 therapy is the standard treatment for advanced prostate cancer. However, almost all patients with lesions will gradually develop castration-resistant prostate cancer (CRPC), which is an incurable disease that develops after continued hormone therapy for one or two years. Therefore, currently no effective treatment options exist for metastatic or recurrent tumors [3]. Advanced PCa is often associated with metastatic characteristics, typically to the bones [4], and in 50-70% of castration-resistant prostate cancer patients bone metastases have been observed [5]. However, little is known about the molecular mechanisms of selective metastasis of PCa cells. In previous studies, it has been suggested that unique molecular signatures or biomarkers exist that predispose a PCa patient to metastasis. Activation of the rat sarcoma (Ras) signaling pathway is among the most commonly observed characteristics during metastasis in advanced PCa [6]. In a recent study, it was demonstated that bone metastasis and anti-apoptotic effects were found in Ras signaling-activated prostate cancer cells [7]. Upregulation of Ras-mediated signaling may be associated with various pathways, including the mitogen-activated protein kinase (MAPK) pathway [8], phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (AKT) cascades [9], and WNT (named as a hybrid of Wingless and Int) signaling [10].
MicroRNAs (miRNAs) are a class of single-stranded non-coding small RNAs, approximately 22 nucleotides (nts) in length, that can bind target mRNAs via partial or complete complementary base pairing. MiRNAs regulate gene expression of oncogenes or tumor-suppressor genes at the post-transcriptional level to control a wide range of biological processes [11], such as cell cycle regulation, differentiation, metabolism, apoptosis, invasion, tumorigenesis, angiogenesis, and metastasis [12]. This suggests that miRNAs have become important mediators of cancer development and therapy. Previous studies have demonstrated that one single miRNA can regulate the expression of over two hundred target genes, and that the expression of certain target mRNAs can be regulated by several miRNAs [13]. Overall, more than one third of structural human genes resulted by regulation of miRNAs [14]. Considering their roles, it is not surprising that abnormal miRNA expression is related to several cancer pathologies, thereby making miRNAs potential clinical biomarkers of cancer patients. For example, miRNAs can be used as specific biomarkers for diagnosis, treatment, and prognosis of tumors [15]. Consequently, in many studies it has been demonstrated that miRNAs play an important role in the occurrence and development of cancer. Based on the above-mentioned new findings, the function and potent application of miRNAs have attracted increased interest in cancer biology.
Several miRNAs, such as miR-21, miR-34a, miR-203, miR-205 and miR-331, have been reported to affect cell proliferation, and metastasis in PCa. Up-regulation of miR-21 increased invasiveness of LNCaP PCa cells [16], however, several other miRNAs, such as miR-34a [16], miR-205 [17], and miR-16 [18], have been reported to inhibit the metastatic ability, and may act as targets of potential therapeutic agents for treating of metastatic PCa. The miRNA-34 family, a highly-conserved family among different species, is comprised of miR-34a, miR-34b and miR-34c. MiR-34a is located on chromosome 1p36, whereas miR-34b and miR-34c are clustered as homologous genes, and share a common primary transcript. In the human genome, miR-34b and miR-34c are located on chromosome 11q23 [19]. Loss of heterozygosity within the 11q23 region has been observed in several solid cancers, including breast cancer [20], lung cancer [21], and PCa [22]. Several studies have indicated that miR-34a and miR-34c, despite their seed regions being identical, exert different regulatory abilities due to their target genes [23,24]. Although the family has a homology of >80% and a share similar set of target genes, their different and important roles in carcinogenesis and the development of tumors need to be further investigated [25]. In a previous study, it was shown that miR-34a-c was a direct transcriptional target of tumor suppressor p53 [26]. In response to DNA damage, hypoxia, and oncogenic stress, p53 activates the miR-34 family of genes and induces cell cycle G1-arrest and/or apoptosis [27], inhibits cell proliferation, and epithelial to mesenchymal transition, migration, invasion, and metastasis of various cancer cell entities [28].
