Study On The Relationship Between TMEM16A And Proliferation, Migration And Apoptosis Of TE-1 And A549 Cells

The Ca²⁺ activated Cl^- channels (CaCCs) are one of anionic channels which can be activated by intracellular Ca2+. CaCCs are robustly expressed in many mammalian cell types, which are fundamental mediators in numerous physiological processes including controlling phototransduction, olfaction, gustation and somaesthetic sensations. CaCCs also contribute to cardiac excitation, smooth muscle contraction, glands and transepithelial secretion and fertilization. In addition, CaCCs may participate in cell division cycle and cell proliferation. Because of their pathophysiological significance, many efforts have been made to clone CaCCs. The molecular identity of CaCC remained controversial till 2008 when three groups independently identified a member of the transmembrane protein 16A (TMEM16A) protein family as a CaCC subunit.Cell proliferation and apoptosis both occur under normal physiological conditions and play an important role in tissues development, embryogenesis, and tissue homeostasis. As with all critical processes, an imbalance between cell proliferation and apoptosis can cause various disease states such as arteriosclerosis, cancer, AIDS etc. Cell proliferation is stimulated by a wide variety of mitogenetic factors. Apoptosis may be triggered by a variety of stimuli including bacterial toxins, radiation, gross increase of extracellular osmolarity, oxidative stress, etc. It is suggested recently that chloride channels may exert tremendous impact on the process of cell proliferation and apoptosis. In spite of the mechanism by which they mediate cell activity remains inconclusive, various hypotheses have been proposed, including regulation of cell volume and regulation of membrane potential and so on.Metastasis is one of the biological characteristics of malignant tumor, and also a difficulty in clinical treatment. Migration is a prerequisite for invasion and metastasis. It was reported that chloride channels also play important roles in migration by participating in the changing of the cell morphology.Esophageal squamous cell carcinoma (ESCC) is a genetically complex tumor type, and a major cause of cancer related mortality, which overall 5-year survival rate remains only 40 %. Lung cancer is the leading category of cancer threatening the health of the people in many countries. China ranks the first in the world with most population of lung cancer. Lung cancer mortality stands the first within all kinds of cancer death. Moreover, among all types of lung cancer, adenocarcinoma occurs most commonly and its incident rate is the highest. The objective of this study is to use human esophageal squamous cell cancer epithelial TE-1 cells and human pulmonary adenocarcinoma epithelial A549 cells, and use methods of molecular biology, electrophysiology and cell biology to: (1) identify the expression of TMEM16A and the calcium activated chloride current in TE-1 and A549 cells. (2) observe the effect of chloride channel blockers on the proliferation, migration and apoptosis of TE-1 and A549 cell lines. (3) study effect of overexpression of TMEM16A on the proliferation, migration and apoptosis of TE-1 and A549 cell lines. (4) study effect of siRNA for TMEM16A on the proliferation, migration and apoptosis of TE-1 and A549.Part 1 The expression of TMEM16A and recording of Ca2+ activated Cl- currents in TE-1 and A549 cellsObjective: To observe the expression of TMEM16A and record the calcium activated chloride current by patch clamp in TE-1 and A549 cell lines.Methods: RT-PCR was used to detect TMEM16A mRNA expression in TE-1 and A549 cells. The expression of TMEM16A protein in TE-1 and A549 cells was identified by western-blot. TMEM16A protein expression in human esophageal squamous cell carcinoma (ESCC) and pulmonary adenocarcinoma was examined by immuno-histochemistry. The current was recorded by patch clamp.Results: (1) TMEM16A mRNA was detected in A549 and TE-1 cell lines. The mRNA quantity of TMEM16A in TE-1 was significantly higher than that in A549 cells when same amount of total RNA was used(P<0.01). (2) Western blot results showed expression of TMEM16A protein in A549 and TE-1cells. The