ClC-3 siRNA抑制鼻咽癌细胞调节性容积回缩

摘要

目的 用siRNA沉默ClC-3氯通道,探讨C1C-3氯通道在鼻咽癌CNE-2Z细胞调节性容积回缩(regulatory volume decrease,RVD)中的作用.方法 使用终浓度100 nmol/L的ClC-3 siRNA转染鼻咽癌CNE-2Z细胞,流式细胞术检测siRNA的转染率,Western blotting检测C1C-3蛋白表达,活细胞图像系统分析对照组和ClC-3 siRNA组细胞在低渗(160mOsm/L)刺激时的容积凋节能力.结果 ClC-3 siRNA转染率为(63.8±3.8)%(n=3,P＜0.01),与对照组相比,C1C-3氯通道蛋白的表达减少(60.9±4.0)%(n=3,P＜0.01).对照组细胞的RVD为(42.6±2.8)%(n=20),ClC-3 siRNA组细胞在低渗液中的最大容积与对照组没有显著性差异,但RVD仅为(10.5±4.8)%,RVD抑制率达75.4%(P＜0.01).结论 ClC-3 siRNA成功转染鼻咽癌CNE-2Z细胞,特异性抑制ClC-3蛋白表达,显著降低细胞RVD能力,提示ClC-3氯通道在鼻咽癌细胞调节性容积回缩中起重要作用.%To investigate the role of C1C-3 chloride channels in regulatory volume decrease (RVD) of nasopharyngeal carcinoma (NPC) CNE-2Z cells. Methods C1C-3 siRNA was transfected into CNE-2Z cells in the presence of the transfection reagent HiPerFect Reagent1". The transfection efficiency of C1C-3 siRNA was detected by flow cytometry. The expression of C1C-3 protein was detected by Western blotting, and the changes of cell volume in 160 mOsmol/L hypotonic solution were determined by image analysis. Results The transfection efficiency of C1C-3 siRNA was (63.8 ±3.8)% (n=3, P<0.01), and compared with the control group, C1C-3 siRNA transfection resulted in a reduction of C1C-3 expression by (60.9 ± 4.0)% (n=3, PO.01). The hypotonic challege (160 mOsmol/L) caused cell swelling and induced RVD. In the control group, hypotonic solution bath for 35 min resulted in a RVD of (42.6±2.8)% (w=20), which was significantly decreased to (10.5 ±4.8)% (n=16) in C1C-3 siRNA-transfected cells, demonstrating a reduction of RVD capacity by 75.4% (P<0.01). Conclusion The capacity of RVD is significantly reduced in CNE-2Z cells by C1C-3 chloride channel protein knock-down via C1C-3 siRNA transfection, indicating an important role of C1C-3 chloride channels in the RVD of CNE-2Z cells.