Introduction
Prostate cancer (PCa) is the most frequently diagnosed cancer and the second leading cause of cancer death among men in developed countries (1). Most patients are initially responsive to androgen-deprivation therapy (ADT); however, PCa can eventually become resistant to ADT and progress to castration-resistant prostate cancer (CRPC). Currently, CRPC is difficult to treat, and most clinical trials for advanced PCa have shown limited benefits, with disease progression and metastasis to the bone or other sites (2,3). Therefore, understanding the molecular mechanisms of CRPC and the metastatic pathways underlying PCa using currently available genomic approaches would help to elucidate for this disease.
The discovery of non-coding RNAs (ncRNAs) in the human genome was an important conceptual breakthrough in the post-genome sequencing era (4). Further improving our understanding of ncRNAs is necessary for continued progress in cancer research. MicroRNAs (miRNAs) are endogenous small ncRNA molecules (19–22 bases in length) that regulate protein-coding gene expression by repressing translation or cleaving RNA transcripts in a sequence-specific manner (5). Numerous studies have shown that miRNAs are aberrantly expressed in many human cancers and that they play significant roles in the initiation, development and metastasis of those cancers (6–9). Moreover, normal regulatory mechanisms can be disrupted by the aberrant expression of tumor-suppressive or oncogenic miRNAs in cancer cells (10,11). Therefore, identification of aberrantly expressed miRNAs is an important first step toward elucidating miRNA-mediated oncogenic pathways.
Based on these data, we have sought to elucidate the miRNA expression signatures of PCa clinical specimens and have investigated the specific roles of miRNAs in PCa oncogenesis using differentially expressed miRNAs (12). Recently, we demonstrated that several miRNAs are downregulated in PCa and that miR-1/133a and miR-143/145 clusters function as tumor suppressors, targeting several oncogenic genes (13,14). Data from our analysis of the miRNA signature of PCa showed that miR-29s (including miR-29a/b/c) are significantly downregulated in cancer tissues, suggesting that miR-29s may act as tumor suppressors.
The aim of the present study was to investigate the functional significance of miR-29s and to identify the molecular pathways and targets regulated by these miRNAs in PCa cells. Our data demonstrated that restoration of mature miR-29s inhibited cancer cell proliferation. Moreover, gene expression data and in silico database analysis showed that laminin γ1 (LAMC1), which is involved in the ‘focal adhesion’ pathway, was a potential target of miR-29-mediated regulation. Silencing of the LAMC1 gene significantly inhibited cell migration and invasion in cancer cells. Thus, our study revealed that tumor-suppressive miR-29s regulated cancer pathways and cancer-related target molecules, providing new insights into the potential mechanisms of PCa oncogenesis and metastasis.
Materials and methods
Clinical prostate specimens
Clinical specimens were obtained from patients at the Teikyo University Chiba Medical Center from 2012 to 2013. All patients had elevated levels of prostate-specific antigen (PSA) and had undergone transrectal prostate needle biopsy. Non-cancerous prostate tissue (n=33) was obtained from patients who were negative for malignancy without indurations on the prostate. Prostate cancer tissues (n=37) contained 90–100% malignant cells in the biopsy cores.
The patient backgrounds and clinicopathological characteristics are summarized in Table 1A and B. Before tissue collection, all patients provided written informed consent of tissue donation for research purposes. The protocol was approved by the Institutional Review Board of Teikyo University.
Cell culture and RNA extraction
Human prostate cancer cells (PC3 and DU145) were obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum in a humidified atmosphere of 5% CO2 and 95% air at 37°C.
Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. RNA quality was confirmed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR)
The procedure for PCR quantification was carried out as previously described (12–14). TaqMan probes and primers for LAMC1 (P/N: Hs00267056_m1; Applied Biosystems, Foster City, CA, USA) and for GUSB (the internal control; P/N: Hs00939627_m1; Applied Biosystems) were assay-on-demand gene expression products. The expression levels of miR-29a (Assay ID: 002112), miR-29b (Assay ID: 000413) and miR-29c (Assay ID: 000587) were analyzed by TaqMan quantitative real-time PCR (TaqMan MicroRNA Assay; Applied Biosystems) and normalized to the expression of RNU48 (Assay ID: 001006). All reactions were performed in triplicate, and each assay included negative control reactions that lacked cDNA.
