The Prostate 74:756^767 (2014)

MicroRNA-494-3pTargets CXCR4 to Suppress the Proliferation, Invasion, and Migration of Prostate Cancer Peng-fei Shen,1 Xue-qin Chen,2 Yong-chuan Liao,3 Ni Chen,2 Qiao Zhou,2 Qiang Wei,1 Xiang Li,1 Jia Wang,1** and Hao Zeng1* 1

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Department of Urology,West China Hospital, SiChuan University,Chengdu,China Department of Pathology, Laboratory of Pathology,West China Hospital, SiChuan University,Chengdu,China 3 Department of Ophthalmology,West China Hospital, SiChuan University,Chengdu,China

BACKGROUND. Although SDF-1/CXCR4 pathway is a potential mechanism of tumor proliferation and progression, the mechanism of controlling CXCR4 expression is not fully understood. This study was to confirm that miR-494-3p might be a potentially posttranscriptional regulator of CXCR4 and over-expression of miR-494 might suppress prostate cancer progression and metastasis. MATERIALS AND METHODS. We firstly postulated the post-transcriptional regulation of CXCR4 by miR-494-3p through bioinformatics analysis, and then it was demonstrated that miR-494-3p could regulate the CXCR4 mRNA post-transcriptionally by binding to the predicted site by dual reporter gene assays. The biological effect of miR-494-3p on prostate cancer cells proliferation, apoptosis, migration, and invasion was measured by MTT, TUNEL, flow cytometry, migration, and invasion assays. RESULTS. It was shown that the mRNA and protein expression levels of CXCR4 were significantly up-regulated in PC-3 and DU145, whereas barely detected in LNCaP and RWPE1. However, the CXCR4 protein levels were inversely related to the mature miR-494-3p expression levels in RWPE-1 and prostate cancer cells. The constitutive over-expression of miR-494-3p could down-regulate the protein level of CXCR4 in PC-3 and DU145. MiR-494-3p also could bind to the seed sequences in the 30 -UTR of the CXCR4 gene. Artificial overexpression of miR-494-3p could inhibit the growth, promote the apoptosis, and inhibit the migration and invasion of PC-3 and DU145 cells in vivo. CONCLUSIONS. Our results suggested that miR-494-3p might play crucial role in prostate cancer by post-transcriptional regulation to CXCR4 mRNA. MiR-494-3p/CXCR4 pathway may be a potential therapeutic target to prevent prostate cancer progression and metastasis. Prostate 74:756–767, 2014. # 2014 Wiley Periodicals, Inc. KEY WORDS:

prostate cancer; CXCR4; miR-494; progression

INTRODUCTION The chemokine receptors are a subset of G protein coupled receptors with seven helical membrane-spanning regions and some of them are implicated in the biological behaviors of a variety of neoplasms [1,2]. Among these receptors, CXCR4 is highly expressed in many cancers, such as breast, lung, prostate, gastric, and pancreatic cancer [3–7]. These CXCR4-positive expressed tumors could preferentially spread to organs or tissues that highly express CXCL12/SDF-1, including lung, liver, lymph nodes, and bone marrow. NF-kB, hypoxia, and VEGF, are important factors that ß 2014 Wiley Periodicals, Inc.

Grant sponsor: National Natural Science Foundation of China (NSFC); Grant numbers: 81172439; 81001207; 81101529; 81101628. Peng-fei Shen and Xue-qin Chen contributed equally to this work. 

Correspondence to: Hao Zeng, Department of Urology, West China Hospital, SiChuan University, GuoXueXiang37#, Chengdu 610041, China. E-mail: [email protected]  Correspondence to: Jia Wang, Department of Urology, West China Hospital, SiChuan University, GuoXueXiang37#, Chengdu 610041, China. E-mail: [email protected] Received 11 September 2013; Accepted 10 February 2014 DOI 10.1002/pros.22795 Published online 18 March 2014 in Wiley Online Library (wileyonlinelibrary.com).

