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. 2012 Nov 27;288(1):1–9. doi: 10.1074/jbc.M112.395947

Identification of microRNA-98 as a Therapeutic Target Inhibiting Prostate Cancer Growth and a Biomarker Induced by Vitamin D

Huei-Ju Ting ‡,§, James Messing ‡, Sayeda Yasmin-Karim , Yi-Fen Lee ‡,1
PMCID: PMC3537001  PMID: 23188821

Background: MicroRNAs play an important role in the anti-tumor effect of vitamin D in prostate cancer.

Results: Vitamin D regulates miR-98 which suppresses CCNJ and prostate cancer cell growth.

Conclusion: miR-98 mediates vitamin D anti-proliferative effect and correlates with vitamin D treatment in blood.

Significance: miR-98 can inhibit the growth of prostate cancer and measure vitamin D treatment.

Keywords: Cancer Prevention, MicroRNA, Prostate Cancer, Transcription Regulation, Vitamin D

Abstract

The anti-tumor effect of vitamin D has been well recognized but its translational application is hindered by side effects induced by supra-physiological concentration of vitamin D required for cancer treatment. Thus, exploring the vitamin D tumor suppressive functional mechanism can facilitate improvement of its clinical application. We screened miRNA profiles in response to vitamin D and found that a tumor suppressive miRNA, miR-98, is transcriptionally induced by 1α,25-dihydroxyvitamin D3 (1,25-VD) in LNCaP. Mechanistic dissection revealed that 1,25-VD-induced miR-98 is mediated through both a direct mechanism, enhancing the VDR binding response element in the promoter region of miR-98, and an indirect mechanism, down-regulating LIN-28 expression. Knockdown of miR-98 led to a reduction of 1,25-VD anti-growth effect and overexpression of miR-98 suppressed the LNCaP cells growth via inducing G2/M arrest. And CCNJ, a protein controlling cell mitosis, is down-regulated by miR-98 via targeting 3′-untranslated region of CCNJ. Interestingly, miR-98 levels in blood are increased upon 1,25-VD treatment in mice suggesting the biomarker potential of miR-98 in predicting 1,25-VD response. Together, the finding that growth inhibitive miR-98 is induced by 1,25-VD provides a potential therapeutic target for prostate cancer and a potential biomarker for 1,25-VD anti-tumor action.

Introduction

The anti-tumor effects of vitamin D against advanced prostate cancer (PCa)2 are well recognized in pre-clinical and clinical studies. However, the therapeutic window of vitamin D treatment is extremely narrow and effective doses cannot be administered without inducing hypercalcemia. Therefore, much effort has been directed toward the identification of new analogs with potent cell-regulatory action as well as decreased risk of inducing adverse calcemic side effects (1, 2). One of the most promising synthetic analogs is Seocalcitol (EB1089, Leo Pharmaceutical Products) (3). In addition, combination treatments consisting of vitamin D analogs and other agents such as dexamethasone or Taxotere, a chemotherapy drug, have also been tested on PCa cells and in clinical trials (4, 5). All those compounds or vitamin D combined therapies have decreased side effects, yet their efficacy in human clinical trials has not been validated. The anti-tumor effect of vitamin D includes anti-proliferation through accumulation of cells in the G0/G1 or G2/M phase (6–9), induction of apoptosis (10, 11), inhibition of angiogenesis (12), and anti-metastasis (13, 14). In addition, it appears that PCa cells have diverse sensitivity to vitamin D, and can become resistant to the tumor suppressive effects of vitamin D. In our experimental model system, long-term treatment of PCa cells with the vitamin D active metabolite, 1α,25-dihydroxyvitamin D3 (1,25-VD), can induce vitamin D resistance in PCa cell culture. Our efforts in improving the anti-tumor effects of 1,25-VD led us to explore the potential role of microRNAs (miRNAs) in mediating this effect.

