Effect of AQP9 Expression in Androgen-Independent Prostate Cancer Cell PC3

Abstract

It is known that aquaporin 9 (AQP9) in the prostate was strictly upregulated by androgen and may represent a novel therapeutic target for several cancers, but whether AQP9 plays a role in the regulation of androgen-independent prostate cancer still remains unclear. In the present study, AQP9 was determined in prostate cancer and adjacent cancer tissues; AQP9-siRNA was applied to silencing AQP9 in androgen-independent prostate cancer cell PC3 cell line. Western blot and flow cytometry analysis were employed to detect changes in related-function of control and AQP9-siRNA groups. The results showed that AQP9 is significantly induced in cancer tissues than that in adjacent cancer tissues. Moreover, knockdown of AQP9 in PC3 androgen-independent prostate cancer cell prostate cancer cells increased inhibition rates of proliferation. In addition, knockdown of AQP9 resulted in a significant decrease in the expression of the Bcl-2 and with a notable increase in the expression of Bax and cleaved caspase 3, indicated that AQP9 knockdown promoted apoptosis in prostate cancer cells. From wound healing assay and matrigel invasion, we suggested that AQP9 expression affects the motility and invasiveness of prostate cancer cells. Moreover, In order to explore the pathway may be involved in AQP9-mediated motility and invasion of prostate cancer cells, the phosphorylation of ERK1/2 was significant suppressed in AQP9 siRNA-transfected cells compared with that in control cells, suggesting that AQP9 is involved in the activation of the ERK pathway in androgen-independent prostate cancer cells.

Keywords: ERK signaling pathway; RNA interference; apoptosis; aquaporin 9; invasion; proliferation; prostate cancer.

Figure 1

(A) Expression of AQP9 in PC3 and LNCap cells was analyzed by immunofluorescence; (B) expression of AQP9 in PC3 and LNCap cells was determined by Western blot. The mean of AQP9/β-actin expression in liver set as 1.0. Data were based on three independent experiments, and shown as mean ± SD (standard deviation).

Figure 2

AQP9 expression was significantly increased in prostate cancer tissues when compared with the adjacent tissues of patients from GEO dataset GSE55945 (p < 0.05 as compared with control). Bars represent means, * p < 0.05.

Figure 3

Expression of AQP9 in PC3 cells, AQP9-siRNA and Mock were analyzed by real-time-PCR (A) and Western blot (B) (p < 0.05 as compared with control). △CT AQP9/β-actin (control PC3) was 6.348. Control: wild-type cells; AQP9-siRNA: cells transfected with AQP9 specific small interfering RNA; Mock: cells only treated with Lipofectamine 2000. The mean of AQP9/β-actin expression in PC-3 cells set as 1.0. Data were based on three independent experiments, and shown as mean ± SD. * p < 0.05 as compared with control.

Figure 4

Cell proliferation was detected 24 h after specific small interfering RNA in cells (p < 0.05 as compared with control). Control: wild-type cells; AQP9-siRNA: cells transfected with AQP9 specific small interfering RNA; Mock: cells only treated with Lipofectamine 2000. Data were based on three independent experiments, and shown as mean ± SD, * p < 0.05.

Figure 5

(A) Cells were double-stained with Annexin V-FITC/PI and apoptosis rates was analyzed using flow cytometry (p < 0.05 as compared with control); (B) protein levels of cleaved caspase 3, Bax, and Bcl2 were detected by Western blot. Data were shown as mean ± SD (p < 0.05 as compared with control). The mean of targeted protein/β-actin expression in PC-3 cells set as 1.0.Control: wild-type cells; AQP9-siRNA: cells transfected with AQP9 specific small interfering RNA; Mock: cells only treated with Lipofectamine 2000. * p < 0.05; ** p < 0.01.

Figure 6

(A) Confluent cell monolayers were wounded with a pipette tip. Representative images were captured at 0 h, 24 h, and 36 h. The wound closure was quantified and normalized to that of control cells (p < 0.05); Cell invasion was analyzed in Matrigel-coated transwell chambers. Representative images were shown in (B); invaded cells were counted in four selected fields (total space is 2 mm²) (p < 0.05 as compared with control); the total number of invaded cells from the control group set as 100%; (C) MMP9 protein expression was examined by Western blot. The mean of MMP9 protein/β-actin expression in PC-3 cells set as 1.0. Control: wild-type cells; AQP9-siRNA: cells transfected with AQP9 specific small interfering RNA; Mock: cells only treated with Lipofectamine 2000. Data were based on three independent experiments, and shown as mean ± SD. * p < 0.05; ** p < 0.01, Bars, 100 µm.

Figure 7

Cells were transfected with siAQP9. (A) After 48 h, Western blotting was performed to detect the phosphorylation of ERK1/2 (p < 0.05 as compared with control). The phosphorylation of ERK1/2 was markedly suppressed in AQP9 siRNA-transfected cells compared with that in control cells. Control: wild-type cells; AQP9-siRNA: cells transfected with AQP9 specific small interfering RNA; (B) after PC3 cells were treated with U0126 (20 µM) or DMSO vehicle, an invasion assay were performed to compare the invasion abilities. The mean number of invaded cells in control group set as 1.0. Data were based on three independent experiments, and shown as mean ± SD, ** p < 0.01.