Study On The Functional Property Of TMEM16A And Relationship Between TMEM16A And Proliferation Of Cancer Cells

Calcium activated chlorine channels (CaCCs) are anionic channels, withboth calcium and voltage dependence. CaCCs play a variety of physiologicalroles in many organs and tissues, such as sensory conduction, regulation ofneuronal and cardiac excitability, regulation of vascular tone andtransepithelial Cl-secretion. Moreover, CaCCs may even participate in celldivision cycle and cell proliferation. The molecular identity of CaCCsremained controversial until2008when TMEM16A, a member of thetransmembrane protein16family, was identified as an important subunit ofCaCCs. This discovery allows a better understanding of their physiologicalroles, structure-function relationship, and regulatory mechanisms.After identification of TMEM16A to be an important subunit ofmolecular basis of CaCCs, efforts are being made to find functional regulatorof TMEM16A. However, until now, few specific regulator of TMEM16A hasbeen reported. Thus more efforts are needed to find functional specificregulators of TMEM16A. The discovery of new functional modulators ofTMEM16A would provide supports for the development of drug treatment ofhypertension, pain, diarrhea and other diseases.Cancer has been a major problem plaguing human life and health. Therole of ion channels play in cancer has been the interest of studies. TMEM16Ais highly expressed in many tumor tissues, and has become the marker genefor gastrointestinal stromal tumors (GISTs). The over-expression ofTMEM16A in these tumor tissues may imply that this molecule is importantfor cancer development. However, no solid evidence has been provided for therole and the mechanism of TMEM16A involvement in the process of tumordevelopment. Part oneEstablishment and electrophysiological properties of a stableTMEM16A-expression HEK293A cell line.Objective: To establish the stable TMEM16A-expression HEK293A cellline and to study its electrophysiological properties.Methods:(1)TMEM16A gene was transfected into HEK293A cells withLipofectamine2000, and cell clones were screened in the presence of G418.(2) mRNA and proteins of TMEM16A were identified by real-time PCR,Western blot techniques.(3) The calcium-activated chloride currents fromTMEM16A-stably-transfected HEK293A cells were studied using patch clamptechnique.(4) The effects of chloride channel regulators on the currents ofTMEM16A from the stably cell lines were studied using patch clamptechnique.Results:(1) mRNA and protein levels of TMEM16A were significantly higher inTMEM16A-stably-transfected HEK293A cell lines compared with that in theuntransfected cells.(2) The currents recorded from TMEM16A-stably-transfected HEK293Acell lines showed properties of intracellular Ca~2+([Ca~2+]i)dependence andoutwardly rectification. The concentration-dependence of TMEM16A currentson [Ca~2+]iwere manifested by the larger outwardly rectification currents in thepresence of600nM [Ca~2+]ithan that in the presence of lower concentrationsof225nM and17nM [Ca~2+]i.(3) When TMEM16A currents were recorded using perforated-patchclamp technique, ATP(100μM) and ionomycin(2μM), a Ca~2+ionophore,evoked large currents, and the currents were blocked by a suggested Cl-channel blocker, tannic acid. The currents amplitudes were affected when Cl-in the external bath solution was replaced by F-, Br-, or I-.(4) The proposed activator of TMEM16A, Eact, strongly increased theTMEM16A currents in the absence of [Ca~2+]i, and the EC50was1.18μM. The CaCCs inhibitors tannic acid and CaCCinh-A01significantly reduced theTMEM16A currents, with IC50of13.87μM and7.11μM, respectively.Conclusion: TMEM16A-expressioned HEK293A cell line wassuccessfully established and the currents from TMEM16A showed propertiessimilar to that of the endogenous CaCC. This cell line therefore provides aplatform for the further studies of TMEM16A modulation and screening ofTMEM16A modulators.Part twoExpression of TMEM16A in A549cells and relationship betweenTMEM16A and proliferation of cancer cells.Objective: To identify the expression of TMEM16A and theCa~2+-activated chloride current (CaCC) in A549cells, and to study therelationship between proliferation of cancer cells and function of TMEM16A.Methods:(1) The functional expression of TMEM16A andcharacterization of the CaCC currents in A549cells were studied usingmethods of real-time PCR, western blot and patch clamp techniques.(2)CaCC/TMEM16A currents in A549cells were recorded using gramicidinperforated-patch technique.(3) CaCC/TMEM16A currents were studied underconditions of different external anions.(4) Proliferation of A549cell lines wasevaluated by MTT assay.Results:(1) The results showed the expression of mRNA and protein ofTMEM16A in A549cells. CaCC-like currents were recorded from A549cellsusing whole-cell patch technique.(2) When CaCC/TMEM16A current from A549cells were recorded usingperforated-patch clamp technique, ATP(100μM) and ionomycin(2μM), a Ca~2+ionophore, evoked large currents, and the currents were blocked by asuggested Cl-channel blocker, tannic acid. The currents amplitudes wereaffected when Cl-in the external bath solution was replaced by F-, Br-, or I-. (3) MTT assay showed that the proliferation of A549cells wassignificantly inhibited by siRNA against TMEM16A. siRNA for TMEM16Atime-dependently suppressed the proliferation of A549cell line compared withthe control siRNA.Conclusion: TMEM16A is possibly the molecular basis of the CaCC inA549cells. CaCC/TMEM16A plays an important role in proliferation ofhuman pulmonary adenocarcinoma epithelial A549cells.