miRNAs are small non-coding single-stranded RNAs of ∼22 nucleotides that destabilize and inhibit the translation of target messenger RNAs (mRNAs). The fact that one single miRNA can control hundreds of target mRNAs is striking and makes miRNAs as powerful as transcription factors in regulating whole cell proteomes. The multifaceted roles of miRNAs in cancer, including PCa, have gained increasing appreciation after recently accumulated evidence. More importantly, miRNA roles in influencing cancer biology, including proliferation, apoptosis, and invasiveness, have proven indispensable. Recently, reports provide evidence supporting the role of certain miRNAs in PCa progression, such as loss of miR-15/16 and miR-101 are often associated with advanced PCa (15, 16). Indeed, introduction of miR-15/16 into PCa inhibits cell growth, while knockdown induces tumorigenicity and invasiveness of normal prostate cells (15). Introduction of miR-101 into PCa cells could suppress growth, invasiveness, and xenograft tumor growth, while knockdown increases invasiveness of benign cells (16). In addition, miR-221 and miR-222 are found to promote PCa cell growth while knockdown of both decrease PCa clonogenicity (17), suggesting oncogenic properties of miR-221 and miR-222. The idea of targeting miRNAs in PCa therapy has been pre-clinically tested and showed promising results. Exploiting miRNAs in improving the efficacy of current therapy could broaden the strategies for battling advanced PCa.

The roles of miRNAs in mediating the vitamin D anti-tumor action remain unclear and demand investigation. It was reported that VDR level is repressed by miR-125b, thus overexpression of miR-125b attenuates the anti-tumor effect of 1,25-VD (18). Vitamin D and testosterone can regulate miRNAs in an additive or synergistic manner in LNCaP (19). Among these miRNAs, the role of miR-22 in mediating the anti-proliferative and anti-migratory effect of vitamin D has been proven important in colon cancer (20). To identify which miRNAs participate in the anti-tumor effect of vitamin D, we profiled miRNAs expression by microarray in the 1,25-VD-treated PCa cell line, LNCaP, and validated that miR-98, a tumor suppressive miRNA, is regulated by 1,25-VD. We further discovered the dysregulation of miRNAs expression in vitamin D resistant LNCaP sublines. Modulation of miR-98 affects the anti-tumor effect of vitamin D suggesting that miR-98 can be a potential target for developing therapies for PCa.

EXPERIMENTAL PROCEDURES

Plasmids and Reagents

Ambion® Pre-miRTM control and miR-98 precursors, and Anti-miRTM control and miR-98 inhibitors were purchased from Invitrogen (Carlsbad, CA). Transfection of anti-miRNA and miRNA precursors was performed using siPORTTM NeoFXTM (Invitrogen). The plasmid pcDNA3.1(-)miR-98 was constructed by PCR amplification of miR-98 from genomic DNA of LNCaP cells using primer pairs XhoI-miR98-s: 5′-CCGCTCGAGTTTGCCTGCTGCCCTTATTAG-3′ and BglII-miR98-as: 5′-CGAGATCTCAACACTGCTAAGACTAAGTG-3′ then inserted into the XhoI and BglII sites of pcDNA3.1(-) vector. The plasmid pGL3-p-LUC-CCNJ 3′-UTR is constructed by inserting CCNJ 3′-UTR, amplified by PCR using primers CCNJ-3′UTR-f: 5′-GCTCTAGATGGGGGAAAGTAGGCTCAAAAGAGA-3′ and CCNJ-3′ UTR-r 5′-GCTCTAGAAAGTTACCACAGGCACTGGCTG-3′, into XbaI site of pGL3-p-LUC (Promega, Madison, WI). LIN 28A and LIN28B antibodies were purchased from Abcam (Cambridge, MA).

Establishing Stable Cell Lines

LNCaP stable cell lines expressing shRNA-targeting VDR (shVDR) and control shRNA (SC) were generated as described in a previous publication (21). LNCaP stable cell line expressing miR-98 (LNCaPmiR98) was established by transfecting LNCaP with pcDNA3.1(-)miR-98 plasmid. Two days after transfection, cells were selected by 350 μg/ml G418 (Invitrogen) for 10 days. Single clones were selected by series dilution. The overexpression of miR-98 was confirmed by Q-PCR.

