MicroRNA-185 and 342 inhibit tumorigenicity and induce apoptosis through blockade of the SREBP metabolic pathway in prostate cancer cells

Abstract

MicroRNA (miRNA or miR) inhibition of oncogenic related pathways has been shown to be a promising therapeutic approach for cancer. Aberrant lipid and cholesterol metabolism is involved in prostate cancer development and progression to end-stage disease. We recently demonstrated that a key transcription factor for lipogenesis, sterol regulatory element-binding protein-1 (SREBP-1), induced fatty acid and lipid accumulation and androgen receptor (AR) transcriptional activity, and also promoted prostate cancer cell growth and castration resistance. SREBP-1 was overexpressed in human prostate cancer and castration-resistant patient specimens. These experimental and clinical results indicate that SREBP-1 is a potential oncogenic transcription factor in prostate cancer. In this study, we identified two miRNAs, miR-185 and 342, that control lipogenesis and cholesterogenesis in prostate cancer cells by inhibiting SREBP-1 and 2 expression and down-regulating their targeted genes, including fatty acid synthase (FASN) and 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR). Both miR-185 and 342 inhibited tumorigenicity, cell growth, migration and invasion in prostate cancer cell culture and xenograft models coincident with their blockade of lipogenesis and cholesterogenesis. Intrinsic miR-185 and 342 expression was significantly decreased in prostate cancer cells compared to non-cancerous epithelial cells. Restoration of miR-185 and 342 led to caspase-dependent apoptotic death in prostate cancer cells. The newly identified miRNAs, miR-185 and 342, represent a novel targeting mechanism for prostate cancer therapy.

Figure 1. MiR-185 and 342 inhibition of SREBP mRNA and protein and expression patterns of miR-185 and 342 in prostate cancer cells.

A, Both miR-185 and 342 inhibited mRNA expression of SREBP-1, SREBP-2, FASN, HMGCR and AR in LNCaP and C4-2B prostate cancer cells determined by qRT-PCR. -: non-transfected; NC: miR-negative control. The relative mRNA expression (fold) was assigned as 1.0 in non-transfected cells. Data were normalized to 18S rRNA and represent the mean ± SD of three independent duplicate experiments. **, P < 0.005 significant differences from NC. B, MiR-185 and 342 inhibited precursor (125 kDa) and mature (68 kDa) forms of SREBP-1 and SREBP-2, FASN, HMGCR and AR expression in LNCaP and C4-2B cells assayed by Western blot. β2-microglobulin (β2M) was used as a loading control. C, MiR-185 and 342 inhibitors (antisense oligonucleotides against miR-185 and 342) increased SREBP-1, SREBP-2, FASN, HMGCR and AR expression in LNCaP and C4-2B cells determined by qRT-PCR. The relative mRNA expression (fold) was assigned as 1.0 in non-transfected cells. Data were normalized to 18S rRNA and represent the mean ± SD of three independent duplicate experiments. **, P < 0.005 significant differences from NC. D, Expression of intrinsic miR-185 and 342 in RWPE-1, LNCaP and C4-2B cells. qRT-PCR results showed that relative expression of miR-185 and 342 was significantly decreased in prostate cancer cells compared with normal/non-cancerous RWPE-1. Lower expression of both miRNAs was observed in aggressive C4-2B compared with LNCaP cells. The relative miRNA expression (fold) was assigned as 1.0 in RWPE-1 cells. **, P < 0.005 significant differences from RWPE-1. Data were normalized to RNU6B control and represent the mean ± SD of three independent experiments performed in quadruplicate.

Figure 2. MiR-185 and 342 suppress cell proliferation, clonogenicity, migration and invasion.

A, MiR-185 and 342 inhibited cell proliferation in LNCaP and C4-2B cells compared with non-transfected (−) and miR-negative control (NC) transfected cells 3 d following miRNA transfection. The relative cell proliferation (%) was assigned as 100% in non-transfected (−) cells. **, P < 0.005 significant differences from NC. Data represent the mean ± SD of two independent quadruplicate experiments. B, MiR-185 and 342 suppressed colony formation in LNCaP and C4-2B cells compared with the control groups after 14 d miRNA transfection. *, P < 0.05 and **, P < 0.005 significant differences from NC. Data represent the mean ± SD of two independent triplicate experiments. C, Cell migration and D, invasion were significantly decreased by miR-185 and 342 in LNCaP and C4-2B cells compared with the control groups. **, P < 0.005 significant differences from NC. Data represent the mean ± SD of two independent quadruplicate experiments.

Figure 3. MiR-185 and 342 induce caspase-dependent apoptotic death.

A, An Annexin V-FITC/PI staining apoptotic assay and flow cytometry were performed in LNCaP and C4-2B cells transfected with miRNAs. Both miR-185 and 342 increased the apoptotic cell fractions (both early and late apoptotic cell fractions; P<0.005) compared with the control groups. B, Caspase 3/7 activities were significantly increased by miR-185 and 342 in LNCaP and C4-2B cells. Caspase 3/7 activities (fold) were assigned as 1.0 in non-transfected (−) cells. **, P < 0.005 significant differences from NC. Data represent the mean ± SD of two independent triplicate experiments. C, MiR-185 and 342 decreased pro-caspase 9, 3 and PARP, and activated cleaved caspase 3 and PARP expression in LNCaP cells as assayed by Western blot. β2M was used as a loading control. C: cleaved.

Figure 4. Intratumoral delivery of miR-185 and 342 leads to regression of prostate tumors in a mouse xenograft model.

A, Subcutaneous C4-2B tumor growth was assayed by tumor volume after intratumoral delivery of miR-185, 342 or NC every 3 d for a 21-d treatment in mice. Both miR-185 and 342 significantly inhibited the growth of C4-2B tumors compared with NC-treated tumors. *, P<0.05; **, P<0.005 significant differences from NC-treated tumors (N = 5 for each group). B, The relative expression of miR-185 or 342 in C4-2B tumors. qRT-PCR results showed that the relative miR-185 or 342 levels were greatly increased in the subcutaneous C4-2B tumors injected with miR-185 or 342 compared with the control tumors. The relative miRNA expression (fold) was assigned as 1.0 in NC. *, P < 0.05 significant differences from NC. Data were normalized to RNU6B and represent the mean ± SD. C, IHC results showed that down-regulation of SREBP-1, SREBP-2, FASN, HMGCR, AR and PSA was observed in the miR-185 and 342-treated subcutaneous C4-2B tumors in comparison with the NC-treated tumors. Scale bars = 25 µm.

Figure 5. MiR-185 and 342 inhibit cell proliferation and induce apoptosis in subcutaneous xenografts.

Quantification of A, Ki67 (cell proliferation) and B, cleaved PARP (C-PARP; apoptosis) positive cells in subcutaneous C4-2B tumor specimens collected from the miR-NC, 185 and 342 treated groups. One hundred cells at 5 randomly selected areas were counted and positively staining cells were recorded. **, P < 0.005 significant differences from the control NC group. Data represent the mean ± SD. Arrowheads indicate the C-PARP positive cells. Scale bars = 100 µm.