protein quantity of TMEM16A in TE-1 was significantly higher than that in A549 cells when same amount of membrane proteins was used (P<0.01). (3) TMEM16A protein was positively identified using immuno-histochemistry in the tissues of ESCC and pulmonary adenocarcinoma, and the expression rate was 100%. We also found the expression of TMEM16A protein in renal clear cell carcinoma and breast cancer. (4) Exchange of extracellular NMDG-Cl and Na-gluconate solutions manifested Cl- sensitive currents in A549 cells. Ionomycin, a Ca2+ ionophore, evoked large currents, and the currents were blocked by a Cl- channel blocker, 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS). Angâ…¡, when applied to activate AT1 receptor, transiently expressed in A549 cells, evoked large currents, which was also blocked by DIDS and enhanced by ionomycin. These results suggested existence of calcium activated chloride channel, possibly with molecular identity of TMEM16A, in A549 cells.Conclusions: (1) Human esophageal squamous cell carcinoma (ESCC) and lung adenocarcinoma expressed TMEM16A channel. (2) A549 cells expressed calcium activated chloride channel, possibly with molecular identity of TMEM16A.Part 2 The Effects of Chloride Channel Blockers on the Proliferation, Migration and Apoptosis of TE-1 and A549 Cell LinesObjective: To study the effects of Cl- channel blockers NFA, DIDS, and NPPB on the proliferation, migration and apoptosis of TE-1 and A549 cell lines.Methods: Proliferation of TE-1 and A549 cell lines was evaluated by MTT assay, BrdU method and Colony-forming unit assay. Cell-cycle distribution of TE-1 and A549 was determined by Fluorescence activated cell sorting (FACS). Transwell chambers were used to study migration of TE-1 and A549. The apoptosis of TE-1 and A549 cells was observed by TUNEL. Results: (1) The morphology of TE-1 and A549 cells was observed by invert microscope after treated with 400μM NFA or 400μM DIDS. After the treatment, the cell volume was decreased, the intercellular gaps were enlarged and the cell proliferation was inhibited. (2) MTT assay showed NFA, DIDS and NPPB time-dependently and concentration-dependently suppressed the proliferation of TE-1 and A549 cells. The proliferation was also detected by BrdU assay. NFA and DIDS inhibited the proliferation of TE-1 and A549 cells. IC50 of NFA for TE-1 and A549 cells were 512μM and 400μM, respectively and IC50 of DIDS for TE-1 and A549 cells were 748μM and 32μM, respectively. (3) Both the morphology of the colony and the colony formation rate are different between TE-1 and A549. The colony of TE-1 is large and with more cells whereas A549 colony is small and with few cells. NFA 400, 800, 1000μM completely abolished the colony formation of TE-1; lower concentrations of NFA had no effect. The colony formation rate of A549 cell was high (up to 117 %), which was also inhibited significantly by NFA. We also made a comparison between TE-1 and A549 on the colony formation rate, and the results showed that A549 had a higher colony formation rate which was inhibited to greater extent by NFA than TE-1. (4) NFA treatment of TE-1 and A549 cells for 48h significantly increased the population of cells in G0/G1 phase (first peak) and decreased those in S (plateau) phase and G2/M phase. Compared with untreated control group, PI of TE-1 cells decreased from 57.4±6.72 % to 23.15±2.05 % with 800μM NFA (P <0.01), and SPF also decreased from 48.6±2.59 % to 23.47±1.5 % with 800μM NFA (P <0.01). NFA had the same effects on A549 cell-cycle progression with PI decreasing from 59.6±8.14 % to 13.24±1.79 % with 800μM NFA (P <0.01), and SPF decreasing from 54.8±1.83 % to 13.6±1.37 % with 800μM NFA (P <0.01). From FCM we found that NFA inhibited the proliferation of TE-1 and A549 cells by stagnating the cells at G0/G1 phase. (5) Transwell-migration cell assay results showed that NFA significantly inhibited the migration of TE-1 and A549 cells. The number of the migrating cells decreased obviously and the shape of TE-1 cells became small and slim. The A549 cells morphology also became small and thin and the cell number were decreased significantly afer treated with 800μM NFA. (6) DeadEnd colorimetric TUNEL system was used to detect the effects of NFA