Regulation of CXCR4 by MicroRNA-494-3p in Prostate Cancer promote and regulate the expression of CXCR4 and affect the behavior of tumor cells [8–10]. Furthermore, increasing evidences suggest that inhibition of CXCR4 could repress tumor proliferation and directing migration [11–13]. Therefore, CXCR4 might be a potential therapeutic target for cancer treatment. However, to date, the accurate mechanism of CXCR4 transcriptional regulation is still not fully understood and our aim is to demonstrate the post-transcriptional regulation mechanism of CXCR4 in prostate cancer. MicroRNAs (miRNAs, miRs), a class of small nonprotein-coding RNAs about 22 nucleotides, could widely influence the signaling networks leading to physiological or pathological responses by binding to the 30 -untranslated region (30 -UTR) of their target gene [14,15]. Recent evidences indicated that miRNAs might function as tumor suppressor genes and oncogenes, and they might regulate tumor proliferation, apoptosis, invasion, and migration by complex pathways [16,17]. Hsa-miR-494 (miR-494), has recently been identified and garnered attention. Several experimental studies have identified the association between miR-494 and malignancies. It was reported that miR494 was up-regulated in human retinoblastoma, Waldenstrom Macroglobulinemia, and lung cancer [18,19]. In addition, over-expression of miR-494 was also observed in acute myeloblastic leukemia to regulate cell differentiation and apoptosis [20]. On the other hand, miR-494 was down-regulated in prostate cancer, breast cancer, lung cancer, and head and neck squamous cell carcinoma [21–23]. Furthermore, the latest experimental studies have demonstrated that miR-494 plays different roles in different malignancies. However, what are the roles of miR-494-3p and its functional target genes in prostate cancer need to be explored. According to bioinformatics analysis, we proposed that CXCR4 might be a potential target gene of miR494-3p. In the present study, we focused on analyzing the mechanism and the role of miR-494-3p regulating CXCR4 post-transcriptionally in human prostate cancer cell lines. Our results confirmed the hypothesis, CXCR4 protein could be regulated post-transcriptionally by miR-494-3p in prostate cancer cell lines, and miR-494-3p/CXCR4 signaling pathway was significantly associated with prostate cancer proliferation, invasion, and migration in vivo. This pathway may be a potential therapeutic target to prevent prostate cancer directing metastasis.

MATERIALS AND METHODS Cell Lines,Tissues, and General Reagents Normal human prostate epithelial cell line RWPE-1 and human prostate cancer cell lines PC-3, DU145, and

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LNCaP from the American Type Culture Collection (ATCC) were maintained in RPMI 1640 with 10% fetal calf serum (FCS, Life Technologies) at 37°C under 5% CO2. The adenovirus immortalized human embryonic kidney epithelial cell HEK-293 was maintained in DMEM with 10% FCS. Human prostate cancer tissues were got from prostate biopsy specimen, which were stored in 80°C refrigerator. Tris–base, Tween 20, DTT, and EDTA were from Amresco. Phenylmethylsulfonyl fluoride, leupeptin, pepstatin, and aprotinin were from Roche Diagnostics. Total RNA Extraction, Stem^Loop Reverse Transcription-PCR and Conventional Reverse Transcription-PCR Total RNA was extracted by using the Trizol reagent (Invitrogen) according to the manufacturer’s instructions. PCR was carried out as previously described [17,24]. The stem–loop reverse transcriptionPCR (RT-PCR) technique was used to examine mature miR-494-3p. The stem–loop RT primer of miR-494-3p was designed as 50 -GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGAGGTT-30 . RT was carried out in 20 ml reaction mixtures at 16°C for 30 min, 42°C for 30 min, and 85°C for 5 min. The PCR primers for mature miR-494-3p were designed as follows: forward, 50 -CATAGCCCGTGAAACATACAC G-30 and reverse, 50 -GTGCAGGGTCCGAGGT-30 (65 bp). The U6 gene (50 -TGGAACGATACAGAG AAGATTAGCA-30 , 50 -AACGCT TCACGA ATTTGC GT-30 , 66 bp) was used as an internal control. Standard conventional RT-PCR was used for other genes and the PCR primers were designed according to their respective cDNA sequences (Genbank) as Table I. Products were resolved by 2% agarose gel or 15% PAGE and were visualized by staining with fluorescent dye Goldview (SBS, Beijing, China) or ethidium bromide (EB). Images were captured by scanning with Typhoon 8600 Multi-Imager (Molecular Dynamics) under fluorescence mode or with Bio-Rad Gel Doc XR (Bio-Rad). Semi-quantitative analysis was performed with the Quantity One Software (Bio-Rad). Real-Time Quantitative PCR Real-time quantitative PCR (Q-PCR) was performed to determine the accurate expression levels of mature miR-494-3p and CXCR1-7 in prostate cancer cells or tissues on Light Cycler 2.0 (Roche). Data were recorded and analyzed by the Light Cycler software 4.05 (Roche). The gene expression DCt values of target genes from each sample were calculated by normalizing with internal control and relative quantitative values were plotted [17,24,25]. The Prostate