The Target Scan method showed that miR-34a, 34b, 34c and miR-449a can bind to mesenchymal-epithelial transition (MET) in PCa cells. Moreover, the migration of cancer cells was controlled by regulating miR-34c through the MET target in vitro and in vivo [29]. This will provide insight on predicting cancer cell migration using miRNAs as specific targets or biomarker in the diagnosis, treatment, and prognosis of advanced cancer. We observed a significant difference in miR-34b and miR-34c expression between the prostate cancer cell lines PC3 and DU145 (data are described in the "Results" section), however, PC3 and DU-145 are PCa cell lines with a moderate to high metastatic potential. This was based on their capacity to invade the extracellular matrix, an established tumor invasion assay, which indicated that expression of miR-34b and 34c correlated with the metastatic potential of PCa cells. An increasing number of studies have indicated that miR-34c may be involved in a novel molecular mechanism that uses miR-34c as a key tumor suppressor by targeting MET regulation in PCa. However, at present, the novel molecular mechanism of miR-34b has not yet been elucidated. In this study, we aimed to investigate the targets and preliminary molecular mechanism of action of miR-34b in migration and invasion of metastatic PCa cells. Using ectopic expression of miR-34b in vitro, followed by gene expression array, we found that the TGF-β signaling pathway was a target of miR-34b in PCa cells. Moreover, we demonstrated that members of TGF-β signal pathway, such as TGF-β, TGF-βR1 and p53 expression, and phosphorylation of mothers against decapentaplegic 3 (SMAD3) inversely correlated with miR-34b levels in PCa cells. Our working hypothesis was that miR-34b plays a role in the initiation, progression, metastases, and transition to castration resistance of PCa, and that some of effects may be mediated by targeting SMAD3 phosphorylation in the TGF-β signaling pathway.

Analysis of the Metastatic Potential of Different Prostate Cancer Cells In Vitro
Several studies have confirmed that DU145 and PC3 are PCa cell lines with moderate and high metastatic potential [30], respectively. In this study, we used the migration and invasion assay to demonstrate the metastatic capacity of the cells to invade the extracellular matrix. Thus, we established a tumor invasion assay to verify the metastatic capacity of androgen-independent PCa cells. The data showed that in PC3 cells, the number of migrating and invading cells increased by 3-fold (p < 0.01) and 1.5-fold (p < 0.05) when compared to that of DU145 cells ( Figure 1). Moreover, the results indicated that DU145 and PC3 are PCa cell lines with a moderate and high metastatic potential, respectively. This suggested that the cultured cells were suitable for use in other experiments.

Expression of miR-34 Family Members in Different Metastatic Potential Prostate Cancer Cells
To identify the differential expression of miRNAs in PCa cells, miRNA sequencing analysis was performed in DU145 and PC3 cells. The miRNAs expression profile in DU145 and PC3 cells by heat map analysis indicated that expression of miR-34b and miR-34c in DU145 cells were significantly higher compared to that in PC3 cells (Figure 2A). The expression level of three members of the miR-34 family was investigated in DU145 and PC3 cells using miRNA sequencing ( Figure 2B) and by qRT-PCR ( Figure 2C). MiRNA sequencing analysis demonstrated that miR-34b was 5000-fold higher expressed in DU145 cells (p < 0.01) compared to that in PC3 cells, whereas for miR-34a, the expression was only 4-fold higher. Therefore, our data showed that there was no significant difference in the expression level of miR-34a between PC3 and DU145 cells. We also determined that miR-34c had a higher level of expression in DU145 cells and was about 10,000-fold higher expressed compared to that in PC3 cells. These findings were consisted with the results of a previous report [31,32]. To confirm these findings, we evaluated the expression of miR-34 family members in two cancer cell lines using qRT-PCR analysis. We found that the expression level of three members of the miR-34 family showed the same trend that was found by miRNA sequencing, suggesting that miR-34b and miR-34c may be involved in prostate cancer metastasis. Our data demonstrated that the expression level of miR-34b and miR-34c negatively correlated with the metastatic potential in PCa. Moreover, our findings were in line with the wound healing assay results of a previous study [32]. Expression levels of miR-34a, miR-34b, and miR-34c in prostate cancer cell lines DU145 and PC3. (A) Heat-map of miRNAs with differential expression comparing DU145 and PC3 cells. Up-regulated miRNAs are in red, whereas down-regulated genes are shown in green. Expression of miR-34b and miR-34c is up-regulated in DU145 cells and downregulated in PC3 cells; (B) Expression of miR-34a, miR-34b and miR-34c in DU145 and PC3 cells by miRNA sequencing analysis; (C) The relative expression of