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Introduction

Prostate cancer (PCa) is the most frequently diagnosed cancer among men in developed countries (1). Although PCa is initially responsive to androgen-deprivation therapy (ADT), most patients experience disease relapse and develop castration-resistant prostate cancer (CRPC) (2). The survival rate of patients with CRPC is poor owing to the occurrence of metastasis (3) and patients with metastatic CRPC cannot be effectively treated using recently developed molecular-targeted therapies (2,3). Therefore, new treatment strategies are needed for these patients. The molecular mechanisms underlying the aggressiveness of CRPC cells and the acquisition of treatment resistance are still unclear.

MicroRNAs (miRNAs) are noncoding RNAs that act as sequence-specific fine tuners for regulating the expression levels of proteins and RNAs (4,5). Notably, a single miRNA can regulate a large number of RNA transcripts in human cells (6). Accordingly, dysregulation of miRNAs can disrupt the tightly regulated RNA networks in cancer cells, leading to cancer cell initiation, development, metastasis, and drug resistance (7,8). The discovery of miRNAs has complicated the analysis of intracellular RNA networks in cancer.

We recently constructed an RNA-sequence based miRNA expression signature using autopsy specimens from patients with CRPC (9). To elucidate the miRNA-mediated RNA networks in metastatic CRPC, we have sequentially identified antitumor miRNAs and oncogenic genes regulated by these miRNAs based on our miRNA expression signatures (9,10). Analysis of our miRNA signatures, including that in CRPC, has shown that both strands of pre-miR-150, i.e., miR-150-5p (the guide strand) and miR-150-3p (the passenger strand), are significantly reduced in cancer tissues (9).

In miRNA biogenesis, pre-miRNA is cleaved by Dicer into the miRNA duplex, containing the guide and passenger strands. The mature guide strand miRNA is incorporated into the RNA-induced silencing complex (RISC) and represses mRNA translation or cleaves mRNA (11). In contrast, the passenger strand of miRNA was previously thought to be degraded and to have no function (12–14). However, our expression data have contradicted this established miRNA theory. We hypothesized that the passenger strand of miR-150-3p may have functions in naïve PCa and CRPC cells by targeting novel oncogenic genes. Accordingly, in this study, we aimed to investigate the functional significance of miR-150-5p and miR-150-3p in naïve PCa and CRPC cells and to identify oncogenic genes involved in the pathogenesis of the disease.

Materials and methods

Clinical prostate specimens

Clinical prostate specimens were obtained from patients admitted to Teikyo University Chiba Medical Center from 2014 to 2015. All patients had elevated prostate-specific antigen (PSA) and had undergone trans-rectal biopsies for definitive cancer diagnoses. Particularly, in patients with CRPC, neuroendocrine differentiation was excluded. The patient backgrounds are summarized in Table I. Samples were staged according to the UICC TNM classification (15).

Table I Characteristics of patients with non-PCa and naïve PCa and CRPC.

Table I

Characteristics of patients with non-PCa and naïve PCa and CRPC.