Transfection with mature miRNA and small-interfering RNA (siRNA)
The following mature miRNA species were used in this study: mirVana miRNA mimic for hsa-miR-29a-3p (product ID: MC12499; Applied Biosystems), hsa-miR-29b-3p (product ID: MC10103), and hsa-miR-29c-3p (product ID: MC10518). The following si-RNAs were used: Stealth Select RNAi si-RNA, si-LAMC1 (P/N: HSS105959, HSS180591; Invitrogen), and negative control miRNA/siRNA (P/N: AM17111; Applied Biosystems). RNAs were incubated with OPTI-MEM (Invitrogen) and Lipofectamine RNAiMax reagent (Invitrogen). The transfection efficiencies of miRNA in PC3 and DU145 cells were confirmed based on downregulation of TWF1 (PTK9) mRNA following transfection with miR-1 as previously reported (15).
Cell proliferation, migration and invasion assays
To investigate the functional significance of miR-29s, we performed cell proliferation, migration and invasion assays using PC3 and DU145 cells. The experimental procedures were performed as described in our previous studies (12–16).
Western blotting
Cells were harvested 72 h after transfection, and lysates were prepared. Fifty micrograms of protein from each lysates was separated on Mini-Protean TGX gels (Bio-Rad, Hercules, CA, USA) and transferred to PVDF membranes. Immunoblotting was performed with mouse anti-LAMC1 antibodies (1:250; HSA001909; Sigma-Aldrich, St. Louis, MO, USA), and anti-GAPDH antibodies (1:1000; ab8245, Abcam, Cambridge, UK) were used as an internal loading control.
Genome-wide gene expression and in silico analysis for the identification of genes regulated by miR-29s
To identify target genes of miR-29s, we used in silico analysis and genome-wide gene expression analysis. First, we screened genes using TargetScan release 6.2 (http://www.targetscan.org/). These genes were then categorized into KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways using GeneCodis analysis (http://genecodis.cnb.csic.es/). To identify upregulated genes in PCa, we analyzed a publicly available gene expression data set in GEO (accession no.: GSE29079).
Plasmid construction and dual-luciferase reporter assay
Partial sequences of the LAMC1 3′-untranslated region (UTR) of putative miR-29s binding sites (180 bp) were inserted between the XhoI-PmeI restriction sites in the 3′-UTR of the hRluc gene in the psiCHECK-2 vector (C8021; Promega, Madison, WI, USA). PC3 cells were transfected with 5 ng of the vector and 10 nM miR-29s using Lipofectamine 2000 (Invitrogen). The activities of firefly and Renilla luciferases in cell lysates were determined with a dual-luciferase assay system (E1910; Promega). Normalized data were calculated as the ratio of Renilla/firefly luciferase activities as previously described (17).
Statistical analysis
The relationships between 2 groups and the numerical values obtained by real-time RT-PCR were analyzed using paired t-tests. The relationships among 3 variables and numerical values were analyzed using the Bonferroni-adjusted Mann-Whitney U test. All analyses were performed using Expert StatView software (version 4, SAS Institute Inc., Cary, NC, USA).
Results
Expression levels of miR-29s (miR-29a/b/c) in PCa specimens
The chromosomal locations of miR-29s in the human genome are shown in Fig. 1. These miRNAs were clustered at 2 different human genomic loci; miR-29b-1 and miR-29a were located at 7q32, and miR-29b-2 and miR-29c were located at 1q32.
First, we evaluated the expression levels of miR-29s (miR-29a/b/c) in normal prostate tissues (n=23) and PCa tissues (n=27). In patients from whom normal prostate tissues were collected, the median PSA level was 7.3 ng/ml (range, 4.3–22 ng/ml). In contrast, in patients from whom PCa tissues were collected, PSA levels were quite high, with a median of 212 ng/ml (range, 3.5–3750 ng/ml). Twenty-one PCa patients had progressive disease with N1 or M1 according to TNM classification (Table IA).
To validate our previous miRNA profiling results in PCa, we evaluated expression of miR-29s in clinical PCa specimens. The expression levels of miR-29a, miR-29b and miR-29c were significantly lower in tumor tissues than in corresponding non-cancerous tissues (P<0.0001, P= 0.0111 and P= 0.0004, respectively; Fig. 2A). Spearman’s rank test showed a positive correlation between the expression of miR-29a and that of miR-29b (R=0.752 and P<0.0001, Fig. 2B). The expression of miR-29a was positively correlated with that of miR-29c (R= 0.804 and P<0.0001, Fig. 2B). Similarly, the expression of miR-29b was positively correlated with that of miR-29c (R=0.753 and P<0.0001, Fig. 2B).
