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目的:探讨小干扰RNA(small interference RNA,siRNA)沉默水通道蛋白-5(aquaporin-5,AQP-5)的表达对人结肠癌HT-29细胞增殖、凋亡及化疗药敏感性的影响。方法:以合成的AQP-5-siRNA序列转染HT-29细胞,采用Western blotting检测AQP-5-siRNA的转染效率,磺酰罗丹明(sulphorhodamine B,SRB)染色法检测各组细胞的增殖抑制率,流式细胞术检测HT-29细胞的凋亡。分光光度法检测HT-29细胞caspase-3活性,real-time PCR和Western blotting检测AQP-5-siRNA转染后HT-29细胞中PCNA和P53在mRNA和蛋白水平的表达。选用5-氟尿嘧啶(5-fluorouracil,5-FU)和顺铂(cisplatin,DDP)刺激AQP-5-siRNA转染细胞,SRB染色法检测细胞的增殖抑制率,以金正均法计算两药联合作用的Q值。结果:与NC-HT-29细胞相比,AQP-5-siRNA-HT-29细胞中AQP-5蛋白的表达显著下降(P<0.05)。SRB检测显示,AQP-5-siRNA-HT-29细胞的增殖抑制率显著增加[(9.23±0.51)%vs 0,P<0.05]。流式细胞术检测显示,AQP-5-siRNA-HT-29细胞的凋亡率显著升高[(10.81±1.32)%vs(0.99±0.18)%,P<0.05];caspase-3活性显著升高[(0.19±0.03)vs(0.09±0.01),P<0.05]。Real-time PCR和Western blotting结果显示,AQP-5-siRNA-HT-29细胞中PCNA mRNA和蛋白表达明显下降(P<0.05),同时,P53 mRNA和蛋白表达明显上升(P<0.05)。AQP-5-siRNA+5-FU组细胞的增殖抑制率显著高于AQP-5-siRNA组和5-FU组[(44.93±2.28)%vs(9.11±0.32)%、(25.68±1.71)%,均P<0.05],AQP-5-siRNA+DDP组细胞的增殖抑制率显著高于AQP-5-siRNA组和DDP组[(39.01±1.76)%vs(9.11±0.32)%、(18.47±1.25)%,P<0.05],而且,AQP-5-siRNA与5-FU或DDP联用的Q值分别为1.38和1.51,均表现为协同作用。结论:AQP-5-siRNA能抑制HT-29细胞增殖、促进其凋亡、并提高HT-29细胞对5-FU和DDP的化疗敏感性。
OBJECTIVE: To investigate the effect of silencing expression of aquaporin-5 (AQP-5) on the proliferation, apoptosis and chemosensitivity of human colon cancer HT-29 cells with small interfering RNA (siRNA). Methods: HT-29 cells were transfected with the synthetic AQP-5-siRNA. The transfection efficiency of AQP-5-siRNA was detected by Western blotting. The proliferation of HT-29 cells was detected by sulborhodamine B (SRB) Inhibition rate, flow cytometry HT-29 cells apoptosis. Caspase-3 activity in HT-29 cells was detected by spectrophotometry. The expression of PCNA and P53 mRNA and protein in HT-29 cells were detected by real-time PCR and Western blotting after AQP-5-siRNA transfection. 5-fluorouracil (5-FU) and cisplatin (DDP) were used to stimulate the AQP-5-siRNA transfected cells. The proliferation inhibition rate of the cells was detected by SRB staining, and the combination of the two drugs Q value. Results: Compared with NC-HT-29 cells, AQP-5-siRNA-HT-29 cells AQP-5 protein expression was significantly decreased (P <0.05). SRB assay showed that the proliferation inhibition rate of AQP-5-siRNA-HT-29 cells was significantly increased [(9.23 ± 0.51)% vs 0, P <0.05]. Flow cytometry showed that the apoptotic rate of AQP-5-siRNA-HT-29 cells was significantly increased (10.81 ± 1.32)% vs (0.99 ± 0.18)%, P <0.05] High [(0.19 ± 0.03) vs (0.09 ± 0.01), P <0.05]. The results of Real-time PCR and Western blotting showed that the expression of PCNA mRNA and protein in AQP-5-siRNA-HT-29 cells was significantly decreased (P <0.05), while the expression of P53 mRNA and protein was significantly increased (P <0.05). The inhibition rate of AQP-5-siRNA + 5-FU group was significantly higher than that of AQP-5-siRNA group and 5-FU group (44.93 ± 2.28% vs 9.11 ± 0.32%, 25.68 ± 1.71% (P <0.05). The inhibition rate of AQP-5-siRNA + DDP group was significantly higher than that of AQP-5-siRNA group and DDP group (39.01 ± 1.76% vs 9.11 ± 0.32%, 18.47 ± 1.25)%, P <0.05]. Moreover, the Q values of AQP-5-siRNA combined with 5-FU or DDP were 1.38 and 1.51, respectively, showing synergistic effects. CONCLUSION: AQP-5-siRNA can inhibit HT-29 cell proliferation, promote apoptosis and increase chemosensitivity to 5-FU and DDP in HT-29 cells.