No.	Diagnosis	Age (years)	PSA (ng/ml)	Gleason score	T	N	M	Stage
1	Non-PCa	57	5.71	–	–	–	–	–
2	Non-PCa	74	9.45	–	–	–	–	–
3	Non-PCa	70	8.58	–	–	–	–	–
4	Non-PCa	73	4.8	–	–	–	–	–
5	Non-PCa	67	6.91	–	–	–	–	–
6	Non-PCa	50	7.05	–	–	–	–	–
7	Non-PCa	74	9.91	–	–	–	–	–
8	Non-PCa	76	20.9	–	–	–	–	–
9	Non-PCa	59	4.5	–	–	–	–	–
10	Non-PCa	75	1.1	–	–	–	–	–
11	Non-PCa	60	7.29	–	–	–	–	–
12	Non-PCa	73	38.7	–	–	–	–	–
13	Non-PCa	69	11.9	–	–	–	–	–
14	Non-PCa	77	23.3	–	–	–	–	–
15	Non-PCa	61	4.57	–	–	–	–	–
16	Non-PCa	59	7.37	–	–	–	–	–
17	Non-PCa	65	5.06	–	–	–	–	–
18	PCa	70	75.7	4+5	4	1	1	D2
19	PCa	78	1,800	4+5	4	1	1	D2
20	PCa	75	68.4	5+4	4	1	0	D1
21	PCa	62	38.7	4+5	2b	1	0	D1
22	PCa	70	25.5	4+5	3b	0	0	C
23	PCa	75	1,260	4+5	3b	1	1	D2
24	PCa	88	888	4+5	3b	1	1	D2
25	PCa	69	33.9	4+5	4	0	1	D2
26	PCa	62	62.3	4+5	3b	1	0	D1
27	PCa	78	5	4+5	2c	0	1b	D2
28	PCa	64	449	4+5	3b	1	1	D2
29	PCa	81	365	4+5	4	1	1	D2
30	PCa	76	715	5+4	4	1	1	D2
31	PCa	79	555	4+5	3	1	1	D2
32	PCa	63	1,120	4+5	2c	0	1b	D2
33	PCa	67	4.95	4+5	4	1	1b	D2
34	PCa	70	19.5	5+5	4	1	1c	D2
35	CRPC	69	15.8	5+4	3b	1	1	D2
36	CRPC	72	212	5+4	4	1	1	D2
37	CRPC	71	4.4	4+5	4	1	1	D2
38	CRPC	66	295	4+5	4	1	1	D2
39	CRPC	68	7.54	4+5	4	1	1b	D2

All patients in this study provided written informed consent for tissue donation for research purposes. The protocol was approved by the Institutional Review Boards of Chiba University and Teikyo University.

Tissue collection and cell culture

Prostate tissues were immersed in RNAlater (Thermo Fisher Scientific, Waltham, MA, USA) and stored at 4°C until RNA extraction. Human prostate cancer cells (PC3 and PC3M cells) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Quantitative real-time reverse transcription polymerase chain reaction (RT-q-PCR)

Stem-loop RT-PCR (TaqMan MicroRNA assays; product ID: 000473 for miR-150-5p and 002637 for miR-150-3p; Applied Biosystems, Foster City, CA, USA) was used in this assays. TaqMan probes and primers for SPOCK1 (product ID: Hs00270274_m1; Applied Biosystems) were assay-on-demand gene expression products. We used GUSB (product ID: Hs00939627_m1; Applied Biosystems), GAPDH (product ID: Hs02758991_g1; Applied Biosystems), and RNU48 (product ID: 001006; Applied Biosystems) as internal controls.

Cell proliferation, migration, and invasion assays

Cell proliferation, migration, and invasion assays were carried out as previously described (9,10).

Argonaute 2 (AGO2)-bound miRNA isolation by immunoprecipitation

PC3 cells were transfected with 10 nM miRNA by reverse transfection and plated in 10-cm plates at 1×105 cells/ml. After 48 h, immunoprecipitation was performed using a microRNA Isolation kit, Human Ago2 (Wako, Osaka, Japan) according to the manufacturer's protocol. Expression levels of miRNAs bound to Ago2 were measured by TaqMan RT-qPCR. miRNA expression data were normalized to the expression of miR-26a (product ID: 000404; Applied Biosystems), which was not affected by miR-150-5p and miR-150-3p.

Western blot analysis

Immunoblotting was conducted with diluted monoclonal anti-SPOCK1 antibodies (1:100 dilution; sc-398782; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and with diluted anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies (1:1,000 dilution; ab8245; Abcam, Cambridge, UK) as a loading control. The procedures were performed as previously described (16).