Effects of restoring miR-29s expression levels on cell proliferation, migration, and invasion in PC3 and DU145 PCa cells
To investigate the functional effects of miR-29s, we performed gain-of-function studies using miRNA transfection in PC3 and DU145 cell lines.
As observed using XTT assays, cell proliferation was not inhibited in miR-29s transfectants, as compared with mock- or miR-control-transfected cells (Fig. 3A). However, cell migration activity was significantly inhibited in miR-29s transfectants, as compared with mock- or miR-control-transfected cells (Fig. 3B). Moreover, in Matrigel invasion assays, transfection with miR-29s significantly inhibited cell invasion as compared with mock- or miR-control-transfected cells (Fig. 3C).
Identification of candidate genes targeted by miR-29s
To identify genes targeted by miR-29s, we analyzed a combination of in silico and gene expression data in PCa. First, we screened miR-29s-targeted genes using the TargetScan database and identified 2,627 genes, which were then categorized into KEGG pathways using GeneCodis analysis, yielding 83 pathways identified as significantly enriched (Table II). Among these pathways, we focused on the focal adhesion pathway because this pathway has been implicated in cancer cell migration and invasion. A total of 58 genes were identified in this pathway. The gene set was then analyzed with available gene expression data from the GEO (accession no.: GSE29079), and genes upregulated in PCa were chosen (Fig. 4).
From this selection, 17 candidate genes were identified as miR-29s targets (Table III). Among these candidates, we focused on the LAMC1 gene and examined LAMC1 function and characteristics in further analyses.
LAMC1 is a direct target of miR-29s in PCa cells
We performed quantitative real-time RT-PCR and western blotting in PC3 and DU145 cells to investigate whether restoration of miR-29s altered LAMC1 gene and LAMC1 protein expression. The mRNA and protein expression levels of LAMC1/LAMC1 were significantly repressed in miR-29s transfectants as compared with mock- or miR-control transfected cells (Fig. 5A and B).
Therefore, we next performed luciferase reporter assays in PC3 cells to determine whether LAMC1 mRNA had target sites for miR-29s. The TargetScan database predicted that the putative miR-29s binding site existed in the 3′-untranslated region (UTR) of LAMC1 (position 1463–1470). We used vectors encoding a partial wild-type sequence of the 3′-UTR of LAMC1 mRNA, including predicted miR-29s target sites. We found that the luminescence intensity was significantly reduced by transfection of miR-29s with the vector carrying the wild-type 3′-UTR of LAMC1 (Fig. 5C).
Effects of downregulating LAMC1 on cell proliferation, migration and invasion in PCa cell lines
To investigate the functional role of LAMC1 in PCa cells, we performed loss-of-function studies using si-LAMC1 transfectants. First, we evaluated the knockdown efficiency of si-LAMC1 transfection in PC3 and DU145 cells. Quantitative real-time RT-PCR and western blotting indicated that the siRNA effectively downregulated LAMC1/LAMC1 expression in both cell lines (Fig. 6A and B).
In our functional analyses, XTT assays demonstrated that cell proliferation was not inhibited in si-LAMC1 transfectants, as compared with mock- or miR-control-transfected cells (Fig. 7A). In contrast, transfection with si-LAMC1 inhibited both cell migration and invasion, as compared with mock- or miR-control-transfected cells (Fig. 7B and C), similar to the results observed for restoration of miR-29s.
LAMC1 expression in PCa specimens
Finally, we evaluated the mRNA expression levels of LAMC1 in normal prostate tissues (n=10) and PCa tissues (n=10). The expression of LAMC1 was significantly higher in PCa tissues compared with normal tissues (P=0.0041), as demonstrated by RT-PCR (Fig. 8). In this analysis, an independent set of clinical specimens was used (Table IB).
Discussion
Emerging evidence has demonstrated that aberrantly expressed miRNAs upset the tightly regulated miRNA/protein-coding gene networks and cause initiation, progression and metastasis of human cancers (11). Therefore, identification of aberrantly expressed miRNAs is an important initial step in elucidating miRNA-mediated oncogenic pathways. Based on this strategy, we analyzed the miRNA expression signature of PCa and identified tumor-suppressive miRNAs and their associated PCa oncogenic pathways (12–14,16). The past studies supported the legitimacy of our strategy for miRNA analysis and contributed to the discovery of novel tumor-suppressive miRNAs in PCa.
