Silencing of miR-193a-5p increases the chemosensitivity of prostate cancer cells to docetaxel

Abstract

Background: Docetaxel-based chemotherapy failure in advanced prostate carcinoma has partly been attributed to the resistance of prostate cancer (PC) cells to docetaxel-induced apoptosis. Hence, there is an urgent need to identify mechanisms of docetaxel chemoresistance and to develop new combination therapies.

Methods: miR-193a-5p level was evaluated by qPCR in prostate tissues and cell lines, and its expression in the tissues was also examined by in situ hybridization. PC cell line (PC3 cell) was transfected with miR-193a-5p mimic or its inhibitor, and then cell apoptosis and the expression of its downstream genes Bach2 and HO-1 were detected by TUNEL staining and Western blotting. Luciferase reporter assay was used to detect the effect of miR-193a-5p and Bach2 on HO-1 expression. Xenograft animal model was used to test the effect of miR-193a-5p and docetaxel on PC3 xenograft growth.

Results: miR-193a-5p was upregulated in PC tissues and PC cell lines, with significant suppression of PC3 cell apoptosis induced by oxidative stress. Mechanistically, miR-193a-5p suppressed the expression of Bach2, a repressor of the HO-1 gene, by directly targeting the Bach2 mRNA 3'-UTR. Docetaxel treatment modestly decreased Bach2 expression and increased HO-1 level in PC3 cells, whereas a modest increase of HO-1 facilitated docetaxel-induced apoptosis. Notably, docetaxel-induced miR-193a-5p upregulation, which in turn inhibits Bach2 expression and thus relieves Bach2 repression of HO-1 expression, partly counteracted docetaxel-induced apoptosis, as evidenced by the increased Bcl-2 and decreased Bax expression. Accordingly, silencing of miR-193a-5p enhanced sensitization of PC3 cells to docetaxel-induced apoptosis. Finally, depletion of miR-193a-5p significantly reduced PC xenograft growth in vivo.

Conclusions: Silencing of miR-193a-5p or blockade of the miR-193a-5p-Bach2-HO-1 pathway may be a novel therapeutic approach for castration-resistant PC.

Keywords: Bach2; Chemoresistance; Docetaxel; HO-1; Prostate cancer; miR-193a-5p.

Fig. 1

miR-193a-5p is upregulated in PC tissues and PC cell lines. a qRT-PCR detected the expression of miR-193a-5p in matched benign prostatic hyperplasia (BPH, n = 40) and PC tissues (PC, n = 40). *P < 0.001 vs BPH. Normalized against an internal control U6 RNA. b Fluorescence in situ hybridization (FISH) for detection of miR-193a-5p in PC and BPH tissues. Blue staining represents the nucleus and green staining indicates miR-193a-5p. Scale bar = 64 μm. c The expression levels of miR-193a-5p were detected in cancer cell lines (LNCaP, PC3, DU145, T24 and UM-UC-3) and normal prostate epithelial cells (RWPE-1); *P < 0.05 vs. RWPE-1

Fig. 2

miR-193a-5p upregulation suppresses PC3 cell apoptosis. a Western blotting detected PCNA and cleaved caspase-3 proteins in PC3 cells treated with or without H₂O₂ (100 μM, 12 h). Right panel shows densitometric analysis of three independent experiments. **P < 0.01 vs. vehicle control. b qRT-PCR detected the expression of miR-193a-5p in PC3 cells treated with or without H₂O₂. *P < 0.05 vs. vehicle control. c Western blotting detected PCNA and cleaved caspase-3 in PC3 cells transfected with miR-193a-5p mimic or control (Ctl) mimic, and then treated with H₂O₂. Bottom panel shows densitometric analysis. ***P < 0.001 vs. Ctl mimic. d PCNA and cleaved caspase-3 were determined by western blotting in PC3 cells transfected with anti-miR-193a-5p or anti-miR-Ctl, and then treated with H₂O₂. Bottom panel shows densitometric analysis. **P < 0.01 vs. anti-miR-Ctl. e The TUNEL staining detected H₂O₂–induced apoptosis in PC3 cells transfected with miR-193a-5p mimic or anti-miR-193a-5p. Blue staining represents the nucleus, and red staining indicates TUNEL-positive cells. Right panel shows the number of TUNEL-positive cells of three independent experiments. Scale bar = 100 μm. *P < 0.05, **P < 0.01 vs. their corresponding control