Selection of putative target genes regulated by miR-150-5p and miR-150-3p in PCa cells

To identify miR-150-5p and miR-150-3p target genes, we analyzed gene expression profile (Gene Expression Omnibus database; accession no. GSE29079). Gene expression data were obtained from miR-150-5p and miR-150-3p transfectant PC3 cells (A SurePrint G3 Human 60K, Agilent Technologies, Santa Clara, CA, USA). We merged these datasets and selected putative miR-150-5p and miR-150-3p target genes using the TargetScan database (Release 7.1; http://www.targetscan.org/vert_71/).

Plasmid construction and dual-luciferase assays

The partial wide-type sequences of the 3′-untranslated region (UTR) of SPOCK1 or those with a deleted miR-150-5p or miR-150-3p target site were inserted in the psiCHECK-2 vector (C8021; Promega, Madison, WI, USA). Alternatively, we used sequences that were missing the miR-150-5p target site (position 182–188) or miR-150-3p target sites (position 1477–1483, position 1749–1756, or position 2593–2599). The procedure for dual-luciferase reporter assays was described previously (17).

Immunohistochemistry using tissue microarrays

We used a tissue microarray of prostate cancer samples obtained from Provitro (Berlin, Germany; cat. no. 401–2209, Lot no. 146.1P), which contained a total of 78 prostate samples (PCa specimens: n=58, prostatic intraepithelial neoplasia: n=10, and normal prostate samples: n=10). Detailed information on these samples is summarized in Table II. The tissue microarray was immunostained following the manufacturer's protocol, as described previously (10,18).

Table II Characteristics of patients, as evaluated using immunohistochemistry.

Table II

Characteristics of patients, as evaluated using immunohistochemistry.

No.	Diagnosis	Age (years)	Gleason score	T	N	IHC score of SPOCK1
1	PCa	64	4+3	3b	0	5
2	PCa	67	3+4	2b	0	3
3	PCa	58	3+4	2b	0	5
4	PCa	63	7	3b	0	6
5	PCa	65	3+3	2b	0	5
6	PCa	61	4+4	3b	×	4
7	PCa	62	3+4	2b	×	2
8	PCa	66	4+4	2b	×	4
9	PCa	61	3+4	3a	×	4
10	PCa	74	4+3	2b	×	5
11	PCa	59	2+3	2b	×	4
12	PCa	69	3+4	3a	0	4
13	PCa	54	3+4	2c	×	6
14	PCa	68	3+4	3a	0	5
15	PCa	58	3+4	3a	0	5
16	PCa	67	3+4	3a	0	4
17	PCa	65	3+3	2a	0	4
18	PCa	77	3+4	4	0	5
19	PCa	64	4+3	2b	0	4
20	PCa	58	3+4	3a	0	6
21	PCa	50	4+3	2b	0	6
22	PCa	53	3+3	2b	0	3
23	PCa	59	4+5	3a	0	5
24	PCa	70	2+3	2b	0	5
25	PCa	65	5+4	3a	0	4
26	PCa	57	3+5	2b	0	5
27	PCa	68	4+4	2b	0	6
28	PCa	58	3+3	2b	0	5
29	PCa	66	3+3	2b	0	3
30	PCa	63	3+4	2b	0	6
31	PCa	56	3+4	2b	0	4
32	PCa	63	5+3	3a	0	3
33	PCa	64	3+5	3a	0	5
34	PCa	60	3+4	2b	0	5
35	PCa	60	3+3	3a	0	5
36	PCa	57	3+2	2b	0	4
37	PCa	50	3+3	2a	0	6
38	PCa	68	3+3	3a	0	5
39	PCa	65	3+4	3b	1	4
40	PCa	69	5+5	3a	1	5
41	PCa	63	3+4	2b	0	5
42	PCa	51	2+3	2b	0	4
43	PCa	62	3+3	3a	0	5
44	PCa	61	3+4	3a	0	5
45	PCa	53	4+4	3b	1	4
46	PCa	56	4+3	2b	0	5
47	PCa	59	2+3	2b	0	5
48	PCa	61	3+4	2b	0	5
49	PCa	51	3+4	3a	0	6
50	PCa	62	3+4	3b	1	5
51	PCa	66	3+3	3a	0	5
52	PCa	62	3+3	2b	0	4
53	PCa	56	3+3	2b	0	5
54	PCa	58	3+3	3a	0	5
55	PCa	66	5+4	3a	0	6
56	PCa	55	3+4	3a	0	5
57	PCa	67	2+3	2b	0	5
58	PCa	61	3+5	2b	0	6
59	PIN	59	–	–	–	3
60	PIN	58	–	–	–	5
61	PIN	62	–	–	–	4
62	PIN	51	–	–	–	6
63	PIN	58	–	–	–	3
64	PIN	68	–	–	–	4
65	PIN	64	–	–	–	5
66	PIN	56	–	–	–	5
67	PIN	61	–	–	–	3
68	PIN	51	–	–	–	5
69	Normal	70	–	–	–	4
70	Normal	63	–	–	–	3
71	Normal	62	–	–	–	4
72	Normal	81	–	–	–	0
73	Normal	67	–	–	–	3
74	Normal	76	–	–	–	4
75	Normal	66	–	–	–	4
76	Normal	69	–	–	–	3
77	Normal	63	–	–	–	5
78	Normal	71	–	–	–	4