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TABLE I. The RT-PCR Primer Sequences of CXCR Subfamily Name CXCR1 CXCR2 CXCR3 CXCR4 CXCR5 CXCR6 CXCR7 b-Actin

RT-PCR primer sequences 0

Sense: 5 -CTGAGCCCCAAGTGGAACGAGACA-3 Anti-sense: 50 -GCACGGAACAGAAGCTTTATTAGGA-30 Sense: 50 -ACATGGGCAACAATACAGCA-30 Anti-sense: 50 -TGAGGACGACAGCAAAGATG-30 Sense: 50 -ACACCTTCCTGCTCCACCTA-30 Anti-sense: 50 -GTTCAGGTAGCGGTCAAAGC-30 Sense: 50 -GGTGGTCTATGTTGGCGTCT-30 Anti-sense: 50 -TGGAGTGTGACAGCTTGGAG-30 Sense: 50 -TCCCCTCCTCACTCCCTTCCCAT-30 Anti-sense: 50 -CCTGCGGTTCCATCTGAGTGACATC-30 Sense: 50 -CTGGTGGTGTTTGTCTGTGG-30 Anti-sense: 50 -AGGTGAGGATGAGCATGGAC-30 Sense: 50 -CTACTTCACCAACACCCCCAG-30 Anti-sense: 50 -GGCAAAGCCCAAGACAACGG-30 Sense: 50 -CTGGCACCACACCTTCTACAATG-30 Anti-sense: 50 -CCTCGTAGATGGGCACAGTGTG-30

Western Blot Analysis Total proteins were extracted, resolved by SDS– polyacrylamide (Sigma) gel electrophoresis and then electroblotted to polyvinylidene difluoride membrane (Amersham Biosciences). Western blot was carried out as previously described [17,24]. The following antibodies were used for Western blot: anti-CXCR1 (mouse monoclonal, 1:3,000, Santa Cruz Biotechnology), anti-CXCR2 (mouse monoclonal, 1:800, Santa Cruz Biotechnology), anti-CXCR3 (rabbit polyclonal, 1:1,000, GeneTex), anti-CXCR4 (rabbit polyclonal, 1:1000, Abcam, Inc.), anti-CXCR5 (rabbit polyclonal, 1:1,000, Abcam, Inc.), antiCXCR6 (goat polyclonal, 1:500, Abcam, Inc.), antiCXCR7 (rabbit polyclonal, 1:200, Abcam, Inc.), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; mouse monoclonal, 1:10,000, Kangcheng, China). Horseradish peroxidase-labeled secondary antibodies were from Zymed Laboratories, Inc. Immunocytochemistry Prostate cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and incubated overnight at 4°C with anti-CXCR4 antibody (rabbit polyclonal, 1:100, Abcam, Inc.). Immunostaining was carried out as previously described [17,24]. Recombinant Adenoviral Vectors for OverExpression of miR-494-3p The pri-miR-494-3p sequence was amplified from HEK-293 cell genomic DNA with the primers as follows: forward, 50 -GGTACCGGACACGCAAATAGAAGCC-30 and reverse, 50 -GTCGACCAACCTCCTTCAACCACAG-30 (434 bp). PCR product was ligated The Prostate