In this study, we focused on the miR-29s (miR-29a, miR-29b and miR-29c) because we observed downregulation of these miRNAs in our PCa expression signature. Our data confirmed that all members of the miR-29 family were significantly downregulated in PCa tissues. Recently, we also observed that miR-29s were downregulated in head and neck and cervical squamous cell carcinomas (HNCSSs) (17,18). Moreover, downregulation of miR-29-family miRNAs has been described by other groups in several types of cancers (19); these studies are all consistent with our results. Although the molecular mechanisms through which miR-29s are silenced in PCa are still unknown, recent data have suggested that transforming growth factor (TGF)-β1 inhibits the expression of miR-29s and promotes the expression of extracellular matrix (ECM) components (20–22). The ECM functions as a critical source for growth, survival, motility and angiogenic factors that significantly affect tumor biology and progression (23–25). Additionally, TGF-β1 signaling is known to contribute to the epithelial-to-mesenchymal transition (EMT), an important step in cancer progression and metastasis (26). Our data demonstrated that restoration of miR-29s significantly inhibited cancer cell migration and invasion. Therefore, upregulation of ECM proteins caused by TGF-β-dependent silencing of miR-29s is an important step for metastasis in PCa cells.
Full understanding of the targets and signaling pathways in PCa that are regulated by the miR-29s family may contribute to our knowledge on PCa metastasis. We categorized miR-29s-target genes into known pathways using KEGG pathways (17,18). From our data in this study, we focused on the ‘focal adhesion’ pathway because restoration of miR-29s inhibited cancer cell migration and invasion in PCa cell lines. Furthermore, we combined the gene expression data of upregulated genes in PCa, generating 17 candidate target genes for miR-29s in focal adhesion pathways. The upregulation of collagen genes in primary tumors with metastatic potential was consistent with recent observations that epithelial-mesenchymal interactions are critical determinants of tumor cell behavior (27,28). High levels of type 1 collagen in metastatic lesions and in the serum of individuals with metastatic disease have also been reported (29,30).
In this study, we selected LAMC1, a member of the laminin super family as a target gene that contributes to cancer cell migration and invasion in PCa cells. Laminins are trimeric proteins that contain α-, β-, and γ-chains and are important ECM regulators that are and biologically active part of the basal lamina, influencing cell differentiation, migration and adhesion, as well as phenotype and survival (31). During tumor progression, several members of the laminin family have been shown to be upregulated in cancer cells and involved in cancer cell migration and invasion (32,33). Our recent studies in HNSCC have demonstrated that laminin-integrin signaling promotes cancer cell migration and invasion and that these signal pathways are regulated by tumor-suppressive miR-218 and miR-29s (17,34).
This is the first study demonstrating that miR-29s directly regulated LAMC1 in cancer cells and that silencing of LAMC1 inhibited cancer cell migration and invasion. The LAMC1 chain is the most widely expressed laminin chain, found predominantly in the basement membrane (35,36). Upregulation of LAMC1 in meningiomas correlates with shorter times to tumor recurrence and decreased progression-free survival (37). Furthermore, treatment with the specific LAMC1 peptide enhances pulmonary metastasis of B16 melanoma cells and induces the production of matrix metalloproteinase (MMP)-9 from B16 cells (38). Some studies have suggested that LAMC1 functions to promote metastasis and might be a novel therapeutic target in the treatment of human cancer. Further studies are needed to determine whether LAMC1-mediated molecular cascades contribute to PCa metastasis. A complete understanding of tumor-suppressive miR-29s and their regulation of LAMC1 signaling should shed light on the mechanisms of PCa metastasis and facilitate the development of more effective strategies for treating PCa.
Our data showed that all members of the miR-29-family were frequently downregulated in PCa cells. Moreover, these miRNAs functioned as tumor suppressors and inhibited cancer cell migration and invasion through regulation of focal adhesion pathways, especially via LAMC1. Elucidation of cancer pathways regulated by tumor-suppressive miR-29s should shed light on PCa metastasis and facilitate the development of more effective strategies for future therapeutic interventions for this disease.