[i] PCa, prostate cancer; PIN, prostatic intra-epithelial neoplasia.

Statistical analysis

The relationships between 2 groups and expression values obtained by RT-PCR were analyzed using Mann-Whitney U tests. The correlations between miR-150-5p and miR-150-3p expression were evaluated using Spearman's rank test. The relationships among more than 3 variables and numerical values were analyzed using Bonferroni-adjusted Mann-Whitney U tests. We used Expert StatView software (version 5.0 SAS Institute Inc., Cary, NC, USA) for these analyses.

Results

The expression levels of miR-150-5p and miR-150-3p in naïve PCa and CRPC specimens and cell lines

We evaluated the expression levels of miR-150-5p and miR-150-3p in PCa tissues (PCa: n=17, CRPC: n=5), normal tissues (non-PCa: n=17), and PCa cell lines (PC3 and PC3M cells). The patient backgrounds are summarized in Table I. The expression levels of miR-150-5p and miR-150-3p were significantly lower in PCa and CRPC tissues than normal tissues (miR-150-5p: P=0.0266 and P=0.018, respectively; miR-150-3p: P=0.0025 and P=0.0002, respectively; Fig. 1A). There was a positive correlation between expression levels of miR-150-5p and miR-150-3p (r=0.854, P<0.0001; Fig. 1B).

Figure 1 Expression levels of miR-150-5p and miR-150-3p and functional assays in PCa cell lines (PC3 and PC3M cells) following miR-150-5p and miR-150-3p transfection. (A) Expression levels of miR-150-5p and miR-150-3p in naïve PCa and CRPC clinical specimens and cell lines were determined by qRT-PCR. Data were normalized to RNU48 expression. (B) Correlation between expression levels of miR-150-5p and miR-150-3p. (r=0.854, P<0.0001). (C) Cell proliferation was determined by XTT assays 72 h after transfection with 10 nM miR-150-5p and miR-150-3p. *P<0.0001. (D) Cell migration activity was determined by migration assays. *P<0.0001. (E) Cell invasion activity was determined using Matrigel invasion assays. *P<0.0001.

Effects of ectopic expression of miR-150-5p and miR-150-3p on cell proliferation, migration, and invasion assays in PCa cell lines

To examine the functional roles of miR-150-5p and miR-150-3p, we performed gain-of-function studies by using PC3 and PC3M cells transfected with mature miRNAs.

XTT assays revealed that proliferation was significantly inhibited in PC3 and PC3M cells transfected with miR-150-5p and miR-150-3p in comparison with that of mock or miR-control-transfected cells (P<0.0001; Fig. 1C). Wound-healing and Matrigel invasion assays demonstrated significant inhibition of cell migration and invasion in both miR-150-5p and miR-150-3p transfectants (P<0.0001; Fig. 1D and E).

