.