miR-124 targets the androgen receptor (AR) transcript, acting as a tumor suppressor to broadly limit the growth of prostate cancer. In this study, we unraveled the mechanisms through which miR-124 acts in this setting. miR-124 inhibited proliferation of prostate cancer cells in vitro and sensitized them to inhibitors of androgen receptor signaling. Notably, miR-124 could restore the apoptotic response of cells resistant to enzalutamide, a drug approved for the treatment of castration-resistant prostate cancer. We used xenograft models to examine the effects of miR-124 in vivo when complexed with polyethylenimine-derived nanoparticles. Intravenous delivery of miR-124 was sufficient to inhibit tumor growth and to increase tumor cell apoptosis in combination with enzalutamide. Mechanistic investigations revealed that miR-124 directly downregulated AR splice variants AR-V4 and V7 along with EZH2 and Src, oncogenic targets that have been reported to contribute to prostate cancer progression and treatment resistance. Taken together, our results offer a preclinical rationale to evaluate miR-124 for cancer treatment. Cancer Res; 75(24); 5309–17. ©2015 AACR.

Prostate cancer is the most frequently diagnosed malignant tumor and the second leading cause of cancer death in American men (1). Our recent study showed that for the past 20 years, the survival for patients presenting in California with metastatic prostate cancer has not improved (2). The androgen receptor (AR) is critical for the development and progression of prostate cancer. Until now, androgen deprivation therapy (ADT), that inhibits AR signaling, represents the primary therapy for patients with hormone-sensitive metastatic prostate cancer. Although it is initially effective, patients invariably relapse and their tumors progress to castration-resistant prostate cancer (CRPC; ref. 3). Because CRPC is commonly associated with aberrant AR signaling that is sufficient to overcome ADT (4), the AR signaling inhibitor (ARSI) enzalutamide has been developed for treating the disease (5). This novel ARSI exhibits greater affinity than bicalutamide for the AR and dramatically inhibits AR function (6). Regrettably, recent studies demonstrated that enzalutamide provides only a modest improvement of survival in patients with prostate cancer due to rapid development of drug resistance (7, 8). Therefore, discovering new therapeutics for enhancing the efficacy of enzalutamide is urgently needed.

microRNAs (miRNA) are endogenous noncoding small RNAs and negatively regulate expression of multiple genes via sequence-specific interactions with the 3′-untranslated regions (UTR) of cognate mRNA targets, leading to inhibiting translation or mRNA degradation. It is estimated that miRNAs can regulate about 60% of protein-coding genes (9). Unlike siRNAs, miRNAs do not require perfect base pairing, and one miRNA has multiple different mRNA targets (10). Therefore, alteration in a single miRNA may change the expression levels of different genes and subsequently affect the signaling pathways involved in a number of physiologic as well as pathologic conditions including cancer. Many miRNAs that act as tumor suppressors or as oncogenes were reported to be aberrantly expressed in various cancer types (11). These findings have generated significant interest in using miRNAs as therapeutic targets for cancer treatment. Indeed, a number of miRNAs were found to inhibit in vivo growth of different human cancer xenografts (12).

Of the known aberrantly expressed and cancer-related miRNAs, miR-124 represents an ideal candidate for therapeutic development. Accumulating evidence indicates that miR-124 is a tumor suppressive miRNA in several types of human cancer (13–15), including prostate cancer. In previous studies, we reported that miR-124 directly targets the AR transcript, that increasing its expression inhibits growth of prostate cancer xenografts, and that it is significantly downregulated in clinical prostate cancer specimens (16), which is consistent with a previous observations by Hellwinkel and colleagues (17). We defined a molecular pathway in which miR-124 targets AR, leading to decreased miR-125 levels and an increased expression of p53. Thus, miR-124 was determined to drive prostate cancer cells toward apoptosis (16). These previous data suggest that miR-124 is involved in the pathogenesis of prostate cancer. In this study, the role of miR-124 was further explored by using synthetic miR-124 mimics. We found that miR-124 directly downregulates the levels of AR transcript variants, as well as enhancer of Zeste homolog 2 (EZH2) and Src tyrosine kinase (Src). Systemic administration of miR-124 not only potently inhibited growth of prostate cancer xenografts but also sensitized prostate cancer tumors to enzalutamide treatment, inducing increased apoptosis in vivo. These findings provide proof-of-concept support for systemic delivery of miR-124 as an adjuvant therapeutic agent for prostate cancer treatment.

Reagents

Bicalutamide was obtained from AstraZeneca. Enzalutamide (marketed as Xtandi and formerly known as MDV3100) was obtained from Medivation, Inc. For in vitro studies, enzalutamide was dissolved in dimethyl sulfoxide. For animal studies, enzalutamide was mixed with 0.5% Methocel A4M suspension (Kremer Pigments Inc.). Ambion pre-miR-124 precursors (in vitro study) and mirVana miR-124 mimics (in vivo study), as well as miRNA negative control (miR-NC), were purchased from Ambion. Both pre-miR-124 precursors and mirVana miR-124 mimics are small double-stranded RNA molecules that mimic endogenous miR-124 and upregulate miR-124 activity. Polyethylenimine (in vivo-jetPEI), a delivery vehicle used in laboratory and clinical trials, was purchased from Polyplus-transfection, Inc. PEI-miR-124 complexes were prepared following the manufacturer's protocol. The anti-AR-V7 monoclonal antibody was purchased from Precision Antibody Store.

Cell lines and culture

Prostate cancer cell lines (LNCaP, C4-2B, 22Rv1, and VCaP) were maintained in RPMI-1640 medium supplemented with 10% FBS (FBS medium) or in RPMI-1640 medium containing 10% charcoal-stripped serum (androgen-deprived medium). To generate the enzalutamide-resistant 22Rv1 subline, 22Rv1 cells were cultured in androgen-deprived medium containing gradually increased concentrations of enzalutamide (from 5 to 40 μmol/L over 4 months). The resultant enzalutamide-resistant 22Rv1 subline was termed 22Rv1-EnzR and maintained in androgen-deprived medium containing 10 μmol/L enzalutamide.

Cell proliferation assay

Prostate cancer cells (3 × 103/well) were seeded in 96-well plates in FBS medium or androgen-deprived medium. After being cultured for 24 hours, cells were transfected with 50 nmol/L miR-124. After 5 hours, cells were treated with fresh medium without or with 10 μmol/L enzalutamide or bicalutamide. A tetrazolium-based cell proliferation assay (WST-1, Promega) was carried out according to the manufacturer's protocol.