Both miR-150-5p and miR-150-3p bound to Ago2

We hypothesized that both miR-150-5p and miR-150-3p may be incorporated into and function as part of the RISC. To test this hypothesis, we performed immunoprecipitation with antibodies targeting Ago2, which plays a central role in the RISC. After transfection with miR-150-5p or miR-150-3p, Ago2-bound miRNAs were isolated, and RT-qPCR was carried out to determine whether miR-150-5p and miR-150-3p bound to Ago2 (Fig. 2A).

Figure 2 Both miR-150-5p and miR-150-3p bound to Ago2. (A) Schematic illustration of miRNA detection method. Isolation of RISC incorporated miRNAs by Ago-2 immunoprecipitation. (B) Expression levels of miR-150-5p and miR-150-3p after transfection with miR-150-3p following immunoprecipitation by Ago2 (P<0.0001).

After transfection with miR-150-5p and immunoprecipitation by anti-Ago2 antibodies, miR-150-5p levels were significantly higher than those of mock- or miR control-transfected cells and those of miR-150-3p-transfected PC3 cells (P<0.0001; Fig. 2B). Similarly, after transfection with miR-150-3p and immunoprecipitation by anti-Ago2 antibodies, miR-150-3p levels were significantly higher than those of mock- or miR control-transfected cells and those of miR-150-5p-transfected PC3 cells (P<0.0001; Fig. 2B).

Screening of target genes regulated by miR-150-3p in PCa cells

To obtain further insights into the molecular mechanisms regulated by antitumor miR-150-3p in PCa cells, we screened these miRNA-regulated genes by using in silico and genome-wide gene expression analyses. First, we undertook genome-wide gene expression analysis using PC3 cells.

Analysis of the TargetScan database showed that 2,558 genes had putative target sites for miR-150-3p in their 3′-UTRs. Next, we screened 328 of these genes using genome-wide gene expression analysis. Finally, we found 14 genes that were upregulated (fold-change log2 >0.5) in cancer tissues by GEO database analyses (GEO accession no. GSE29079). Our strategy for analysis is shown in Fig. 3. Putative target genes of miR-150-3p are summarized in Table III. Among these candidate genes, SPOCK1 had a putative binding site for miR-150-5p according to the TargetScan database. Therefore, we focused on SPOCK1 as a candidate target gene of the dual strands of pre-miR-150 and performed further investigations of this target in PCa.

Figure 3 The strategy for analysis of miR-150-3p target genes in PCa cells. A total of 2,558 genes were annotated as miR-150-3p putative target genes in the TargetScan database (release 7.1). Next, we merged the data for gene expression analysis data in miR-150-3p transfectants, and we analyzed gene expression with the available GEO data set (GSE29079). The analyses showed that 14 genes were putative targets of miR-150-3p regulation in PCa cells.

Table III Putative target genes regulated by miR-150-3p in PCa cells.

Table III

Putative target genes regulated by miR-150-3p in PCa cells.

Entrez gene ID	Gene symbol	Gene name	PC3 miR-150-3p transfectant (log2 ratio)	Site counts	GEO Fold change
55771	SPOCK1	Sparc/osteonectin, cwcv and kazal-like domains proteoglycan (testican) 1	−2.718465	3	1.112207
11113	LIG3	Ligase III, DNA, ATP-dependent	−1.013958	2	0.833472
9448	MLEC	Malectin	−1.05939	1	0.817763
1017	NETO2	Neuropilin (NRP) and tolloid (TLL)-like 2	−1.639355	2	0.808991
51400	HIST1H3B	Histone cluster 1, H3b	−1.070237	1	0.803539
22877	PHF12	PHD finger protein 12	−1.531983	1	0.779377
4615	NCALD	Neurocalcin δ	−1.345623	2	0.772963
6695	SIM2	Single-minded family bHLH transcription factor 2	−2.060616	1	0.707018
79071	TAB3	TGF-β activated kinase 1/MAP3K7 binding protein 3	−1.341698	1	0.642641
8358	MAP4K4	Mitogen-activated protein kinase kinase kinase kinase 4	−1.043328	1	0.580977
10186	HNRNPAB	Heterogeneous nuclear ribonucleoprotein A/B	−1.501292	1	0.571322
11113	FARP1	FERM, RhoGEF (ARHGEF) and pleckstrin domain protein 1 (chondrocyte-derived)	−1.379558	1	0.536826
54475	SPECC1L	Sperm antigen with calponin homology and coiled-coil domains 1-like	−1.168633	1	0.506925
6548	NFIB	Nuclear factor I/B	−1.189804	1	0.505127