Product (bp) 0

152 180 191 227 224 250 226 248

into pMD18/T (TaKaRa), identified by sequencing, and cloned into shuttle plasmid pAd-Track-CMV. After linealized with PmeI, pAdTrack-miR-494-3p was transformed into BJ5183 bacterial cells with adenoviral pAdeasy-1 vector (Stratagene) for homologous recombination. The identified recombinant adenovirus plasmid pAdeasy-miR-494-3p was digested with PacI and transfected into HEK-293 cells for package of recombinant miR-494-3p adenovirus (AD-miR-494-3p). ADmiR-494-3p was amplified by repeated infection and verified by PCR. The pAdTrack-CMV empty vector was used as control (AD-control). The titers and the multiplicity of infection (MOI) were determined according to the manufacturer’s protocols. Luciferase Reporter Constructs and Dual Reporter Gene Assay The seed sequences (173–179 nt) of CXCR4 30 -UTR with flanking sequences were amplified from HEK293 cell genomic DNA with the primers CXCR4-XbaIP1: 50 -TCTAGAGACTGACCAATATTGTACAG-30 and CXCR4-XbaI-P2: 50 -TCTAGATGCCATCTTCTACAGCAA-30 (323 bp). PCR products were ligated into pMD18/T and the pGL3-Promoter vector (Promega, Madison, WI) was used to construct reporter plasmids (designated as pGL3-site). Overlapping PCR was used for site-directed mutation and deletion of the seed sequences (designated as pGL3-Mut and pGL3-Del). The PCR primers were used as follows: CXCR4-Mut 1 (50 -TTTTGTCGACGTATTGATGTGT-30 ), CXCR4-Mut 2 (50 -AATACGTCGACAAAAAAATTTA-30 ), CXCR4Del 1 (50 -TTTTTTTTATTGATGTGTGTCTAGGC-30 ), and CXCR4-Del 2 (50 -CATCAATAAAAAAAATTTATATAAATAAGTC-30 ). PC-3 cells were cultured in 24well plates and transfected with plasmids by using

Regulation of CXCR4 by MicroRNA-494-3p in Prostate Cancer Lipofectamine 2000 (Invitrogen). Each reporter construct and the pRL-CMV plasmid (Promega) containing the renilla luciferase gene (as internal control) were co-transfected into PC-3 cells. Cells were infected with AD-miR-494-3p and AD-control (MOI 100) 24 hr after transfection and collected 24 hr later. The firefly and renilla luciferase activities were assayed on Luminometer TD-20/20 (Turner Design, Sunnyvale, CA). Cell Viability Assay,Terminal Deoxynucleotidyltransferase-Mediated Biotinylated dUTP Nick End-Labeling (TUNEL), and Flow Cytometry Cells were cultured in 96-well plates and measured by tetrazolium-based MTT (Sigma) cell proliferation assay. TUNEL was performed by using in situ cell death detection kit (Roche Diagnostics) as previously described [17,24]. Prostate cells were incubated with Annexin V-APC, propidium iodide (PI), or both (BD Pharmingen) in AnnexinV binding buffer for 30 min at 4°C in dark and then analyzed on BD FACS Aria flow cytometer (BD Pharmingen). Data were analyzed with the manufacturer’s software and Annexin V-APC(þ)/ PI() cells were gated as the apoptotic cell population. Migration and Invasion Assays Transwell—polycarbonate filter membrane with a diameter of 6.5 mm and pore size of 8 mm (Millipore) and Matrigel (BD Biosciences) were used to assess the migration and invasion abilities of PC-3 and DU145 cells after transfection of AD-miR-494-3p. After 24 hr, cells were re-suspended in RPMI 1640 without FCS. 15 ml of Matrigel was added into the upper chamber of a transwell plate and incubated at 37°C for 4 hr until solidified. Triplicate transwell chambers were placed into a 24-well plate and the lower chamber contained RPMI 1640 with 10% FCS. About 10  104 cells were added into the upper chamber (RPMI 1640 without FCS) for migration assays and about 5  104 cells for invasion assays. The incubation was carried out 8, 16, and 24 hr for migration assays and 24 and 48 hr for invasion assays. The medium in the top and bottom chambers was carefully aspirated. The membrane was washed with PBS and fixed in 4% paraformaldehyde, together with any residual cells were removed off the upper surface of the membrane. The cell invasion was quantified by counting the cells stained by crystal violet (average cell count of five random visual fields). Migration and invasion assays were carried out as previously described [26,27]. Statistical Analysis All values were expressed as means  SD. Comparison between two mean values was made by an

