Effects Of Silence Of ClC-3 Gene On The Cell Cycle In Nasopharyngeal Carcinoma Cells

Objective:To investigate the role of ClC-3 chloride channels in the cell cycle of nasopharyngeal carcinoma CNE-2Z cells by silencing the expression of ClC-3 gene using the small interference RNA (siRNA) technology.Methods:ClC-3 gene was silenced by the siRNA technique in nasopharyngeal carcinoma CNE-2Z cells. Transfection efficiency of ClC-3 siRNA was detected by the flow cytometry. The patch-clamp technique was employed to record Cl- currents. The changes of cell volume were determined by image analysis using the Q500MC software. The expressions of ClC-3 and cyclin D1 proteins were detected by immunofluorescence and Western blot techniques. Cell cycle distribution was detected by the flow cytometry.Results:(1) Transfection efficiency of ClC-3 siRNA was (63.8±3.8)%(n= 3). Compared with the control group, ClC-3 expression was reduced by (60.9±4.0)%(n= 3, P<0.05) in the cells treated with ClC-3 siRNA. (2) A chloride current was activated by extracellular applications of 47% hypotonic solution, with current density of (74.2±6.5) pA/pF (n= 5) in control cells and (13.7±4.1) pA/pF (n= 5) in the cells treated with ClC-3 siRNA at+80 mV. The hypotonicity-activated current in the siRNA group was reduced by (81.5±4.7)%(P< 0.05). (3) The capacity of regulatory volume decrease (RVD) was reduced by siRNA treatments. The RVD induced by 47% hypotonic solution was (42.6±2.8)%(n= 20) in the control cells and (10.5±4.8)%(n= 16) in the cells treated with ClC-3 siRNA. RVD capacity in the siRNA group was decreased by 75.4%(P< 0.05). (4) Compared with the control group, the percentages of cells in G0/G1 phases was increased from (56.8±2.8)%to (69.9±3.0)% and those in S phase was decreased from 35.4% to 24.0% in ClC-3 siRNA group (n= 3, P< 0.05), indicating the arrest of cells at G0/G1 phases by ClC-3 siRNA treatments. (5) cyclin D1 expression was reduced by (40.3±2.0)%(n= 3, P< 0.05) in the cells treated with ClC-3 siRNA, while that in the transfaction agent or negative siRNA control group was not changed significantly.Conclusion:Nasopharyngeal carcinoma CNE-2Z cells express ClC-3 chloride channel proteins. ClC-3 chloride channel proteins are involved in regulation of the cell cycle and play an important role in regulating entry of cells into S phase from G0/G1 phases. ClC-3 chloride channels may control cell cycle progress by regulating RVD and cyclin D1 expression.