Regulation of SPOCK1 expression by miR-150-5p and miR-150-3p in PCa cells

Our studies revealed that SPOCK1 mRNA was significantly reduced in both miR-150-5p and miR-150-3p transfectants in comparison with those in mock or miR-control transfectants (P<0.0001 and P<0.0001; Fig. 4A). Expression of SPOCK1 protein was also repressed in these miRNAs transfectants (Fig. 4B).

Figure 4 Direct regulation of SPOCK1 by miR-150-5p and miR-150-3p in PCa cell lines. (A) SPOCK1 mRNA expression in PCa cell lines was evaluated by qRT-PCR 72 h after transfection with 10 nM miR-150-5p and miR-150-3p. GUSB was used as an internal control. *P<0.0001. (B) SPOCK1 protein expression in PCa cell lines was evaluated by western blot analyses 72 h after transfection with miR-150-5p and miR-150-3p. GAPDH was used as a loading control. (C) miR-150-5p or miR-150-3p binding sites in the 3′-UTR of SPOCK1 mRNA. (D) Dual-luciferase reporter assays using vectors encoding putative miR-150-5p and miR-150-3p target sites of the SPOCK1 3′-UTR (positions 182-188, 1477-1483, 1749-1756, and 2593-2599) for both wild-type and deleted regions. Normalized data were calculated as ratios of Renilla/firefly luciferase activities. *P<0.05.

Target prediction databases indicated that miR-150-5p had one putative target site in the 3′-UTR of SPOCK1 (Fig. 4C). Likewise, miR-150-3p had three putative target sites (Fig. 4C). To determine whether SPOCK1 mRNA contained functional target sites, we performed a dual-luciferase reporter assay.

The TargetScan database identified one putative target site in the 3′-UTR of SPOCK1 for miR-150-5p (position 182-188) and three target sites of SPOCK1 for miR-150-3p (positions 1477-1483, 1749-1756, and 2593-2599). We used vectors encoding a partial wild-type sequence of the 3′-UTR of SPOCK1 mRNA, including the predicted miR-150-5p and miR-150-3p target site, or a vector lacking the miR-150-5p and miR-150-3p target sites. We found that the luminescence intensity was significantly reduced by co-transfection with miR-150-5p or miR-150-3p and the vector carrying the wild-type 3′-UTR of SPOCK1 (P<0.05; Fig. 4D).

Effects of silencing SPOCK1 on cell proliferation, migration, and invasion in PCa cells

We evaluated the knockdown efficiency of si-SPOCK1 transfection in PC3 cells. Our present data showed that si-SPOCK1 transfection effectively downregulated SPOCK1 expression in PC3 and PC3M cells (Fig. 5A and B).

Figure 5 SPOCK1 mRNA and SPOCK1 protein expression after si-SPCOK1 transfection and the effects of SPOCK1 silencing in PCa cell lines. (A) SPOCK1 mRNA expression in PCa cell lines was evaluated by qRT-PCR 72 h after transfection with 10 nM si-SPOCK1_1 or si-SPOCK1_2. GAPDH was used as an internal control. (B) SPOCK1 protein expression in PCa cell lines was evaluated by western blot analysis 72 h after transfection with siRNAs. GAPDH was used as a loading control. (C) Cell proliferation was determined using XTT assays 72 h after transfection with si-SPOCK1_1 or si-SPOCK1_2. *P<0.0001. (D) Cell migration activity was determined by migration assays. *P<0.0001. (E) Cell invasion activity was determined using Matrigel invasion assays. *P<0.0001.