Reporter plasmid construction and luciferase assay

To construct reporter plasmids, approximately 0.5-kb DNA fragments containing the putative miR-124 binding sites were prepared by high-fidelity PCR from the 3′UTRs of individual genes of interest. The corresponding fragments of 3′UTRs lacking the miR-124–binding site were used as negative controls. DNA fragments were cloned into the pMIR-REPORT luciferase vector (Ambion) downstream of the luciferase gene. The sequences and cloning direction of these PCR products were validated by DNA sequencing. For luciferase assays, cells (3 × 104/well) were seeded into 24-well plates and cultured for 24 hours. The cells were then cotransfected with reporter plasmids and 25 to 100 nmol/L miR-124 mimics or miRNA-negative control (miR-NC). The pRL-SV40 Renilla luciferase plasmid (Promega) was used as an internal control. Two days later, cells were harvested and lysed with passive lysis buffer (Promega). Luciferase activity was measured using a dual-luciferase reporter assay (Promega). The activities of the pMIR-REPORT firefly test reporters were normalized by Renilla luciferase activity.

Western blot assay

Total protein was extracted from cultured cells or xenograft tumors, and the concentrations were estimated using the Coomassie (Bradford) Protein Assay Reagent (Pierce). Equal amounts of denatured protein samples were loaded on a 10% SDS-PAGE. After electrophoresis, proteins were transferred to Immobilon PVDF membrane. Immunoblotting was conducted using individual specific primary antibodies and appropriate horseradish peroxidase-conjugated secondary antibodies following standard protocols.

Clonogenic assay

Six-well plates were seeded with 3 × 104 22Rv1-EnzR cells per well and incubated overnight. Cells were transfected with miR-124 or transfected with miR-124 for 3 days followed by treatment with 10 μmol/L enzalutamide. After 2 weeks, cells were stained with crystal violet (0.4% crystal violet in 20% methanol). For quantitative clonogenic assays, the colonies were solubilized with 30% acetic acid, and the absorbance was read at a wavelength of 540 nm.

Animal experiments

Animal studies were performed according to the protocols approved by the Institutional Animal Care and Use Committee of the University of California, Davis (Sacramento, CA). Male athymic nude mice (4–6 weeks old) were purchased from Harlan Laboratories and housed in pressurized, ventilated cages with standard rodent chow and water and a 12-hour light/dark cycle. The CWR22 xenograft tumor was a gift from Dr. Thomas A. Pretlow (Case Western Reserve University, Cleveland, OH). Xenografts were implanted by subcutaneous injection into the flanks of the mice with CWR22 cell suspensions (∼2 × 106 cells) in a mixture (1:1 vol/vol) of culture medium and Matrigel (Becton Dickinson). When tumor volume reached about 50 mm3, mice were randomized into four treatment groups (n = 8 mice per group): negative control, miR-124, enzalutamide, and miR-124 + enzalutamide. Treatment was conducted by intravenous injection of 10 μg jetPEI/miR-NC or 10 μg jetPEI/miR-124 complexes (three times weekly for 5 weeks) or by oral administration of enzalutamide (20 mg/kg/wk, once weekly for 5 weeks). During the treatment period, tumor volumes were monitored twice weekly and tumor volume was calculated according to the following formula: ½ (length × width × height). Mice were either sacrificed before or on day 35 after the first treatment due to having tumors that reached the upper limit for acceptable size according to the criteria of the IACUC or euthanized on day 42 because of planned termination of the experiment. Tumors were removed and immediately snap-frozen for RNA isolation or Western blot analysis.

TUNEL assay

Apoptosis was detected on 4-μm-thick formalin-fixed, paraffin-embedded tumor specimens using the TUNEL Assay Kit (Roche) following the manufacturer's protocol. In brief, paraffin sections were dewaxed in xylene, rehydrated in serially graded ethanol steps, and treated with proteinase K and H2O2. After washing with PBS, the slides were incubated in buffer containing TUNEL-peroxidase for 1 hour at 37°C. The reaction was stopped by rinsing the slides in stop wash buffer. The sections were then incubated with 3,3′-diaminobenzidine solution. After counterstaining with hematoxylin, the sections were covered and apoptosis was assessed by light microscopic examination. Apoptosis was quantitated by measuring the TUNEL-positive areas in three randomly chosen fields in each of three tumors.

miR-124 inhibits proliferation of prostate cancer cells alone or in combination with ARSIs

Our previous studies revealed that miR-124 was downregulated in clinical prostate cancer samples and lentivirally expressed miR-124 (lenti-miR-124) inhibited growth of prostate cancer cells, indicating that miR-124 acts as a tumor suppressor (16). Hence, to explore miR-124 as a potential therapeutic modality, we determined whether synthetic miR-124 is able to decrease resistance of prostate cancer cells to enzalutamide. For this purpose, both androgen-independent 22Rv1 and C4-2B cells grown in androgen-deprived medium and androgen-dependent LNCaP cells in FBS medium were treated with miR-124 and enzalutamide, alone or in combination. As shown in Fig. 1A–C, combination treatment resulted in significant inhibition of proliferation compared with the single-agent treatment (P < 0.01). In addition, C4-2B cells were treated with miR-124 and bicalutamide. Similarly, the combination treatment significantly increased growth inhibition of C4-2B cells (P < 0.01, Fig. 1D), which is accompanied with obvious cell morphologic changes, characterized by cellular shrinking or displaying an appearance of dying cells (Supplementary Fig. S1). These in vitro data provide evidence that miR-124 increases therapeutic efficacy of ARSIs.

Figure 1.

WST-1 analyses of proliferation of prostate cancer cells. 22Rv1 cells (A) and C4-2B cells (B) grown in androgen-deprived medium, and LNCaP cells (C) grown in FBS medium were treated with miR-124 and enzalutamide (Enz), alone or in combination. D, C4-2B cells grown in androgen-deprived medium were treated with miR-124 and bicalutamide (Bic), alone or in combination. These experiments were repeated three times with similar results obtained each time. The representative results are shown as mean ± SD (n = 3). The bars represent SDs. untreat., untreated; miR-NC, miRNA-negative control; both, combination of miR-124 and enzalutamide.

Figure 1.

WST-1 analyses of proliferation of prostate cancer cells. 22Rv1 cells (A) and C4-2B cells (B) grown in androgen-deprived medium, and LNCaP cells (C) grown in FBS medium were treated with miR-124 and enzalutamide (Enz), alone or in combination. D, C4-2B cells grown in androgen-deprived medium were treated with miR-124 and bicalutamide (Bic), alone or in combination. These experiments were repeated three times with similar results obtained each time. The representative results are shown as mean ± SD (n = 3). The bars represent SDs. untreat., untreated; miR-NC, miRNA-negative control; both, combination of miR-124 and enzalutamide.

