TRPM7离子通道在口腔鳞癌细胞增殖和迁移中的作用

摘要

目的:探讨TRPM7离子通道在口腔鳞癌细胞增殖和迁移中的作用.方法:Western blotting,RT-PCR和间接免疫荧光法检测口腔鳞癌细胞OC2中TRPM7的表达和细胞内定位.MTT法和Transwell试验分别检测抑制TRPM7通道(2-APB、siRNATRPM7)对OC2细胞增殖和迁移的影响.Western blotting检测2-APB或siRNA TRPM7阻断PI3K/AKT通路对OC2细胞中TR-PM7的表达影响.采用膜片钳技术初步探索干预TRPM7通道对OC2细胞阳离子通透的影响.结果:OC2细胞中TRPM7过表达,主要表达在细胞质中.阻断TRPM7通道后,OC2细胞增殖和迁移能力受抑制,且呈浓度依赖性和时间依赖性.阻断TR-PM7通道后,抑制OC2细胞TRPM7可下调磷酸化AKT以及磷酸化ERK的表达,而对组成性AKT以及ERK的表达无明显影响.膜片钳技术显示在TRPM7过表达的OC2细胞中TRPM7样电流激活,2-APB阻断TRPM7通道后电流明显减弱,而采用TRPM7通道活化剂Bradykinin后,电流明显增强.结论:TRPM7在口腔鳞癌细胞OC2中过表达,可能通过影响PI3 K/AKT和MAPK-ERK信号转导途径以及阳离子内流发挥对细胞增殖、迁移的调节作用.%Objective:To investigate the role of the transient receptor potential melastatin subfamily member 7 (TRPM7) ion channel in the proliferation and migration of oral squamous cancer cells OC2.Methods:The expression of TRPM7 in OC2 cells was examined by western blotting,RT-PCR and indirect immunofluorescence assay.The influence of TRPM7 on cell proliferation and migration under the inhibition of TRPM7 ion channel with 2-APB or siRNA TRPM7 in OC2 cells was examined by MTT and Transwell respectively.The influence of PI3K/AKT signal pathway on the expression of TRPM7 in OC2 cells was tested by Western blotting.The change of positive ion current when the OC2 cells were treated with TRPM7 inhibitor and activator was explored with patch clamp technique.Results:TRPM7 overexpression in OC2 cells mainly in the cytoplasm was observed,siRNA TRPM7 downregulated the expression of TRPM7.Inhibition of TRPM7 (2-APB or siRNA TRPM7) inhibited the growth and proliferation of OC2 cells in a time-and dose-dependent manner.Blockage of TRPM7 ion channel inhibited the migration capacity of OC2 cells.Inhibition of TRPM7 downregulated the expression of phosphorylated AKT and phosphorylated ERK.Patch clamp assay showed the activation of TRPM7-like current in OC2 cells.2-APB could weaken this current while Bradykinin could enhance the current.Conclusion:TRPM7 may regulate the proliferation and migration of oral cancer cells by the regulation of PI3K/AKT and MAPK/ERK signal pathways and cell positive current.