Functional assays demonstrated that cell proliferation, migration, and invasion were inhibited in si-SPOCK1 transfectants compared with those in mock- or miR control-transfected cells (P<0.0001, Fig. 5C–E).

Analysis of SPOCK1 expression in naïve PCa and CRPC clinical specimens by immunohistochemistry

Next, we examined the expression levels of SPOCK1 in naïve PCa specimens by immunohistochemical staining. SPOCK1 was strongly expressed in several cancer tissues, while low expression was observed in normal tissues (Fig. 6A). Moreover, the expression score for SPOCK1 protein was significantly higher in PCa tissues than in normal tissues (P<0.001, Fig. 6B). The patient backgrounds and clinicopathological characteristics are summarized in Table II.

Figure 6 Immunohistochemical staining of SPOCK1 protein in naïve PCa and CRPC specimens. (A) Immunohistochemical staining of SPOCK1 in naïve PCa using a tissue microarray. SPOCK1 was strongly expressed in several cancer tissues, while low expression was observed in normal tissues. (B) Quantification of SPOCK1 expression in PCa (n=58), PIN (n=10), and normal prostatic tissue (n=10) by tissue microarray. (C) Expression of SPOCK1 in CRPC specimens. SPOCK1 was strongly expressed in cancer lesions, similar to PSA.

To analyze SPOCK1 protein expression, immunohistochemistry was performed with CRPC specimens. Immunohistochemical staining demonstrated high expression of SPOCK1 in CRPC tissues. SPOCK1 was strongly expressed in the cytoplasm of the PCa cells almost in the same area where PSA was expressed (Fig. 6C).

Discussion

Androgen-dependent PCa initially responds to ADT, which can result in disease control. However, most PCa cells eventually acquire ADT-resistance mechanisms. Moreover, there are no curative treatments for patients with metastatic CRPC (19). One of the main challenges in treating CRPC is controlling aggressive, lethal metastatic PCa cells. We believe identification of the genes and pathways responsible for metastasis may lead to the development of new therapeutic strategies. Accordingly, we have focused on identifying antitumor miRNAs and oncogenic RNA networks mediated by these miRNAs in naïve PCa and CRPC cells (9,20,21). For example, antitumor miR-223 inhibits cancer cell migration and invasion by targeting ITGA3 and ITGB1 (18). Antitumor miR-218 suppresses migration and invasion by regulating LASP1 (22). Moreover, antitumor miRNAs (miR-26a/b, miR-29a/b/c and miR-218) function cooperatively to suppress metastasis-promoting LOXL (23).

In this study, we demonstrated that the dual strands of pre-miR-150, i.e., miR-150-5p and miR-150-3p, acted as antitumor miRNAs in naïve PCa and CRPC cells. According to the miRNA database (miRBase: http://www.mirbase.org/), miR-150-5p is a guide strand of pre-miR-150, and miR-150-3p is the corresponding passenger strand. Previous studies have shown that miR-150-5p (the guide strand) is frequently downregulated in cancer tissues and functions as an antitumor miRNA in several types of cancer (24–26). In this study, we focused on miR-150-3p (the passenger strand) and investigated the antitumor roles of this miRNA in naïve PCa and CRPC cells because no prior studies had evaluated the functions of miR-150-3p in cancer cells.