on the apoptosis of TE-1 and A549 cells. The quantitative results showed that there were no apoptosis appeared either in control or in NFA treated groups.Conclusions: (1) 400μM NFA and 400μM DIDS decreased the cell volume of TE-1 and A549 cells, enlarged the intercellular gaps and inhibited the proliferation of TE-1 and A549 cells. (2) NFA, DIDS and NPPB time-dependently and concentratione-dependently suppressed the proliferation of TE-1 and A549 cells. IC50 of NFA for TE-1 and A549 cells were 512μM and 400μM, respectively and IC50 of DIDS for TE-1 and A549 cells were 748μM and 32μM, respectively. (3) NFA inhibited the colony formation of TE-1 and A549 cells. (4) NFA increased the population of cells in G0/G1 phase (first peak) and decreased those in S (plateau) phase and G2/M phase of TE-1 and A549 cells. NFA inhibited the proliferation of TE-1 and A549 cells by stagnating the cells at G0/G1 phase. (5) NFA significantly inhibited the migration of TE-1 and A549 cells. The morphology of TE-1 and A549 cells were also became small and thin. (6) NFA had no effect on the apoptosis of TE-1 and A549 cell lines. (7) Ca2+ activated Cl- currents, possibly through TMEM16A, may play important role in cancer cell growth and migration.Part 3 The Effects of Overexpression of TMEM16A on the Proliferation, Migration and Apoptosis of TE-1 and A549 Cell LinesObjective: To study the effects of overexpression of TMEM16A on the proliferation, migration and apoptosis of TE-1 and A549 cells.Methods: With Lipofectamine? 2000, TMEM16A gene was transfected into TE-1 and A549 cells, then cell clones were picked up in the presence of G418. TMEM16A proteins were identified by confocal fluorescent microscope, RT-PCR, Western-blot and Immuno-histochemistry. Proliferation of TE-1-TMEM16A and A549-TMEM16A cells was evaluated by MTT assay. Cell-cycle distribution of TE-1-TMEM16A and A549-TMEM16A was determined by Fluorescence activated cell sorting (FACS). Transwell chambers were used to study migration of TE-1-TMEM16A and A549-TMEM16A cells. The apoptosis of TE-1-TMEM16A and A549-TMEM16A cells was observed by TUNEL.Results: TMEM 16A overexpression cell lines of TE-1 and A549 were first established. (1) The best screening concentration of G418 for TMEM16A expression stable cell line was determined to be 600μg/ml. (2) The stable TMEM16A-expression TE-1 and A549 cell lines were first observed by fluorescence microscopy showing signal of GFP co-transfected. (3) The results of RT-PCR showed that TMEM16A mRNA expressed in both control and stable TMEM16A-expression TE-1 and A549 cells, and the expression was significantly increased in TMEM 16A stable cell lines. (4) Western blot results showed that TMEM16A protein expressed in control and stable TMEM16A-expression TE-1 and A549 cells, and the expression was significantly increased in TMEM 16A stable cell lines. (5) Immunohistochemical analysis was used to detect TMEM16A protein expressions in TE-1 and A549 cells and the results showed that TMEM16A proteins were overexpressed in the membrane and cytoplasm of TE-1-TMEM16A and A549-TMEM16A cells. (6) MTT assay showed that the proliferation of TE-1 and A549 cells was time-dependently increased by overexpression of TMEM16A. (7) The population of TE-1-TMEM16A and A549-TMEM16A cells in G0/G1 phase (first peak) significantly decreased and those in S (plateau) phase and G2/M phase increased. Compared with control group, PI of TE-1-TMEM16A cells increased from 16±1.39 % to 32.4±1.85 % in 24 h and 53.7±2.69 % in 48 h(P <0.01), and SPF also increased from 13.5±1.56 % to 17.4±1.88 % (P <0.05) and 43.9±2.49 % (P <0.01). Overexpression of TMEM16A had the same effect on A549 cell-cycle progression with PI increasing from 12.7±2.31 % to 28.7±3.42 % and 45.1±3.76 % (P <0.01), and SPF increasing from control group of 10±0.96 % to 21.7±2.53 % and 34±3.41 % (P <0.01). (8) The transwell-migration cell assay showed that overexpression of TMEM16A significantly stimulated the migration of TE-1 (data not shown) and A549 cells. The A549 migrating cells increased obviously and the cell numbers of control group were 176±13, 408±15, and increased to 453±38, 606±9 after transfected with TMEM16A for 24 and 48h, respectively. (9) DeadEnd colorimetric TUNEL