Fig. 3

miR-193a-5p inhibits PC3 cell apoptosis by increasing HO-1 expression. a PC3 cells were transfected with the indicated RNA constructs and then treated with H₂O₂, HO-1, p47^phox and p22^phox expression was determined by Western blotting. b Immunohistochemical staining of HO-1 at different stages of PC progression. 1, Benign prostatic hyperplasia (BPH); 2, Gleason grade 2; 3, Gleason grade 4; 4, Gleason grade 5 PC. HO-1 expression increases with high Gleason score. Bars = 40 μm. c Fluorescence in situ hybridization (FISH) staining was performed on sections from BPH and PC tissues. Green, red and blue staining indicates miR-193a-5p, HO-1 and DAPI, respectively. Bar = 64 μm. d HO-1 mRNA and miR-193a-5p were measured by RT-qPCR, and Pearson correlation analysis shows a positive correlation between miR-193a-5p and HO-1. (P = 0.0157; R = 0.5325). e PC3 cells were treated with the different concentrations of Docetaxel (Doc), and the expression of miR-193a-5p was detected by qRT-PCR. *P < 0.05, **P < 0.01 vs. vehicle control. f Following transfection with the indicated RNA constructs, PC3 cells were treated with Doc (10 nM). HO-1 and cleaved caspase-3 were determined by Western blotting. g PC3 cells were treated with Doc (10 nM) alone or together with 20 μM Hemin (sigma, #51280) or Znpp (Protoporphyrin IX zinc, sigma, #282820) for 24 h, and cell apoptosis was assessed by flow cytometry. h PC3 cells were treated as in (G), TUNEL staining detected cell apoptosis. Right panel shows the number of TUNEL-positive cells of three independent experiments. Bar = 100 μm. *P < 0.05 vs. Doc alone; **P < 0.01 vs. vehicle control. i PC3 cells were treated as in (g), Western blotting detected HO-1 and cleaved caspase-3. Experiments were repeated three times

Fig. 4

HO-1 upregulation leads to resistance of PC3 cells to docetaxel-induced apoptosis via the Bcl2/Bax pathway. a and c Immunohistochemical staining of Bcl-2 (a) and Bax (c) at different stages of PC progression. 1, BPH; 2, Gleason grade 2; 3, Gleason grade 4; 4, Gleason grade 5 PC. Bars = 40 μm. b and d Bcl-2, HO-1 and Bax mRNAs were measured by qRT-PCR, the correlation between Bcl-2 and HO-1 or between Bax and HO-1 was evaluated by Pearson correlation analysis, respectively. (b, P < 0.05, R = 0.4502; (d), P < 0.05, R = −0.5529). e PC3 cells were treated with the different concentrations of Doc, and Western blot analysis detected Bcl-2 and Bax expression. Right panel shows densitometric analysis of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle control; ^# P < 0.05, ^## P < 0.01, ^### P < 0.001 vs. vehicle control. f PC3 cells were treated with Doc alone or together with Hemin or Znpp for 24 h, HO-1, Bcl-2 and Bax were determined by Western blot analysis. Right panel shows densitometric analysis of three independent experiments. *P < 0.05, **P < 0.01 vs. their corresponding control

Fig. 5

Bach2 is a direct target of miR-193a-5p. a Prediction of miR-193a-5p binding site at Bach2 3′-UTR. Red color indicates the sequence of the mutated miR-193a-5p binding site. b Luciferase reporter assays were performed in PC3 cells co-transfected cells with miR-193a-5p mimic and wild-type (WT) or mutant (mut) Bach2 3′-UTR-luciferase reporter **P < 0.01 vs. Ctl mimic. c and d PC3 cells were transfected with miR-193a-5p mimic (c) or anti-miR-193a-5p (d), Bach2 and Nrf2 expression was analyzed by Western blotting. Bottom panel shows densitometric analysis of three independent experiments. **P < 0.01, ***P < 0.001 vs. their corresponding control. e Bach2 mRNA was detected by qRT-PCR in BPH (n = 21) and PC tissues (n = 21). *P < 0.01 vs. BPH. f Pearson correlation was used to analyze the relationships between miR-193a-5p and Bach2. (R = −0.4902, P = 0.0282)

