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Archive > Volume 20 Issue 3 > 2013,20(3):306-311. DOI:10.3872/j.issn.1007-385X.2013.03.009 Prev Next
Basic Research
Silencing AQP-5 on proliferation, apoptosis and chemosensitivity of human colon cancer HT-29 cells
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Abstract:
Objective:To investigate the effect of siRNA (small interference RNA, siRNA) silencing aquaporin-5 ( AQP-5 ) expression on the proliferation, apoptosis, and chemosensitivity of human colon cancer HT-29 cells. Methods: A synthetic AQP-5-siRNA sequence was transfected into HT-29 cells, and the inhibition efficiency was detected by Western blotting. Sulphorhodamine B (SRB) assay were used to detect the cell proliferation inhibition rate. Cell apoptosis of HT-29 cells were detected by flow cytometry (FCM). The activity of caspase-3 in HT-29 cells was measured by spectrophotometry. The mRNA and protein levels of PCNA and P53 in HT-29 cells after AQP-5-siRNA transfection were determined by real-time PCR and Western blotting. 5-fluorouracil (5-FU) and cisplatin (DDP) were selected to treat the AQP-5-siRNA-HT-29 cells, and SRB assay was used to detect the cell proliferation inhibition rate. The “Q” Method of Jin Zhenjun was used to evaluate the interaction of the two drugs. Results: SRB assay showed that compared with NC-HT-29 cells, AQP-5 expression was significantly decreased in AQP-5-siRNA-HT-29 cells (P<0.05). Compared with NC-HT-29 cells，the proliferation inhibition rate was increased significantly in AQP-5-siRNA-HT-29 cells (\[9.23±051\]% vs 0, P<0.05). Flow cytometry analysis showed that compared with NC-HT-29 cells, the cell apoptosis rate increased dramatically in AQP-5-siRNA-HT-29 cells (\[10.81±1.32\]% vs \[0.99±0.18\]%, P<0.05). The activities of caspase-3 were also increased in AQP-5-siRNA-HT-29 cells compared with NC-HT-29 cells (\[0.19±0.03\] vs \[0.09±0.01\], P<0.05). Real-time PCR and Western blotting results indicated that compared with NC-HT-29 cells, the mRNA and protein levels of PCNA were decreased (P<0.05), simultaneously, the mRNA and protein levels of P53 were increased (P<0.05) in AQP-5-siRNA-HT-29 cells. Compared with AQP-5-siRNA-HT-29 cells or 5-FU-HT-29 cells, the proliferation inhibition rate was increased remarkably in AQP-5-siRNA+5-FU-HT-29 cells(\[44.93±2.28\]% vs \[9.11±032\]%, \[25.68±171\]%, P<0.05), respectively. Compared with AQP-5-siRNA-HT-29 cells or DDP-HT-29 cells, the proliferation inhibition rate was increased remarkably in AQP-5-siRNA+DDP-HT-29 cells (\[39.01±176\]% vs \[911±0.32\]%, \[18.47±1.25\]%, P<0.05), respectively. Moreover, the synergistic effects were showed between AQP-5-siRNA and 5-FU or DDP with Q value of 1.38 and 1.51, respectively. Conclusion:AQP-5-siRNA can inhibit the proliferation, promote the apoptosis of HT-29 cells, and increase the chemosensitivity of HT-29 cells to 5-FU and DDP.
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