Close modal

miR-124 restores the response of prostate cancer cells to enzalutamide

In our previous study, we found that miR-124 directly targets full-length AR and downregulates the level of truncated ARs (16) that are derived from AR transcript splice variants (18). Because expression of AR variants mediates the development of resistance to enzalutamide (19), we tested whether miR-124 is able to reduce resistance of prostate cancer cells to enzalutamide. We established an enzalutamide-resistant subline (22Rv1-EnzR) that displays an equivalent growth rate as the parental 22Rv1 cells (data not shown), and an 8-fold increase in the expression of AR-regulated miR-125b (Supplementary Fig. S2A). As miR-124 downregulates miR-125b (16), we first evaluated the effect of miR-124 on miR-125b levels. Treatment with miR-124 induced a 52% reduction of miR-125b (Supplementary Fig. S2B). The effect of miR-124 on proliferation of 22Rv1-EnzR cells was then assessed. Consistent with the results for the parental 22Rv1 cell line, in which miR-124 reduced clonogenic cells by 43%, treatment of 22Rv1-EnzR cells with miR-124 resulted in a clonogenic inhibition by 36% when compared with the miR-NC control (P < 0.01, Fig. 2A and B). Next, we tested whether miR-124 was able to decrease the resistance of 22Rv1-EnzR cells to enzalutamide. In this regard, 22Rv1-EnzR cells were first transfected with miR-124 for 3 days followed by enzalutamide treatment for 5 days. It was found that miR-124 treatment significantly increased the efficacy of enzalutamide (10% in enzalutamide vs. 68% in enzalutamide + miR-124, P < 0.01; Fig. 2C). Data shown in Fig. 2 strongly suggest that miR-124 is able to restore the responsiveness of 22Rv1-EnzR cells to enzalutamide.

Figure 2.

The inhibitory effect of miR-124 on colony formation in enzalutamide-resistant 22Rv1 (22Rv1-EnzR) cells. A, influence of miR-124 on colony-forming cells, as evaluated by quantitative clonogenic assay. B, representative dishes are shown for colony-forming assays of 22Rv1 cells (left) and 22Rv1-EnzR cells (right). C, miR-124 decreases the resistance of 22Rv1-EnzR cells to enzalutamide (Enz). The clonogenic assays were repeated three times, with similar results obtained each time. The results are shown as mean ± SE (n = 3). *, P < 0.01, miR-124 treatment versus miRNA-negative control (miR-NC) treatment. untreat., untreated.

Figure 2.

The inhibitory effect of miR-124 on colony formation in enzalutamide-resistant 22Rv1 (22Rv1-EnzR) cells. A, influence of miR-124 on colony-forming cells, as evaluated by quantitative clonogenic assay. B, representative dishes are shown for colony-forming assays of 22Rv1 cells (left) and 22Rv1-EnzR cells (right). C, miR-124 decreases the resistance of 22Rv1-EnzR cells to enzalutamide (Enz). The clonogenic assays were repeated three times, with similar results obtained each time. The results are shown as mean ± SE (n = 3). *, P < 0.01, miR-124 treatment versus miRNA-negative control (miR-NC) treatment. untreat., untreated.

Close modal

Identification of miR-124 targets

To further understand the role of miR-124 in prostate cancer, we performed miR-124 target prediction using the BIBISERV and TargetScan programs and focused on prostate cancer metastasis- and castration resistance–related genes. Three genes were predicted to be potential miR-124 targets on the basis of the presence of miR-124binding sites in their 3′UTRs: EZH2, Src, and STAT3, which function as oncogenes and contribute to metastasis and castration resistance of prostate cancer. To validate the in silico predictions, luciferase assays of their 3′UTRs were completed. Cotransfection of a luciferase-EZH2 3′UTR reporter and synthetic miR-124 (50 nmol/L) in C4-2B cells resulted in a reduction of luciferase activity by 46% (Fig. 3A). Similarly, cotransfection of the luciferase-Src 3′UTR reporter and synthetic miR-124 induced a 42% decrease of the enzyme activity (Fig. 3B) when compared with transfection with the negative control (miR-NC). Similar results were obtained in PC3 cells (data not shown). We also examined the levels of EZH2 and total Src proteins in miR-124–transfected 22Rv1 and C4-2B cells and found that miR-124 downregulated these two oncogenic proteins (Fig. 3C and D). Although STAT3 was also identified as a high potential target of miR-124, experimental data did not support this, at least in the context of prostate cancer cells tested (data not shown).

Figure 3.

Validation of EZH2 and Src as miR-124 targets. A and B, luciferase reporter assay analyses of the 3′UTRs of EZH2 and Src in C4-2B cells. The top sequences are the miR-124 seed sequence and the predicted miR-124–binding sites in the EZH2 and Src 3′UTR target regions. The 3′UTRs of EZH2 and Src lacking the miR-124 binding site were used as controls in these experiments. The assays were repeated three times, with each assay being performed in triplicate wells and similar results being obtained each time. Representative results are shown as mean ± SD (n = 3). MBS, miR-124–binding site; miR-NC, miRNA-negative control; RLU, relative luciferase units. The numbers (25 and 50) are the concentrations (nmol/L) of miR-124 used in these assays. C and D, Western blot analyses of EZH2 and Src expression levels in C4-2B cells, treated, and untreated controls.

Figure 3.

Validation of EZH2 and Src as miR-124 targets. A and B, luciferase reporter assay analyses of the 3′UTRs of EZH2 and Src in C4-2B cells. The top sequences are the miR-124 seed sequence and the predicted miR-124–binding sites in the EZH2 and Src 3′UTR target regions. The 3′UTRs of EZH2 and Src lacking the miR-124 binding site were used as controls in these experiments. The assays were repeated three times, with each assay being performed in triplicate wells and similar results being obtained each time. Representative results are shown as mean ± SD (n = 3). MBS, miR-124–binding site; miR-NC, miRNA-negative control; RLU, relative luciferase units. The numbers (25 and 50) are the concentrations (nmol/L) of miR-124 used in these assays. C and D, Western blot analyses of EZH2 and Src expression levels in C4-2B cells, treated, and untreated controls.