Fig. 6

Silencing of miR-193a-5p enhances sensitization of PC3 cells to docetaxel-induced apoptosis through upregulating Bach2 expression. a PC3 cells were co-transfected with HO-1 promoter reporter construct and si-Bach2, and then treated with or without Doc. Luciferase reporter assays were performed. *P < 0.01 vs. empty vector or si-Ctl. b ChIP-qPCR detected Bach2 binding to the HO-1 promoter in PC3 cells transfected with anti-miR-193a-5p or anti-miR-Ctl, and then treated with or without Doc. *P < 0.05 vs. anti-miR-Ctl plus PBS. c Agarose gel electrophoresis for PCR products of chromatin immunoprecipitation showing Bach2 binding to the HO-1 promoter. d PC3 cells were transfected with anti-miR-193a-5p or anti-miR-Ctl, and then treated with or without Doc. Immunofluorescence staining detected the expression of HO-1 (green) and Bach2 (red). Nuclei were staining with DAPI (blue). Scale bars = 75 μm. e PC3 cells were treated as in (d), cell apoptosis was assessed by TUNEL staining. Right panel shows the number of TUNEL-positive cells of three independent experiments. *P < 0.01, **P < 0.01 vs. their corresponding control

Fig. 7

miR-193a-5p, Bach2 and HO-1 coordinate docetaxel-induced apoptosis in PC3 cells. a PC3 cells were transfected with miR-193a-5p mimic or Ctl mimic and then treated with or without Doc, and HO-1, Bach2 and cleaved caspase-3 were detected by Western blotting. Bottom panel shows densitometric analysis of three independent experiments. *P < 0.01, **P < 0.01, ***P < 0.001 vs. their corresponding control. b Western blotting detected HO-1, Bach2 and cleaved caspase-3 in PC3 cells transfected with anti-miR-193a-5p or Anti-miR-Ctl and then treated with or without Doc. Bottom panel shows densitometric analysis of three independent experiments. *P < 0.01, **P < 0.01, ***P < 0.001 vs. their corresponding control. c Western blotting detected HO-1, Bach2 and cleaved caspase-3 in PC3 cells transfected with the indicated RNA constructs. Right panel shows densitometric analysis of three independent experiments. *P < 0.01 vs. their corresponding control

Fig. 8

Depletion of miR-193a-5p reduces PC xenograft growth in vivo. a PC3 cells engineered to stably express anti-miR-193a-5p (LV-anti-miR-193a-5p) or negative control (LV-miR-Ctl) were injected subcutaneously in 200 μl PBS/Matrigel (50: 50) into the right forelimb to establish xenograft tumors. From the first day, mice were intraperitoneally injected with 10 mg/kg Doc every three days. Tumor volumes were monitored by direct measurement with calipers and calculated by the formula: (length × width²)/2. **P < 0.01 vs. Doc + LV-miR-Ctl; ^# P < 0.05, ^## P < 0.01 vs. LV-anti-miR-193a-5p (each groups, n = 16). b Representative tumor sizes in each group of mice. c Xenograft tumor wet weight in each group of mice. *P < 0.05, **P < 0.01, ***P < 0.001 vs. Doc + LV-anti-miR-193a-5p. d The xenograft models of nude mice were prepared as in (A), Western blotting detected the expression of Bach2, HO-1, cleaved caspase-3, Bcl-2 and Bax in xenograft tumors. e The xenograft models of nude mice were prepared as in (a), the TUNEL staining detected cell apoptosis in xenograft tumors. Blue staining represents the nucleus, and red staining indicates TUNEL-positive cells