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independent-samples t-test. Statistical significance was set at P  0.05. RESULTS The Expression Prof|les of CXCR1-7 in Prostate Cancer The chemokine receptor CXCR subfamily have been discovered different functions in prostate cancer, and CXCR4 was significantly related to prostate cancer progression [4,5,28,29]. In the present study, CXCR1-7 mRNA, and protein expression levels in RWPE-1 and prostate cancer cell lines were validated by conventional RT-PCR, Q-PCR, and Western blot analysis, respectively (Fig. 1A and B). Copy number of target genes (relative to b-actin) in Q-PCR was determined by the 2DDCt method, with DDCt ¼ DCtcancer  DCtRWPE1 ¼ (Ctcancer-CXCR  Ctcancer-actin)  (CtRWPE-1-CXCR  CtRWPE-1-actin). It was shown that the mRNA and protein expression levels of CXCR4 were significantly upregulated in hormone-independent prostate cancer cell lines PC-3 and DU145, whereas they were barely detected in hormone-dependent cell line LNCaP and normal prostate epithelial cell line RWPE-1. MiR-494-3p Expression Was Inversely Related to CXCR4 Over-Expression in PC-3 and DU145 Cells Several studies have reported the signature of miRs expression profiles in prostate cancer [21,27,30], and some of them were identified as the potential regulatory miRs of CXCR4 by using TargetScan 5.1 (http:// www.targetscan.org/), such as miR-302, miR-373, miR-494-3p, miR-520c, miR-588, and miR-622. In the present study, expressions of probable CXCR4-related mature miRs were investigated by stem loop RT-PCR, and results showed predominately differential expression of miR-494-3p in different prostate cells. Concretely speaking, miR-494-3p was detected to be highly expression in RWPE-1, and moderately expressed in LNCaP cells but barely detectable in prostate cancer tissues, PC-3, and DU145 cells (Fig. 1C). Real-time QPCR was further performed to verify that miR-494-3p was only 1/10 in PC-3 of that in RWPE-1, 1/50 in DU145, and 2–4% in prostate cancer tissues. Copy number of target genes (relative to U6) in Q-PCR was determined by the 2DDCt method with DDCt ¼ DCtcancer  DCtRWPE-1 ¼ (Ctcancer-miR  Ctcancer-U6)  ( CtRWPE-1-miR  CtRWPE-1-U6). These results suggested that the CXCR4 protein levels were inversely related to the mature miR-494-3p expression levels in RWPE-1 and cancer cells/tissues. Therefore, we finally hypothesized miR-494-3p as the major potential post-transcriptional regulator of CXCR4. The Prostate

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Fig. 1. The expression levels of CXCR1-7 in PC-3, DU145, LNCaP, and RWPE-1cells were determined, and miR-494-3p expression was inversely related to CXCR4 over-expression in PC-3 and DU145 cells. A: RT-PCR and Q-PCR showed the different levels of CXCR1-7 mRNA, in which only the levels of CXCR4 mRNAwere significantly higher in PC-3 and DU145 cells than that in LNCaP and RWPE-1cells. B: Western blot analysis showed the protein expression levels of CXCR1-7 and their results were consistent with PCR. C: In contrast to CXCR4, miR-494-3p expression levels (stem loop RT-PCR) in prostate cancer tissues, PC-3, and DU145 cells were also significantly lower than thatin LNCaP and RWPE-1cells and they wereinverselyrelated to CXCR4 over-expression.

Over-Expression of miR-494-3p Led to DownRegulation of CXCR4 Protein in PC-3 and DU145 Cells The expression levels of CXCR4 in PC-3 and DU145 cells treated with artificial AD-miR-494-3p were assessed by Western blot analysis and immunocytochemistry (Fig. 2). These data supported that miR-4943p could repress the protein translation of CXCR4.

Dual Reporter Gene Assays Showed Interaction of miR-494-3p With 30 -UTR of CXCR4 To confirm that miR-494-3p can regulate CXCR4 mRNA post-transcriptionally by binding to the predicted site, luciferase reporter gene constructs containing the potential seed sequences of CXCR4 30 -UTR and synonymous mutants of the site were prepared and cloned into the luciferase reporter vector pGL3 The Prostate

(Fig. 3A). These luciferase reporter vectors were transfected into cells, respectively, and then infected with Ad-miR-494-3p after 24 hr. Subsequently, the luciferase and renilla activity in each well was measured. It was shown that the luciferase reporter gene activity was significantly down-regulated by 63.2% in construct with normal CXCR4 30 -UTR sequences, whereas reporter constructs abnormal CXCR4 30 -UTR sequences were not affected (Fig. 3B). These data suggested that miR-494-3p could bind to the seed sequences in the 30 UTR of the CXCR4 gene, and then further regulate CXCR4 expression. The Functional Roles of miR-494-3p in Prostate Cancer Cell Lines The role of miR-494-3p in the proliferation of prostate cancer cell lines. To explore the biological effect of miR-494-3p on cell proliferation, miR-494-3p