Close modal

Previous study demonstrated that miR-124 downregulated truncated AR (16), suggesting a regulatory link between miR-124 and AR splice variants that lack the AR ligand–binding domain (LBD). In this study, we observed an increased expression of AR-V7 in 22Rv1-EnzR cells compared with 22Rv1 cells (Fig. 4A). We thus asked whether miR-124 directly targets AR-V7. Using the BIBISERV program, we analyzed the 3′UTR sequences of five AR variants deposited in the NCBI database: AR-V1 (also termed AR4, GI:224181615), AR-V3 (also termed AR6 or AR1/2/2b, GI:224181621), AR-V4 (also termed AR5, ARV6 or AR1/2/3/2b, GI:224181619), AR-V7 (also termed AR3, GI:224181613), and AR-V12 (also termed ARv567es, GI:270358641). One miR-124binding site was identified in the 3′UTRs of AR-V3, V4, and V7, which contain an identical sequence of 1.3 kb in length (Fig. 4B, top). However, an miR-124binding site was not identified in AR-V1, or in AR-V12 in which the 3′UTR length is 219 bases without a poly-A tail, indicating an incomplete 3′UTR. To validate this 3′UTR element in AR-V3, -V4, and -V7 as being responsible for their regulation by miR-124, a luciferase reporter vector containing the miR-124binding site was cotransfected with synthetic miR-124 into 22Rv1 cells. As shown in Fig. 4B (bottom), cotransfection resulted in a 62% reduction of the enzyme activity. Similar results were obtained when the assay was performed in 293 cells (data not shown). Using immunoblot analysis with the anti-AR-V7 antibody, we evaluated the effect of miR-124 on the regulation of AR-V7 protein level. Transfection of 22Rv1 cells with miR-124 for 3 days downregulated the expression of AR-V7 by 60%, whereas enzalutamide treatment for 3 days did not alter AR-V7 level (Fig. 4C). In addition, immunostaining of miR-124–treated 22Rv1-EnzR cells for AR-V7 expression demonstrated a dramatic reduction of AR-V7 intensity (Supplementary Fig. S3). Because AR-V3 lacks exon 3 that is necessary for the AR binding to DNA, transactivational function of AR-V3, -V4, and -V7 was assessed using a yeast AR functional assay that can be used to detect constitutive activity of AR spliced variants (Supplementary Fig. S4A and S4B). Both AR-V4 and -V7, but not AR-V3, were able to constitutively activate the expression of the reporter gene ADE2 (Supplementary Fig. S4C), suggesting that miR-124–regulated AR-V4 and -V7, or lack thereof, contribute to prostate cancer pathogenesis. Next, we determined whether miR-124 inhibits growth of prostate cancer cells via its regulation of AR-V7. To this end, both 22Rv1 and VCaP cell lines that express high level of AR-V7 were transfected with 100 nmol/L siAR-V7 for 3 days, followed by treatment with 50 nmol/L miR-124. Western blotting demonstrated that siAR-V7 was able to dramatically downregulate the expression of AR-V7 protein but not the level of full-length AR (Fig. 4D). WST proliferation analysis was then performed on days 3 and 6 after miR-124 treatment. It was found that knockdown of AR-V7 truncates miR-124–mediated growth inhibition in both 22Rv1 and VCaP cell lines (Fig. 4E). Taking the results from Figs. 3 and 4 together, miR-124 directly targets EZH2 and Src, as well as AR-V4 and -V7.

Figure 4.

Validation of AR-V4 and AR-V7 as miR-124 targets. A, Western blot analysis of AR-V7 expression level in 22Rv1 and enzalutamide-resistant 22Rv1 (22Rv1-EnzR) cells. B, top, map of AR-V3, -V4, & -V7 3′UTRs (black) with exon 2 (E2) and/or exon 3 (E3; white) plus an identical miR-124–binding site in their 3′UTRs and the miR-124 seed sequence. TGA is the stop codon. Bottom, luciferase reporter assay analysis of the 3′UTR of AR-V7 in 22Rv1 cells. The assay was repeated three times, with each assay being performed in three wells and similar results were obtained each time. Representative results are shown as mean ± SD (n = 3). MBS, miR-124–binding site; miR-NC, miRNA-negative control; RLU, relative luciferase units. C, Western blot analysis of AR-V7 expression in miR-124–treated 22Rv1 cells. Enz, enzalutamide. D, Western blot analysis of AR-V7 expression in siAR-V7 (siV7)-treated 22Rv1 and VCaP cells. E, WST proliferation analyses of 22Rv1 and VCaP cell lines that were treated with siAR-V7 for 3 days, followed by treatment with 50 nmol/L miR-124.

Figure 4.

Validation of AR-V4 and AR-V7 as miR-124 targets. A, Western blot analysis of AR-V7 expression level in 22Rv1 and enzalutamide-resistant 22Rv1 (22Rv1-EnzR) cells. B, top, map of AR-V3, -V4, & -V7 3′UTRs (black) with exon 2 (E2) and/or exon 3 (E3; white) plus an identical miR-124–binding site in their 3′UTRs and the miR-124 seed sequence. TGA is the stop codon. Bottom, luciferase reporter assay analysis of the 3′UTR of AR-V7 in 22Rv1 cells. The assay was repeated three times, with each assay being performed in three wells and similar results were obtained each time. Representative results are shown as mean ± SD (n = 3). MBS, miR-124–binding site; miR-NC, miRNA-negative control; RLU, relative luciferase units. C, Western blot analysis of AR-V7 expression in miR-124–treated 22Rv1 cells. Enz, enzalutamide. D, Western blot analysis of AR-V7 expression in siAR-V7 (siV7)-treated 22Rv1 and VCaP cells. E, WST proliferation analyses of 22Rv1 and VCaP cell lines that were treated with siAR-V7 for 3 days, followed by treatment with 50 nmol/L miR-124.

Close modal

miR-124 inhibits growth of prostate cancer xenografts and sensitizes prostate cancer tumors to enzalutamide

Having validated that miR-124 negatively regulates the expression of AR variants and multiple oncogenes, we next investigated whether systemic administration of synthetic miR-124 could inhibit growth of CWR22 xenograft tumors. This xenograft model was selected as (i) we detected the expression of AR-V7 in frozen CWR22 tumors (data not shown) and (ii) Sirotnak and colleagues found that the antiandrogen bicalutamide does not inhibit the growth of the CWR22 tumors (20). In this experiment, miR-124 was delivered into CWR22 tumors by using the linear polyethylenimine derivative jetPEI that has been reported to efficiently deliver miRNAs into tumor xenografts (21). In the mice of the negative control treatment group (miR-NC), tumors rapidly grew and all mice were sacrificed before day 35 due to tumors reaching the maximal-acceptable size. In contrast, treatment with miR-124 significantly inhibited tumor growth, with almost a 50% reduction of tumor volume at day 35 compared with that of miR-NC–treated tumors (P < 0.05, Fig. 5A and B). We next determined whether combined administration of miR-124 and enzalutamide would result in enhanced inhibition of prostate cancer cell growth. When compared with enzalutamide treatment, combination treatment inhibited tumor growth by 54% on day 35 posttreatment (P < 0.05, Fig. 5A and B), indicating that miR-124 increased anti–prostate cancer efficacy of enzalutamide. In addition, there were no safety concerns of PEI-miR-124 complexes based on observations that mouse body weights were unchanged throughout the course of the study (data not shown). Therefore, synthetic miR-124 is able to inhibit growth of prostate cancer tumors when given by itself or in combination with enzalutamide.