Colony Formation Assay

Cells were seeded on 6-well dishes at a density of 104 cells/well. Cells were treated with EtOH or 1,25-VD of different concentration every other day. Two weeks after treatment, colonies were fixed and stained by crystal violet. After taking images by Chemidoc (Bio-Rad), colonies were counted by QuantityOne software (Bio-Rad).

ChIP Assay

ChIP assays were performed according to a previous publication (22). DNA fragments containing VDREs in miR-98 promoter were amplified by the following specific primer pairs. VDRE-A: 5′-GGAGCAGCATGTATCTGAGGGGC-3′ (forward) and 5′-GGGGGATGTCTGCTGCAAGGA-3′ (reverse); VDRE-B: 5′-AGGTGCGTGGCAGCTTGGTT-3′ and(forward) and 5′-TGTTTCCTGCGCTTCTCTCGCC-3′ (reverse); VDRE-C: 5′-TGGAGCCAGAAGCAGAATGGCT-5′ and 5′-CCCCACACTGGCAGTTCACCG-3′ (reverse); VDRE-D: 5′-TTCCCCCTCAGCCCCAAACAA-3′ (forward) and 5′-AATGTGGGACTTTGCAAGCACCT-3′ (reverse).

Quantitative PCR (Q-PCR) Assays and Primers

Total RNA was harvested from cells by Trizol (Invitrogen) and converted to cDNA using NCodeTM VILOTM miRNA cDNA synthesis kit (Invitrogen). Q-PCR was performed as in the previous report (22). Primers used are U6: 5′-CTCGCTTCGGCAGCACA-3′; miR-98: 5′-TGAGGTAGTAAGTTGTATTGTT-3′; LIN28A: 5′-AGCGCAGATCAAAAGGAGACA-3′ (forward) and 5′-CCTCTCGAAAGTAGGTTGGCT-3′ (reverse); LIN28B: 5′-TTGAGTCAATACGGGTAACAGGA-3′ (forward) and 5′-TGACAGTAATGGCACTTCTTTGG-3′ (reverse); CCNJ: 5′-CTGGCCGCCGATATTCACC-3′ (forward) and 5′-GGCAATCAAGTCAGCAAAATACC-3′ (reverse).

Cell Cycle Analysis

After indicated treatments, cells were trypsinized, washed by PBS, and fixed in 70% EtOH overnight. Cells were then pelleted and incubated in PBS containing 0.05 mg/ml RNase A for 30 min at room temperature. After washing, the cells were stained with 10 μg/ml propidium iodide for 10 min then washed three times with PBS. Cells were resuspended in PBS at 106 cells/ml. Cell cycle profiles were determined by flow cytometry using FACSCanto-II (BD, Franklin Lakes, NJ) and analyzed by FlowJo software.

Mouse Models

The study was approved by the University of Rochester Committee on Animal Resources, and the mice were kept in a specific pathogen-free environment at the animal facility of the University of Rochester Medical Center. PTEN(−/+) and TRAMP mice were obtained from The Jackson Lab (Bar Harbor, Maine). Adult male mice at 24–26 weeks of age were intraperitoneally injected with 1,25-VD (25 ng/g of mice weight) in corn oil. After 24 h, mice were anesthetized for drawing cardiac blood which was then mixed with EDTA (0.2%) to prevent coagulation. Total blood cells were spun down and RNA extracted by Trizol.

Cell viability assay, Western blotting assay, transient transfection, and luciferase assays were performed according to previous publications (22, 23).