Passenger strands of miRNA are thought to be degraded and are not expected to be incorporated into the RISC (4). However, our data showed that the passenger strand of miR-150 was incorporated into the RISC in PCa cells, and this is the first report of the antitumor function of miR-150-3p in cancer cells. Our recent studies demonstrated that miR-145-3p (the passenger strand of pre-miR-145) acted as an antitumor miRNA targeting oncogenic UHRF1 and MTDH in bladder and lung cancer, respectively (16,27). Similarly, we confirmed the antitumor function of miR-139-3p (a passenger strand of pre-miR-139) in bladder cancer (17). These findings suggested that the passenger strands of miRNAs may have some biological functions in human cells, similar to the guide strands of miRNAs. The involvement of passenger strand miRNAs in the regulation of cellular processes is a novel concept in RNA research.

One of the main challenges in miRNA studies is to seek out miRNA targeting genes and RNA networks mediated by these miRNAs in cancer cells. We revealed that SPOCK1 was a direct target of dual strands of miR-150-5p and miR-150-3p in PCa cells. Moreover, we demonstrated the overexpression of SPOCK1 in naïve PCa and CRPC clinical specimens. SPOCK1/testican-1 belongs to the Ca2+-binding proteoglycan family, which includes SPARC, testican-2, and testican-3 (28). Overexpression of SPOCK1 was observed in several cancers and has been shown to play pivotal roles in cancer cell progression, metastasis, and drug resistance (29–31). SPOCK1 is upregulated in lung cancer and is associated with metastasis and survival (32). Interestingly, ectopic expression of SPOCK1 induces the epithelial-mesenchymal transition (EMT) in lung cancer cells (33). Another study demonstrated that SPOCK1 induces MET-dependent EMT signaling in lapatinib-resistant gastric cancer (34). Several reports have indicated that tyrosine kinase receptor inhibitors (TKIs) can frequently cause the acquisition of TKI resistance in cells and that the EMT is deeply involved in these events (35,36). These findings suggest that SPOCK1 mediates the EMT signaling to regulate cancer cell aggressiveness and drug resistance.

Most patients with PCa exhibit ADT failure and progress to CRPC with metastasis. Moreover, no curative treatments are available for advanced CRPC with metastasis (37). In CRPC cells, the EMT is associated with metastatic processes and is involved in drug resistance (38). Interestingly, androgens and androgen receptor-mediated signaling enhance the EMT and cancer cell aggressiveness (39). Transforming growth factor β (TGFβ) is a pivotal player that induces EMT in cancer cells (40). Increased expression of TGFβ and EMT-related proteins has been observed in CRPC bone metastasis (41). Interestingly, expression of SPOCK1 is elevated by TGFβ treatment in lung cancer cells, suggesting that SPOCK1 mediates downstream TGFβ signaling (33). We suggest that SPOCK1 expression may be related to induction of TGFβ signaling and epigenetic regulation of miR-150-5p and miR-150-3p in naïve PCa and CRPC cells. A recent study showed that overexpression of SPOCK1 in RWPE-1 cells (non-neoplastic adult human prostatic epithelial cells) promotes cell viability and cell migration and invasion abilities (42). These findings were supported by our present data. Thus, the expression of SPOCK1 may be involved in the pathogenesis of naïve PCa and CRPC, and SPOCK1-mediated signaling may be a promising therapeutic target in this disease.

In conclusiom, the dual strands of pre-miR-150, i.e., miR-150-5p and miR-150-3p, were significantly reduced in naïve PCa and CRPC tissues and acted as antitumor mRNAs. The passenger strand of miR-150-3p was found to have a specific function in cancer cells. SPOCK1 was directly regulated by dual strands of miR-150-5p and miR-150-3p in PCa cells. Overexpression of SPOCK1 was confirmed in naïve PCa and CRPC tissues and acted as an oncogene in this disease. Elucidation of miR-150/SPOCK1-mediated molecular networks may lead to a better understanding of the pathogenesis of naïve PCa and CRPC and facilitate the development of new treatment strategies.

Acknowledgments

This study was supported by KAKENHI grants 15K10801(C), 15K20071(C), 16K20125, and 16H05462(B).

References