Figure 5.

Systemic administration of synthetic miR-124 increases the inhibitory efficacy of enzalutamide on growth of prostate cancer xenografts. A, each nude mice was injected subcutaneously with CWR22 cell suspension (∼2 × 106 cells). When tumor volumes reached ∼50 mm3, treatment was conducted by intravenous injection of 10 μg jetPEI/miR-NC or 10 μg jetPEI/miR-124 complexes (three times weekly for 5 weeks) or by oral administration of enzalutamide (20 mg/kg/wk, once weekly for 5 weeks). Tumor sizes were measured and tumor growth curves were obtained. Each time point represents mean ± SD of 8 independent values. B, representative results of mice treated with miR-124 and enzalutamide (Enz), alone or in combination.

Figure 5.

Systemic administration of synthetic miR-124 increases the inhibitory efficacy of enzalutamide on growth of prostate cancer xenografts. A, each nude mice was injected subcutaneously with CWR22 cell suspension (∼2 × 106 cells). When tumor volumes reached ∼50 mm3, treatment was conducted by intravenous injection of 10 μg jetPEI/miR-NC or 10 μg jetPEI/miR-124 complexes (three times weekly for 5 weeks) or by oral administration of enzalutamide (20 mg/kg/wk, once weekly for 5 weeks). Tumor sizes were measured and tumor growth curves were obtained. Each time point represents mean ± SD of 8 independent values. B, representative results of mice treated with miR-124 and enzalutamide (Enz), alone or in combination.

Close modal

miR-124 downregulates AR-V7, EZH2, and Src and induces apoptosis in CWR22 xenografts

Upon termination of the in vivo experiments described above, immunohistochemistry (IHC) was performed to determine the levels of the AR-V7 protein in the xenograft tumors. Treatment with miR-124 alone or in combination with enzalutamide caused a marked decrease in the immunostaining intensity compared with treatment with miR-NC or enzalutamide alone (Supplementary Fig. S5A). We examined protein levels of AR-V7, EZH2, and total Src in tumor tissues using Western blot analysis. It was found that these three miR-124 targets were significantly downregulated in miR-124 + enzalutamide–treated tumors compared with tumors treated with enzalutamide alone (Fig. 6A). Therefore, combined repression of constitutively active AR variants and downregulation of EZH2 and Src contribute to miR-124–mediated tumor inhibition. The observation of miR-124–induced downregulation of these targets provides evidence that PEI nanoparticles can efficiently deliver miR-124 into prostate cancer xenograft cells.

Figure 6.

Systemic administration of miR-124 downregulates its targets and induces apoptosis. A, protein levels of AR-V7, EZH2, and Src, as well as p53, were detected by Western blot analysis in five enzalutamide (Enz)-treated tumors (left) and five miR-124/enzalutamide–treated tumors (right). β-Actin was used as a loading control. B, TUNEL assays of apoptosis in tumor sections. Apoptosis was measured by TUNEL staining in prostate cancer tumors treated with miR-124 and enzalutamide, alone or in combination. Left, the representative positive TUNEL staining. For quantitation of apoptosis, TUNEL-positive areas were measured using the ImageJ program in three randomly chosen fields from three tumors and expressed as the percentage of apoptosis (mean ± SE, n = 3; right).

Figure 6.

Systemic administration of miR-124 downregulates its targets and induces apoptosis. A, protein levels of AR-V7, EZH2, and Src, as well as p53, were detected by Western blot analysis in five enzalutamide (Enz)-treated tumors (left) and five miR-124/enzalutamide–treated tumors (right). β-Actin was used as a loading control. B, TUNEL assays of apoptosis in tumor sections. Apoptosis was measured by TUNEL staining in prostate cancer tumors treated with miR-124 and enzalutamide, alone or in combination. Left, the representative positive TUNEL staining. For quantitation of apoptosis, TUNEL-positive areas were measured using the ImageJ program in three randomly chosen fields from three tumors and expressed as the percentage of apoptosis (mean ± SE, n = 3; right).

Close modal

We previously observed that miR-124 downregulates miR-125b in cultured prostate cancer cells (16). Herein, the expression of miR-125b was measured by qPCR. Similarly, systemic administration of miR-124 induced a reduction of miR-125b abundance in miR-124–treated tumors (Supplementary Fig. S5C). Because miR-125b targets p53 (22, 23), we evaluated the effects of miR-124 on p53 level. As expected, a profound increase in p53 protein was detected in miR-124–treated tumors (Fig. 6A). Having demonstrated that miR-124 inhibits the growth of prostate cancer xenografts and upregulates p53, we next investigated whether systemic administration of miR-124 induces apoptosis in the mouse tumors. TUNEL staining was performed. Positive staining was rare in tumors treated with miR-NC. Different extents of TUNEL-positive areas were detected in tumors treated with enzalutamide and/or miR-124, particularly in combined treated tumors, in which large TUNEL-positive areas were detected. The nuclei of apoptotic cells are small and contain condensed and/or fragmented chromatins. Representative results are shown in Fig. 6B. Quantitative TUNEL assays demonstrated that the combination of miR-124 and enzalutamide resulted in apoptosis in 37% of the cell population, whereas the single-agent treatment induced only 17% apoptosis (P < 0.05, Fig. 6B).

Tumor-suppressive miR-124 is a highly conserved miRNA and has been reported to be downregulated in various human cancers, including cancers of the breast (24), liver (25), stomach (26), colon (13), and kidney (27), as well as leukemia (28) and glioma (29). This miRNA attracted our attention due to (i) its ability to directly target the AR and subsequently induce downregulation of miR-125b and upregulation of p53 and (ii) its significantly reduced expression levels in prostate cancer (16, 17). In this study, we investigated the growth-inhibitory effects of miR-124 on a variety of prostate cancer cell lines and xenograft tumors. Results from in vitro and in vivo experiments demonstrated that miR-124 significantly inhibited growth of prostate cancer cells and sensitized prostate cancer cells to treatment with ARSIs. Moreover, miR-124 can restore the response of 22Rv1-EnzR cells to enzalutamide. Combined administration of miR-124 and enzalutamide induced increased apoptosis in tumor xenografts. These preclinical findings suggest that miR-124 plays an important role in prostate cancer and can be exploited for adjuvant therapeutic application.