RESULTS

Vitamin D Induces miR-98 Expression in LNCaP Cells

In search of miRNAs mediating the anti-proliferative effect of 1,25-VD, we performed systemic miRNA array profiling comparing LNCaP cells treated with ethanol (EtOH) vehicle control and 1,25-VD (LC Sciences). We performed two independent experiments in microarray study. There are 8 miRNAs consistently up- or down-regulated by 1,25-VD with statistical significance in individual experiments (Fig. 1A). Further in-depth analysis provided by LC Science shows that miR-98 is the miRNA showing strong signals (>500) in array; therefore it was chosen for further validation and exploration for its roles in mediating 1,25-VD anti-tumor effects. First, we confirmed 1,25-VD effects on the miR-98 expression by Q-PCR in LNCaP sublines with different vitamin D responsiveness: vitamin D-resistant line (LNCaP-r) versus parental cells (LNCaP-p) (24). As shown in Fig. 1B, miR-98 expression was induced by 1,25-VD in LNCaP-p cell, but this miR-98 induction by 1,25-VD was diminished in the LNCaP-r cells. This suggests the regulation of miR-98 is through 1,25-VD/VDR transcriptional activity. To further verify VDR's involvement in this regulation, we knocked down VDR by shRNA against VDR and examined the miR-98 expression upon 1,25-VD treatment. We found that VDR expression was reduced in VDR shRNA clones as compared with scramble shRNA controls (Fig. 1C), and 1,25-VD induced miR-98 in scramble shRNA expressed control lines, sc8 and sc10, but not in VDR knockdown clones, shVDR3 and shVDR7 (Fig. 1D). In summary, 1,25-VD-induced miR-98 is VDR-dependent.

FIGURE 1.

FIGURE 1.

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The vitamin D induction of miR-98 is dependent on VDR transactivity in LNCaP cells. A, array profiling of miRNAs expression in 1,25-VD treated LNCaP cells showed 8 miRNAs significantly regulated by 1,25-VD treatment. LNCaP cells were treated with EtOH or 1,25-VD (100 nm) for 24 h then harvested for RNA extraction. Total RNAs were submitted to LC Science for array profiling of miRNAs expression. The relative expression of miRNAs in 1,25-VD-treated group compared with EtOH-treated groups with statistical significance (p < 0.01) in two independent experiments was summarized. B, vitamin D-induced miR-98 in LNCaP parental cells but not in vitamin D-resistant cells. Cells were treated with EtOH or 1,25-VD 100 nm for 24 h, then total RNAs harvested from cells were reverse-transcribed to cDNA using VILO NCode kit. The expression of miR-98 and U6 was measured by Q-PCR. The relative miR-98 expression, after being normalized with U6 in 1,25-VD-treated group compared with EtOH-treated group, was calculated and mean ± S.D. plotted. *, p < 0.05 compared with EtOH-treated group in each subline. C, VDR expression in LNCaP cell lines stably expressed with scramble shRNA (subline sc8 and sc10) or shRNA against VDR (subline shVDR3 and shVDR7). Total RNA harvested from cells was reverse-transcribed to cDNA. The mRNAs of VDR and actin were measured by Q-PCR. After being normalized with actin, the relative mRNA expression of VDR compared with LNCaP-sc8 cells was calculated and mean ± S.D. plotted. *, p < 0.05 compared with sc sublines. D, vitamin D induction of miR-98 is diminished in the VDR knockdown LNCaP cells. Cells were treated and harvested for detecting miR-98 expression as in B. The relative expression of miR-98 normalized by U6 was calculated and mean ± S.D. plotted. *, p < 0.05 1,25-VD treated group compared with EtOH-treated group in each subline.

The Induction of miR-98 Contributes to the Anti-proliferative Effect of 1,25-VD

MiR-98 belongs to the Let-7 family which is well categorized as a tumor suppressive miRNA. It has been shown to regulate HMGA2, a gene controlling growth and tumorigenesis (25). Therefore, we suspect that miR-98 is involved in the anti-proliferative mechanism of 1,25-VD. Cells were transfected with antagomirs of miR-98 (anti-miR-98) to knock down miR-98 expression; the 1,25-VD responsiveness was examined. As expected, the 1,25-VD induction of miR-98 was diminished in the anti-miR-98 transfected cells (Fig. 2A); consequently, 1,25-VD-mediated growth inhibition effect was significantly reduced in the anti-miR-98 cells as compared with cells that were transfected with control antagomirs (Fig. 2B). This indicates the importance of miR-98 in mediating the anti-proliferative effect of 1,25-VD. On the other hand, overexpression of miR-98 precursor, pre-miR-98, increased more than 400 fold the expression of miR-98 (Fig. 2C). The overexpression of miR-98 alone can suppress LNCaP cells growth up to 30% and treatment with 1,25-VD in the miR-98 overexpressed cells does not further suppress cell growth (Fig. 2D). We further examined the growth inhibitive effect of miR-98 by colony formation assay detecting cell survival and cell viability and by direct cell counting. In order to perform this study, we established miR-98 stably expressed LNCaP cells (LNCaPmiR98) (Fig. 2E). The result showed dramatic inhibition of cell survival and cell viability and by miR-98 when it was stably expressed in LNCaP cells (Fig. 2, F and G). Treatment of 1,25-VD further suppressed the colony numbers and cell viability. This suggests miR-98 is a key mediator of the anti-proliferative effect of 1,25-VD.

