Involvement of microRNA-21 in mediating chemo-resistance to docetaxel in androgen-independent prostate cancer PC3 cells

Abstract

Aim: To investigate whether microRNA-21 was involved in mediating the chemoresistance of prostate cancer cells to docetaxel.

Methods: A microarray technique was used to determine the miRNA profile in docetaxel-resistant PC3 cells. Real-time PCR was used to confirm the array results. miR-21 mimics and inhibitors were synthesized and introduced to cells using Lipofectamine 2000. Cell proliferation was examined with the CCK-8 assay. Luciferase reporter containing PDCD 3'UTR was constructed and the activity was detected by a dual luciferase assay. PDCD4 protein expression was evaluated using Western blot.

Results: A docetaxel-resistant prostate cancer PC3 cell line (PC3R) was established . Using microarrays, miR-21 was found to be up-regulated in PC3R cells. Ectopic expression of miR-21 increased the resistance to docetaxel in PC3 wild type cells. In contrast, silencing of miR-21 in PC3R cells sensitized the cells to docetaxel. The IC(50) values for miR-21-silencing cells and control cells were 28.31 and 35.89 nmol/L, respectively. PDCD4, a direct target gene of miR-21, could mediate chemoresistance to docetaxel in PC3 cells.

Conclusion: Our findings suggest that miR-21 contributed to the resistance of PC3 cells to docetaxel, and that targeting miR-21 may offer a promising therapeutic approach in sensitizing prostate cancer to docetaxel treatment.

Figure 1

The inhibitory rate of PC3wt and PC3R cells in response to docetaxel treatment.

Figure 2

Relative expression of four miRNA in PC3wt and PC3R cells. (A) Relative expression of miR-21 in PC3wt and PC3R cells. (B) Relative expression of miR-210 in PC3wt and PC3R cells. (C) Relative expression of miR-192 in PC3wt and PC3R cells. (D) Relative expression of miR-296-5p in PC3wt and PC3R cells. The expression was determined by quantitative real-time PCR. U6 snRNA served as the internal control. Error bars represent SEM. n=3. ^bP<0.05 vs PC3wt cells.

Figure 3

Ectopic expression of miR-21 increased the resistance of PC3wt cells to docetaxel. (A) Cell proliferation of PC3wt cells after transfection with miR-21 mimics every 24 h for three days. Error bars represent SEM.n=3. ^bP<0.05. Statistical analysis was performed using Student's t-test. (B) The inhibitory rate of PC3wt cells after transfection with miR-21 mimics or control RNA for 24 h and treatment with different doses of docetaxel for another 48 h. Statistical analysis was performed using Paired t-test, P<0.01

Figure 4

Silencing of miR-21 expression in docetaxel-resistant PC3 cells led to sensitization of the cells to docetaxel. (A) Cell proliferation of PC3R cells after transfection with miR-21 inhibitors every 24 h for three days. Error bars represent SEM. n=3. ^bP<0.05. Statistical analysis was performed using Student's t-test. (B) The inhibitory rate of PC3R cells after transfection with miR-21 inhibitors or control RNA for 24 h and treatment with different doses of docetaxel for another 48 h. Statistical analysis was performed using Paired t-test, P<0.05

Figure 5

PDCD4 is a direct target gene of miR-21 in PC3 cells. (A) Western blot of PDCD4 expression in PC3wt and PC3R cells. (B) Luciferase activity assays of miR-21 co-transfected with various luciferase reporters. The luciferase activity of each sample was normalized to the Renilla luciferase activity. Error bars represent SEM. ^bP<0.05. Statistical analysis was performed using Student's t-test. (C) Western blot of PDCD4 expression in PC3wt transfected with miR-21 or control RNA, and PC3R cells transfected with miR-21 inhibitors or control RNA. n=3.

Figure 6

PDCD4 can mediate chemoresistance to docetaxel in PC3 cells. (A) Western blot of PDCD4 expression in PC3wt transfected with PDCD4 siRNA or control RNA. (B) The inhibitory rate of PC3wt cells after transfection with PDCD4 siRNA or control RNA for 24 h and treatment with different doses of docetaxel for another 48 h. (C) Western blot of PDCD4 expression in PC3R cells transfected with PDCD4 vector or the control vector. (D) The inhibitory rate of PC3R cells after transfection with the PDCD4 vector or control vector for 24 h and treatment with different doses of docetaxel for another 48 h.