Like most other miRNAs, prostate cancer–related targets of miR-124 have not been validated experimentally. In our earlier report, full-length AR was identified and confirmed as a bona fide target of miR-124 (16). In the present study, EZH2 and Src were validated as targets of miR-124. EZH2 functions not only as a histone methyltransferase, silencing tumor suppressor genes and antimetastatic genes (30), but also as an AR coactivator to support prostate cancer growth (31). Overexpression of EZH2 is a common occurrence in prostate cancer and is associated with a poor clinical outcome of patients with prostate cancer (32). Similarly, Src is highly involved in prostate cancer, and inhibition of Src activity inhibits prostate cancer tumor growth (33, 34). Thus, their activities are required for the development of metastasis and castration resistance of prostate cancer. Perhaps, the most striking finding of this study is the detection of miR-124 target sites at the 3′UTRs of AR-V4 and AR-V7 transcripts. AR-V7 is the most studied AR variant. Its expression was obviously enhanced in CRPC cells due to intragenic AR gene rearrangement and altered RNA splicing (35, 36). In this study, restoration of miR-124 level led to significant downregulation of AR-V7 protein in xenograft tumors. The potential mechanisms for decreasing the levels of AR-V7 protein include: (i) miR-124 directly binds to the 3′UTR of AR-V7, leading to degradation of AR-V7 mRNA and inhibition of AR-V7 translation; (ii) miR-124 directly represses the full-length AR (16), whereas AR-V7 RNA splicing is coupled with the full-length AR gene transcription (35). Therefore, miR-124–induced downregulation of the full-length AR accompanies a reduction of AR-V7; and (iii) RNA splicing factor ASF that is a potential miR-124 target is specifically important for AR-V7 splicing (35). Taken together, the finding that miR-124 mediates downregulation of at least three oncoproteins relevant to prostate cancer progression and therapy resistance provides a firm molecular foundation for using miR-124 as novel adjunctive therapeutic agent.

Data presented in this study strongly suggest that miR-124 holds great promise as a therapeutic target. In our previous study, lentivirally expressed miR-124 exhibited significant growth inhibition of prostate cancer tumors (16). Although lentiviral vectors are efficient delivery agents, virus-based miRNA therapy may raise serious safety problems related to toxicity and immunogenicity. In this study, synthetic miR-124 mimics were used because they have the same physical and chemical characters as endogenous miR-124, which avoids many potential safety risks. Like other nucleic acid–based therapies, a major challenge is encountered for the delivery of miR-124 into prostate cancer cells. In this study, we elected to use PEI-based delivery, which has a considerable advantage over lentiviral-based vectors and has been widely studied for gene delivery. The PEI nanoparticles, or “polyplexes,” used in this study are composed of linear polymers that have sufficient stability, favorable pharmacokinetic properties, and higher biocompatibility (e.g., low toxicity relative to virus-based vectors). Under physiologic conditions, PEI molecules are partially protonated and easily form complexes with miRNA. After systemic injection, PEI–miRNA complexes interact with the tumor cell surface and enter cells by endocytosis (37). Notably, PEI increases internalization and facilitates the release of internalized miRNAs from endosomes due to a so-called “proton sponge effect” (21). Our data obtained in animal experiments demonstrated that PEI is able to efficiently deliver miR-124 to prostate cancer xenografts, which is shown by PEI–miR-124 complex–induced antitumor effects and the inhibition of several specific targets. Although PEI as a delivery agent still has limitations (e.g., lower efficiency relative to virus-based vectors), the observed antitumor effects and lack of unwanted side effects suggest that this method of systemic delivery might be more clinically relevant and worthwhile to pursue.

Treatment of advanced prostate cancer has proven extremely challenging, especially in light of the fact that the newest ARSI enzalutamide provides only a modest improvement in survival of patients. Thus, while we search for new agents, improving response to presently available agents could be beneficial in lowering mortality in this disease. Because one miRNA can target multiple cancer-related genes or pathways, combined use of miRNA and enzalutamide may represent a novel and viable therapeutic strategy to prevent or delay recurrence of castration resistance. Over the last several years, we have investigated the role of miRNA in prostate cancer and discovered several miRNAs (miR-124, miR-125b, miR-30, and let-7c) that contribute to the AR signaling network. Using the data presented in this study combined with those in our previous publications (23, 38–41), an miR-124–regulated oncopathway is depicted in Fig. 7. This oncopathway regulates several proapoptotic genes and oncogenes and miR-124 is a key regulator. In this study, we targeted this oncopathway by using miR-124 and enzalutamide. Indeed, we observed that combination treatment inhibited proliferation of cultured CRPC cells in an additive manner and caused significantly greater antitumor activity in mice, when compared with the single treatment. Therefore, our results establish miR-124 as a prostate cancer–relevant miRNA with the promise of being an adjuvant therapeutic agent. Even so, the question can be asked, what will be the mechanism of resistance of CRPC against miR-124? One potential mechanism is through altering its AR-V pattern. On the basis of the fact that AR-V12 is constituently active in prostate cancer and that the 3′UTR in AR-V12 is different from AR-V7 (42), it will be a prime target for bypassing miR-124. Future studies to define the mechanism of resistance need to be conducted.

Figure 7.

Schematic model of miR-124–regulated oncopathway in prostate cancer cells. Data presented in this study and in our previous publications support a miR-124-AR/AR-Vs-miR-125b oncopathway in prostate cancer cells.

Figure 7.

Schematic model of miR-124–regulated oncopathway in prostate cancer cells. Data presented in this study and in our previous publications support a miR-124-AR/AR-Vs-miR-125b oncopathway in prostate cancer cells.

Close modal

In summary, we found that tumor-suppressive miR-124 is an important modifier of an oncopathway in prostate cancer that regulates the expression of EZH2, Src, and AR variants that contribute to pathogenesis and treatment resistance of prostate cancer and that restoration of miR-124 inhibited the growth of enzalutamide-resistant prostate cancer cells and sensitized these cells to enzalutamide. Importantly, we observed that PEI-mediated systemic administration of synthetic miR-124 inhibited the growth of androgen-dependent and -independent and enzalutamide-resistant prostate cancer cells. Therefore, our data presented in this study suggest that miR-124–based therapies have great potential in the design of combination therapy for prostate cancer treatment.

J.C. Yang reports receiving commercial research grant from Medivation. C.P. Evans reports receiving commercial research grant and speakers bureau honoraria from Medivation/Astellas; has ownership interest (including patents) in Medivation; and is also a consultant/advisory board member for Medivation/Astellas. No potential conflicts of interest were disclosed by the other authors.