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Fig. 2. The effects of artificial miR-494-3p over-expression on CXCR4 in PC-3 and DU145 cells. A: Homogenous green fluorescence protein expression showed the infected efficiency of AD-miR-494-3p and AD-control in PC-3 and DU145 cells and the miR-494-3p levels were significantly up-regulated. B and C: Western bolt analysis (semi-quantitative histograms) and immunocytochemistry (brown staining) showed that artificial over-expression of miR-494-3p by AD-miR-494-3p resulted in a significant down-regulation of CXCR4 protein level compared with AD-control.

was artificially over-expressed in PC-3 and DU145 cells and the cellular and molecular changes were measured by MTT, TUNEL, and flow cytometry. The growth of cells was greatly reduced at 96 hr after ADmiR-494-3p infection as measured by MTT analysis

(Fig. 4A). At the same time, cellular apoptosis status was also investigated by flow cytometry (annexin VAPC staining) and TUNEL (Fig. 4B and C). Apoptosis was much higher in cells infected with AD-miR-494-3p than that of with negative AD-control. The Prostate

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Fig. 3. Identifying the seed sequences of miR-494-3p was in the CXCR4 30 -UTR by dual reporter gene assays. A:The potential seed sequences of miR-494-3p was predicted in the 173^179 nt on the CXCR4 30 -UTR. B: The dual reporter gene assays were performed with pGL3 expression constructs containing the potential seed sequences of CXCR4 30 -UTR and synonymous mutants of the site inserted downstream of the luciferase coding sequence. The activity of the basic pGL3 construct (pGL3Promoter) was set as the baseline. After artificial over-expression of miR-494-3p (pre-transfected with luciferase reporter vectors) in PC-3 cells, the reporter gene activity (represented by relative luciferase activity) was significantly decreased when PGL3-site was present in the construct, whereas it was obviously restored when PGL3-Mut or PGL3-Del present. Expression of AD-miR-494-3p alone (without seed sequencespresent) or PGL3-site (withoutinfection of AD-miR-494-3p) hadno effectonreporter gene activity.

The role of miR-494-3p in the migration and invasion of prostate cancer cell lines. Migration and invasion assays were used to further investigate the migratory and invasive activities of PC-3 and DU145 cells. These biological behavior changes were observed after cells infected with AD-miR-494-3p and negative AD-control. As shown in Figure 5A, the migration ability of PC-3 and DU145 cells was significantly inhibited at 8, 16, and 24 hr after infected with AD-miR-494-3p compared to AD-con. The same phenomenon was observed in the invasion assays and the invasiveness of cells was suppressed at 24 and 48 hr with ADmiR494 infection (Fig. 5B). These results suggested that over-expression of miR-494-3p could significantly inhibit migration and invasion of PC-3 and DU145 cells through targeting CXCR4. The Prostate

In the last few years, extending evidences have demonstrated that chemokines (CXCL) and their receptors (CXCR) play important roles in many fundamental cellular processes and in cancer progression [31,32]. CXCRs seem to be expressed in various tumor types, including breast cancer, prostate cancer, pancreatic cancer, lung cancer, and ovarian carcinomas [33–35]. CXCR4 have been identified that it is of importance in the progression of cancer and may function as a prognostic marker in various types of cancer. Recent evidences have highlighted the role of CXCR4 in prostate cancer [34,36,37]. In the present study, we demonstrated the mRNA and protein expression profiles of CXCR1-7 in RWPE-1 and prostate cancer cells. CXCR4 was the only receptor whose expression was higher in PC-3 and DU145 cells than that in LNCaP and RWPE-1. It was reported in several studies that CXCR4 was aberrantly expressed in prostate cancer and participated in directing metastasis of prostate cancer by binding to its ligand SDF-1 [30–40]. Wang and colleagues [41] identified that one of common cell survival signaling pathway, such as MEK/ERK and PI3K/AKT pathways, could be activated by SDF-1/CXCR4 axis, and found that different signal transduction pathways might play pivotal roles in the regulation of prostate cancers progression. Interference with SDF-1/CXCR4 signaling pathway could affect the progression and directing metastasis of prostate cancer. Experiments in vitro and in vivo revealed that CXCR4 antibody inhibited the migration and invasion of PC3 and DU145 cell lines [10,42]. NF-kB, hypoxia, and VEGF could also regulate the expression of CXCR4 to affect the progression and metastasis of prostate cancer [8– 10]. MicroRNAs were potential regulators of SDF-1/ CXCR4 pathway by targeting CXCR4. The roles of miRs in prostate cancer development and progression have been explored by several studies [17,43,44]. For instance, compared to metastatic prostate cancer, miR-let7c, miR-100, and miR-218 were significantly over-expressed in localized high Gleason score, pT3 prostate cancer [45]. MiR-221/222 was detected to directly target the tumor suppressor p27Kip1 contributing to the oncogenesis and progression of prostate cancer through p27Kip1 down-regulation [46]. According to previous literatures and bioinformatics analysis, we identified the signatures of miRs (including miR-302a, miR-373, miR-494-3p, miR520c, miR-588, and miR-622), which might be potential regulators of CXCR4 in RWPE-1 and prostate cancer cells. Further analysis suggested that, in contrast to CXCR4, miR-494-3p was the only one that could take part in post-transcription of CXCR4 in advanced prostate cancer cell lines.

