Objective: To study the role of aquaporin 5(AQP5) in regulating the cell proliferation and apoptosis of human colorectal cancer cells and explore the possible mechanism.
Methods: A small interfering RNA (siRNA) targeting AQP5 was used to suppress endogenous AQP5 expression in the human colorectal cancer cell lines COLO 205 and SW480, and the transfection efficiency of AQP5 siRNA was determined using immunofluorescence assay and PCR. The changes in the proliferation of the transfected cells was evaluated with MTT assay, and the cell apoptosis was analyzed using Annexin V-FITC/PI and TUNEL assays; the changes of Bax and Bcl2 expressions in the cells were determined using RTPCR and Western blotting.
Results: Transfection with AQP-5-siRNA resulted in a significant reduction (up to 90%) of AQP-5 expression in COLO 205 and SW480 cells. MTT assay showed that AQP-5-siRNA transfection significantly inhibited the cell proliferation compared NS siRNA transfection (P<0.05). Flow cytometric analysis revealed significantly increased apoptotic rate of cells following AQP-5-siRNA transfection compared with NS?siRNA transfection(P<0.05). Real-time quantitative RT-PCR and Western blotting demonstrated that AQP-5-siRNA transfection significantly increased Bax and Bcl-2 expressions at both mRNA and protein levels in the cells.
Conclusion: AQP5-siRNA can promote apoptosis of colorectal cancer cells in vitro possibly in relation to its effects on Bax and Bcl expressions.
目的: 探讨水通道蛋白-5(AQP-5) 对细胞增殖及凋亡影响及可能的机制。
方法: 体外常规培养人结直肠癌COLO 205和SW480细胞，取对数生长期的细胞用于实验。通过siRNA技术抑制内源性AQP-5的表达，并经免疫荧光、PCR检测AQP-5-siRNA转染效率，利用MTT法检测细胞增殖能力，Annexin V-FITC/PI和TUNEL检测细胞凋亡情况，RT-PCR和Western blot检测Bax、Bcl-2表达变化。
结果: 在COLO 205和SW480细胞株中，转染AQP-5-siRNA后AQP-5表达下调达90%。MTT分析结果显示，与转染NS-siRNA组相比，转染AQP-5-siRNA组的细胞增殖抑制率显著增加（P < 0.05）；流式细胞分析结果发现，与NSsiRNA组细胞相比，转染AQP-5-siRNA后细胞凋亡率显著增加（P < 0.05）；荧光实时定量RT-PCR和蛋白质印迹法检测结果显示：与转染NS-siRNA组相比，转染AQP-5-siRNA明显提升Bax/Bcl-2 mRNA和蛋白的表达。
结论: AQP-5-siRNA可体外促进结直肠癌细胞凋亡，其机制可能与Bax/Bcl表达有关。