system was used to study the effects of NFA on the apoptosis of TE-1-TMEM16A and A549-TMEM16A. The quantitative results showed that there were no apoptosis appeared either in control or in TMEM16A transfected groups.Conclusions: (1) Stable TMEM16A-expression TE-1 and A549 cell lines were created by liposome method successfully. (2) Overexpression of TMEM16A could stimulate the growth of human ESCC cell line TE-1 and pulmonary adenocarcinoma epithelial A549 cell. (3) Overexpression of TMEM16A could stimulate the proliferation of TE-1 and A549 cells by stagnating the cells at G2/M phase and S phase. (4) The migration activity of TE-1-TMEM16A and A549-TMEM16A cells could be stimulated by overexpression of TMEM16A. (5) Overexpression of TMEM16A had no effects on the apoptosis of TE-1 and A549 cell lines.Part 4 The Effects of siRNA for TMEM16A on the Proliferation, Migration and Apoptosis of TE-1 and A549Objective: To investigate the effects of TMEM16A lose of function on proliferation, migration and apoptosis of TE-1 and A549 cell lines.Methods: RT-PCR and western blot methods were used to detect the efficiency of TMEM16A knocked down in mRNA level and in protein level, respectively. Cell proliferation was evaluated by MTT assay. Cell-cycle distribution was determined by Fluorescence activated cell sorting (FACS). Transwell chambers were used to detect cell migration. Cell apoptosis was observed by TUNEL.Results: (1) RT-PCR was applied to detect the transfection efficiency of siRNA. The TMEM16A mRNA level in TE-1-siRNA and A549-siRNA cells was significantly decreased compared with control(P<0.01). (2) Western blot results showed TMEM16A protein in TE-1 and A549 cells in the two siRNA groups were significantly decreased compared with ssRNA groups(P<0.01). (3) MTT assay showed that the proliferation of TE-1 and A549 cells was significantly inhibited by siRNA for TMEM16A. siRNA for TMEM16A time-dependently suppressed the proliferation of both cell lines compared with ssRNA. (4) After siRNA for TMEM16A transfection for 48h, the population of TE-1 and A549 cells in G0/G1 phase (first peak) significantly increased and those in S (plateau) phase and G2/M phase decreased. Compared with ssRNA group, PI of TE-1 cells decreased from 54.1±3.43 % to 40.1±5.31 % (P <0.05), and SPF also decreased from 44.2±3.24 % to 31.7±2.76 % (P <0.01). siRNA for TMEM16A had the same effects on A549 cell-cycle progression with PI decreasing from 47.6±4.73 % to 36.4±4.12 % (P <0.05), and SPF decreasing from 34.8±2.63 % to 27.9±1.97 % (P <0.05). (5) To examine whether siRNA for TMEM16A influence the migration activity of TE-1 and A549 cells, transwell-migration cell assay was used. The results showed that siRNA for TMEM16A significantly inhibited the migration of TE-1 and A549 cells. The A549 cells number of ssRNA group were 250±15, 404±9, decreased to 130±10, 55±9 afer treated with siRNA for 24 and 48h respectively. (6) The apoptosis of TE-1-siRNA and A549-siRNA cells was investigated by DeadEnd colorimetric TUNEL system. The quantitative results showed that there were no apoptosis appeared either in control or in siRNA treated groups.Conclusions: (1) mRNA and protein expressions of TMEM16A were both decreased in TE-1 and A549 cells after transfection with siRNA for TMEM16A. (2) siRNA for TMEM16A inhibited the proliferation of TE-1-siRNA and A549-siRNA cells. (3) siRNA for TMEM16A inhibited the proliferation of TE-1 and A549 cells by stagnating the cells at G0/G1 phase. (4) The migration activity of TE-1 and A549 cells was decreased by siRNA for TMEM16A. (5) siRNA for TMEM16A had no effect on the apoptosis of TE-1 and A549 cell lines.CONCLUSIONS1 Human esophageal squamous cell carcinoma (ESCC) and lung adenocarcinoma expressed TMEM16A channel and these cancer cells expressed calcium activated chloride channel, possibly with molecular identity of TMEM16A.2 Chloride channel blockers inhibited the proliferation and migration of TE-1 and A549 cells, but had no effect on the apoptosis.3 Overexpression of TMEM16A stimulated the proliferation and migration, but had no effect on the apoptosis TE-1 and A549 cells.4 siRNA for TMEM16A inhibited the proliferation and migration, but had no effect on the apoptosis of TE-1 and A549 cells.