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Fig. 4. Effects of miR-494-3p over-expression on cell growth and apoptosis. A: MTTassays showed that PC-3 (P < 0.001between groups) and DU145 (P < 0.001between different groups) cells growth were significantly inhibited concomitant with the miR-494-3p/CXCR4 expression changes. B: Increased cell apoptosis was shown by flow cytometry analysis with percentage of annexinV-APC-stained apoptotic cells. C: TUNEL assays further demonstrated that significant increase of cells apoptosis was induced by the miR-494-3p/CXCR4 expression changesin PC-3 and DU145 cells.

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Fig. 5. The inhibitive effects of miR-494-3p over-expression on migration and invasion in transwell culture system. A: After miR-494-3p up-regulated, the cells in the upper chamber migrated to the lower chamber and the cells were counted at 8, 16, and 24 hr. The migration function of PC-3 (P < 0.001between different time points, respectively) cells was significantly inhibited. B: Over-expression of miR-494-3p retained the capacity of PC-3 (P < 0.001 between different time points, respectively) and DU145 (P < 0.001 between different time points, respectively) cells to invade matrigel-coated transwell filters at 24 and 48 hr.Cells were stained by crystal violet and this experiment wasrepeated three times. The Prostate

Regulation of CXCR4 by MicroRNA-494-3p in Prostate Cancer Some recent researches have revealed that miR-494 is involved in some human diseases, such as childhood leukemia, lung cancer, cardiac injury, head and neck squamous cell carcinoma [20,22,47,48]. Ohdaira and colleagues found that miR-494 could suppress human lung cancer A549 cell proliferation and colony forming activity, and induce senescence regulate by targeting IGF2BP1 [23]. However, the role of miR-494-3p and its target genes in prostate cancer are still to be elucidated and we hypothesized that the under-expression of miR-494-3p in aggressive prostate cancer cells might increase their aggressive potential ability through the activation of CXCR4. As mentioned above, CXCR4 was a potential target of miR-494-3p according to the result of bioinformatics analysis. There was one effective binding site in the CXCR4 30 -UTR and the hypothesis was strengthened by our findings. In order to demonstrate the hypothesis finally, dual luciferase reporter gene assay was used and the relative luciferase activity decreased up to 63.2% after up-regulation of miR-494-3p in PC-3 cells. So, CXCR4 was a post-transcriptional target of miR-494-3p. In this study, we observed the biological behavior of PC-3 and DU145 cells after artificial over-expression of miR-494-3p. First, we provided functional evidence that miR-494-3p/CXCR4 axis could induce a decrease of cell growth in PC-3 and DU145 cells by MTT. And this property was accompanied by their increased ability of apoptosis, a feature clearly connected to prostate cancer proliferation. Second, we also collected experimental evidences that prostate cancer migration and invasion abilities were inhibited when miR-494-3p was over-expressed. These were powerful proofs that miR-494-3p/CXCR4 signal pathway was involved in the proliferation and directional metastasis of prostate cancer. To our knowledge, regulatory pathways caused by miRNAs are very complex processes in the cell, individual miRNA may regulate several targets by partial base pairing to mRNAs and a particular target is probably modulated by a few miRNAs via different number and types of binding sites in the 30 -UTR of target genes [49]. In previous studies, researchers found that CXCR4 could also be regulated by miR146a in Kaposi’s sarcoma, miR-155 in breast cancer, miR-1 in thyroid adenomas and carcinomas, and miR-302-367 cluster in glioblastoma multiforme [36,50–52]. When we screened miRs, which may be the potential regulators of CXCR4, however, we found that miR-302a was barely detectable in prostate cancer cells and RWPE-1. So, we thought that the expression profiles were different among cancers and miR-494-3p/CXCR4 signal pathway was special in prostate cancer.