Conception and design: X.-B. Shi, A.-H. Ma, L. Xue, H.G. Nguyen, H.-J. Kung

Development of methodology: X.-B. Shi, A.-H. Ma, L. Xue, M. Li, H.G. Nguyen

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): X.-B. Shi, A.-H. Ma, L. Xue, M. Li, H.G. Nguyen, R. Gandour-Edwards

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): X.-B. Shi, A.-H. Ma, L. Xue, M. Li, H.G. Nguyen, C.G. Tepper, C.P. Evans

Writing, review, and/or revision of the manuscript: X.-B. Shi, A.-H. Ma, C.G. Tepper, C.P. Evans, H.-J. Kung

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): X.-B. Shi, A.-H. Ma, J.C. Yang

Study supervision: X.-B. Shi, A.-H. Ma

The authors thank Dr. Melanie C. Bradnam for her editorial assistance.

This work was supported by the Stand Up To Cancer-Prostate Cancer Foundation-Prostate Dream Team Translational Cancer Research Grant SU2C-AACR-PCF DT0812. This research grant is made possible by the generous support of the Movember Foundation. Stand Up To Cancer is a program of the Entertainment Industry Foundation administered by the American Association for Cancer Research. This work was also supported in part by funding from NIH R01CA136597, Department of Defense grants PC080488 and PC111467, and Medivation/Astellas.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Siegel
R
,
Ma
J
,
Zou
Z
,
Jemal
A
. 
Cancer statistics, 2014
.
CA Cancer J Clin
2014
;
64
:
9
29
.
2.
Wu
JN
,
Fish
KM
,
Evans
CP
,
Devere White
RW
,
Dall'era
MA
. 
No improvement noted in overall or cause-specific survival for men presenting with metastatic prostate cancer over a 20-year period
.
Cancer
2014
;
120
:
818
23
.
3.
Egan
A
,
Dong
Y
,
Zhang
H
,
Qi
Y
,
Balk
SP
,
Sartor
O
. 
Castration-resistant prostate cancer: adaptive responses in the androgen axis
.
Cancer Treat Rev
2014
;
40
:
426
33
.
4.
Polkinghorn
WR
,
Parker
JS
,
Lee
MX
,
Kass
EM
,
Spratt
DE
,
Iaquinta
PJ
, et al
Androgen receptor signaling regulates DNA repair in prostate cancers
.
Cancer Discov
2013
;
3
:
1245
53
.
5.
Scher
HI
,
Fizazi
K
,
Saad
F
,
Taplin
ME
,
Sternberg
CN
,
Miller
K
, et al
Increased survival with enzalutamide in prostate cancer after chemotherapy
.
N Engl J Med
2012
;
367
:
1187
97
.
6.
Tran
C
,
Ouk
S
,
Clegg
NJ
,
Chen
Y
,
Watson
PA
,
Arora
V
, et al
Development of a second-generation antiandrogen for treatment of advanced prostate cancer
.
Science
2009
;
324
:
787
90
.
7.
Ning
YM
,
Pierce
W
,
Maher
VE
,
Karuri
S
,
Tang
SH
,
Chiu
HJ
, et al
Enzalutamide for treatment of patients with metastatic castration-resistant prostate cancer who have previously received docetaxel: U.S. Food and Drug Administration drug approval summary
.
Clin Cancer Res
2013
;
19
:
6067
73
.
8.
Nelson
WG
,
Yegnasubramanian
S
. 
Resistance emerges to second-generation antiandrogens in prostate cancer
.
Cancer Discov
2013
;
3
:
971
4
.
9.
Friedman
RC
,
Farh
KK
,
Burge
CB
,
Bartel
DP
. 
Most mammalian mRNAs are conserved targets of microRNAs
.
Genome Res
2009
;
19
:
92
105
.
10.
Thomson
DW
,
Bracken
CP
,
Goodall
GJ
. 
Experimental strategies for microRNA target identification
.
Nucleic Acids Res
2011
;
39
:
6845
53
.
11.
Cortez
MA
,
Ivan
C
,
Zhou
P
,
Wu
X
,
Ivan
M
,
Calin
GA
. 
microRNAs in cancer: from bench to bedside
.
Adv Cancer Res
2010
;
108
:
113
57
.
12.
Bader
AG
,
Brown
D
,
Winkler
M
. 
The promise of microRNA replacement therapy
.
Cancer Res
2010
;
70
:
7027
30
.
13.
Lujambio
A
,
Ropero
S
,
Ballestar
E
,
Fraga
MF
,
Cerrato
C
,
Setien
F
, et al
Genetic unmasking of an epigenetically silenced microRNA in human cancer cells
.
Cancer Res
2007
;
67
:
1424
9
.
14.
Agirre
X
,
Vilas-Zornoza
A
,
Jimenez-Velasco
A
,
Martin-Subero
JI
,
Cordeu
L
,
Garate
L
, et al
Epigenetic silencing of the tumor suppressor microRNA Hsa-miR-124a regulates CDK6 expression and confers a poor prognosis in acute lymphoblastic leukemia
.
Cancer Res
2009
;
69
:
4443
53
.
15.
Mitchell
PS
,
Parkin
RK
,
Kroh
EM
,
Fritz
BR
,
Wyman
SK
,
Pogosova-Agadjanyan
EL
, et al
Circulating microRNAs as stable blood-based markers for cancer detection
.
Proc Natl Acad Sci U S A
2008
;
105
:
10513
8
.
16.
Shi
XB
,
Xue
L
,
Ma
AH
,
Tepper
CG
,
Gandour-Edwards
R
,
Kung
HJ
, et al
Tumor suppressive miR-124 targets androgen receptor and inhibits proliferation of prostate cancer cells
.
Oncogene
2013
;
32
:
4130
8
.
17.
Hellwinkel
OJ
,
Sellier
C
,
Sylvester
YM
,
Brase
JC
,
Isbarn
H
,
Erbersdobler
A
, et al
A cancer-indicative microRNA pattern in normal prostate tissue
.
Int J Mol Sci
2013
;
14
:
5239
49
.
18.
Marcias
G
,
Erdmann
E
,
Lapouge
G
,
Siebert
C
,
Barthelemy
P
,
Duclos
B
, et al
Identification of novel truncated androgen receptor (AR) mutants including unreported pre-mRNA splicing variants in the 22Rv1 hormone-refractory prostate cancer (PCa) cell line