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CONCLUSIONS In summary, our results suggested that over-expression of miR-494-3p might play crucial role in prostate cancer by post-transcriptionally regulating CXCR4 mRNA. We identified CXCR4 as a functional target of miR-494-3p and showed that the abnormalities of the miR-494-3p/CXCR4 pair might contribute to the progression and metastasis of prostate cancer. We believe that our study on miR-494-3p and its target gene CXCR4 would lead to a better understanding of mechanisms mediating the development and progression of prostate cancer. REFERENCES 1. Vandercappellen J, Van Damme J, Struyf S. The role of CXC chemokines and their receptors in cancer. Cancer Lett 2008;267:226–244. 2. Mantovani A, Savino B, Locati M, Zammataro L, Allavena P, Bonecchi R. The chemokine system in cancer biology and therapy. Cytokine Growth Factor Rev 2010;21:27–39. 3. Darash-Yahana M, Pikarsky E, Abramovitch R, Zeira E, Pal B, Karplus R, Beider K, Avniel S, Kasem S, Galun E, Peled A. Role of high expression levels of CXCR4 in tumor growth, vascularization, and metastasis. FASEB J 2004;18:1240–1242. 4. Vindrieux D, Escobar P, Lazennec G. Emerging roles of chemokines in prostate cancer. Endocr Relat Cancer 2009;16:663–673. 5. Hirbe AC, Morgan EA, Weilbaecher KN. The CXCR4/SDF-1 chemokine axis: A potential therapeutic target for bone metastases? Curr Pharm Des 2010;16:1284–1290. 6. Kim J, Yip ML, Shen X, Li H, Hsin LY, Labarge S, Heinrich EL, Lee W, Lu J, Vaidehi N. Identification of anti-malarial compounds as novel antagonists to chemokine receptor CXCR4 in pancreatic cancer cells. PLoS ONE 2012;7:e31004. 7. Chen G, Chen SM, Wang X, Ding XF, Ding J, Meng LH. Inhibition of chemokine (C-X-C motif) ligand 12/chemokine (CX-C motif) receptor 4 axis (CXCL12/CXCR4)-mediated cell migration by targeting mammalian target of rapamycin (mTOR) pathway in human gastric carcinoma cells. J Biol Chem 2012;287:12132–12141. 8. Schutyser E, Su Y, Yu Y, Gouwy M, Zaja-Milatovic S, Van Damme J, Richmond A. Hypoxia enhances CXCR4 expression in human microvascular endothelial cells and human melanoma cells. Eur Cytokine Netw 2007;18:59–70. 9. Salcedo R, Wasserman K, Young HA, Grimm MC, Howard OM, Anver MR, Kleinman HK, Murphy WJ, Oppenheim JJ. Vascular endothelial growth factor and basic fibroblast growth factor induce expression of CXCR4 on human endothelial cells: In vivo neovascularization induced by stromal-derived factor-1alpha. Am J Pathol 1999;154:1125–1135. 10. Shanmugam MK, Manu KA, Ong TH, Ramachandran L, Surana R, Bist P, Lim LH, Prem Kumar A, Hui KM, Sethi G. Inhibition of CXCR4/CXCL12 signaling axis by ursolic acid leads to suppression of metastasis in transgenic adenocarcinoma of mouse prostate model. Int J Cancer 2011;129:1552–1563. 11. Avecilla ST, Hattori K, Heissig B, Tejada R, Liao F, Shido K, Jin DK, Dias S, Zhang F, Hartman TE, Hackett NR, Crystal RG, Witte L, Hicklin DJ, Bohlen P, Eaton D, Lyden D, de Sauvage F, Rafii S. Chemokine-mediated interaction of hematopoietic pro-

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