.
Hum Mutat
2010
;
31
:
74
80
.
19.
Li
Y
,
Chan
SC
,
Brand
LJ
,
Hwang
TH
,
Silverstein
KA
,
Dehm
SM
. 
Androgen receptor splice variants mediate enzalutamide resistance in castration-resistant prostate cancer cell lines
.
Cancer Res
2013
;
73
:
483
9
.
20.
Sirotnak
FM
,
She
Y
,
Lee
F
,
Chen
J
,
Scher
HI
. 
Studies with CWR22 xenografts in nude mice suggest that ZD1839 may have a role in the treatment of both androgen-dependent and androgen-independent human prostate cancer
.
Clin Cancer Res
2002
;
8
:
3870
6
.
21.
Ibrahim
AF
,
Weirauch
U
,
Thomas
M
,
Grunweller
A
,
Hartmann
RK
,
Aigner
A
. 
MicroRNA replacement therapy for miR-145 and miR-33a is efficacious in a model of colon carcinoma
.
Cancer Res
2011
;
71
:
5214
24
.
22.
Le
MT
,
Teh
C
,
Shyh-Chang
N
,
Xie
H
,
Zhou
B
,
Korzh
V
, et al
MicroRNA-125b is a novel negative regulator of p53
.
Genes Dev
2009
;
23
:
862
76
.
23.
Shi
XB
,
Xue
L
,
Ma
AH
,
Tepper
CG
,
Kung
HJ
,
White
RW
. 
miR-125b promotes growth of prostate cancer xenograft tumor through targeting pro-apoptotic genes
.
Prostate
2011
;
71
:
538
49
.
24.
Liang
YJ
,
Wang
QY
,
Zhou
CX
,
Yin
QQ
,
He
M
,
Yu
XT
, et al
MiR-124 targets Slug to regulate epithelial-mesenchymal transition and metastasis of breast cancer
.
Carcinogenesis
2013
;
34
:
713
22
.
25.
Schwabe
RF
,
Wang
TC
. 
Targeting liver cancer: first steps toward a miRacle?
Cancer Cell
2011
;
20
:
698
9
.
26.
Xia
J
,
Wu
Z
,
Yu
C
,
He
W
,
Zheng
H
,
He
Y
, et al
miR-124 inhibits cell proliferation in gastric cancer through down-regulation of SPHK1
.
J Pathol
2012
;
227
:
470
80
.
27.
Gebauer
K
,
Peters
I
,
Dubrowinskaja
N
,
Hennenlotter
J
,
Abbas
M
,
Scherer
R
, et al
Hsa-mir-124-3 CpG island methylation is associated with advanced tumours and disease recurrence of patients with clear cell renal cell carcinoma
.
Br J Cancer
2013
;
108
:
131
8
.
28.
Vazquez
I
,
Maicas
M
,
Marcotegui
N
,
Conchillo
A
,
Guruceaga
E
,
Roman-Gomez
J
, et al
Silencing of hsa-miR-124 by EVI1 in cell lines and patients with acute myeloid leukemia
.
Proc Natl Acad Sci U S A
2010
;
107
:
E167
8
;
author reply E9–70
.
29.
Wei
J
,
Wang
F
,
Kong
LY
,
Xu
S
,
Doucette
T
,
Ferguson
SD
, et al
miR-124 inhibits STAT3 signaling to enhance T cell-mediated immune clearance of glioma
.
Cancer Res
2013
;
73
:
3913
26
.
30.
Yang
YA
,
Yu
J
. 
EZH2, an epigenetic driver of prostate cancer
.
Protein Cell
2013
;
4
:
331
41
.
31.
Xu
K
,
Wu
ZJ
,
Groner
AC
,
He
HH
,
Cai
C
,
Lis
RT
, et al
EZH2 oncogenic activity in castration-resistant prostate cancer cells is Polycomb-independent
.
Science
2012
;
338
:
1465
9
.
32.
Varambally
S
,
Dhanasekaran
SM
,
Zhou
M
,
Barrette
TR
,
Kumar-Sinha
C
,
Sanda
MG
, et al
The polycomb group protein EZH2 is involved in progression of prostate cancer
.
Nature
2002
;
419
:
624
9
.
33.
Cai
H
,
Babic
I
,
Wei
X
,
Huang
J
,
Witte
ON
. 
Invasive prostate carcinoma driven by c-Src and androgen receptor synergy
.
Cancer Res
2011
;
71
:
862
72
.
34.
Yang
JC
,
Ok
JH
,
Busby
JE
,
Borowsky
AD
,
Kung
HJ
,
Evans
CP
. 
Aberrant activation of androgen receptor in a new neuropeptide-autocrine model of androgen-insensitive prostate cancer
.
Cancer Res
2009
;
69
:
151
60
.
35.
Liu
LL
,
Xie
N
,
Sun
S
,
Plymate
S
,
Mostaghel
E
,
Dong
X
. 
Mechanisms of the androgen receptor splicing in prostate cancer cells
.
Oncogene
2014
;
33
:
3140
50
.
36.
Li
Y
,
Alsagabi
M
,
Fan
D
,
Bova
GS
,
Tewfik
AH
,
Dehm
SM
. 
Intragenic rearrangement and altered RNA splicing of the androgen receptor in a cell-based model of prostate cancer progression
.
Cancer Res
2011
;
71
:
2108
17
.
37.
Zhang
Y
,
Wang
Z
,
Gemeinhart
RA
. 
Progress in microRNA delivery
.
J Control Release
2013
;
172
:
962
74
.
38.
Shi
XB
,
Xue
L
,
Yang
J
,
Ma
AH
,
Zhao
J
,
Xu
M
, et al
An androgen-regulated miRNA suppresses Bak1 expression and induces androgen-independent growth of prostate cancer cells
.
Proc Natl Acad Sci U S A
2007
;
104
:
19983
8
.
39.
Amir
S
,
Ma
AH
,
Shi
XB
,
Xue
L
,
Kung
HJ
,
Devere White
RW
. 
Oncomir miR-125b suppresses p14(ARF) to modulate p53-dependent and p53-independent apoptosis in prostate cancer
.
PLoS One
2013
;
8
:
e61064
.
40.
Kao
CJ
,
Martiniez
A
,
Shi
XB
,
Yang
J
,
Evans
CP
,
Dobi
A
, et al
miR-30 as a tumor suppressor connects EGF/Src signal to ERG and EMT
.
Oncogene
2014
;
33
:
2495
503
.
41.
Nadiminty
N
,
Tummala
R
,
Lou
W
,
Zhu
Y
,
Shi
XB
,
Zou
JX
, et al
MicroRNA let-7c is downregulated in prostate cancer and suppresses prostate cancer growth
.
PLoS One
2012
;
7
:
e32832
.
42.
Hu
R
,
Isaacs
WB
,
Luo
J
. 
A snapshot of the expression signature of androgen receptor splicing variants and their distinctive transcriptional activities
.
Prostate
2011
;
71
:
1656
67
.