FIGURE 2.

FIGURE 2.

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Modulation of miR-98 affects the anti-growth effect of 1,25-VD in LNCaP. A, expression of miR-98 was knocked down by antagomir against miR-98. One day after being transfected with antagomirs, LNCaP cells were treated with EtOH or 1,25-VD for 24 h. Total RNA was harvested then reverse-transcribed to cDNA. The relative expression of miR-98 was measured and plotted as described in Fig. 1B. *, p < 0.05. B, anti-growth effect of 1,25-VD in LNCaP was decreased by antagomir of miR-98. LNCaP cells were transfected with control antagomirs and miR-98 antagomirs. After 24 h, cells were treated with EtOH or 100 nm 1,25-VD every 2 days. Viable cells were detected and quantified by MTT assay at 2, 4, 6 days after treatment. The viable cells of the EtOH-treated group at the day of assay was set as 100% then relative viable cells were calculated and mean% ± S.D. plotted (n = 3). *, p < 0.05 and **, p < 0.01 when compared with the EtOH-treated group. #, p < 0.05 when compared with 1,25-VD treated group. C, RNA was harvested as described in A. Amount of miR-98 was quantified by Q-PCR. The relative expression level compared with control pre-miRNA transfected, EtOH-treated group was calculated and mean ± S.D. plotted. **, p < 0.01 compared with EtOH-treated control group. D, LNCaP cells were seeded and transfected with control or pre-miR98 oligonucleotides. After 24 h, cells were treated with EtOH or 1,25-VD (1∼100 nm) every 2 days. At day 6 after treatment, viable cells were measured by MTT assay. The relative percentages of survival cells compared with the group transfected with control pre-miRNA and treated with EtOH were calculated and plotted. **, p < 0.01 compared with control group of same treatment. E, LNCaP stable line overexpressing miR-98 was established by transfecting pcDNA3.1(-)miR-98 followed by neomycine selection. The expression of miR-98 in parental and the stable line LNCaPmiR98 was examined by Q-PCR. F, colony formation assay was performed by seeding 104 LNCaP and LNCaPmiR98 cells in 6-well culture dishes. One day after seeding, cells were treated with EtOH or indicated concentration of 1,25-VD every other day. Two weeks after treatment, cells were stained and counted. The colony forming percentage was calculated by setting colony number of EtOH-treated LNCaP cells as 100% then mean ± S.D. plotted. G, cell viability assay were performed by seeding 25,000 cells/well of 12 wells, and treated with EtOH, or indicated concentration of 1,25-VD every other day. Cells were trypsinized at day 6 for counting by Vi-CELL XR 2.03 (Beckman Coulter, Inc.)

1,25-VD Regulates miR-98 Expression via Direct and Indirect Mechanisms

The transcriptional regulation of miR-98 has not yet been studied. Its transcription start (TSS) site has been predicted to be located at 3070 bp in 5′ upstream, within the host gene HUWE1 (26). We searched for putative VDR response elements (VDREs) within 2 kb flanking TSS sites by ChIP mapper online program, and four putative VDREs were identified (Fig. 3A). We performed a ChIP assay to test the binding between VDR and putative VDREs and found that VDR bound all VDREs in a ligand-independent manner, except VDRE-B (Fig. 3, B and quantified result in C), suggesting that VDR can bind to VDREs in 5′ promoter of miR-98 to induce its expression. In addition to direct regulation of miR-98 by VDR, we also tested the possibility that 1,25-VD induces miR-98 via an indirect mechanism, such as through regulation of LIN28 proteins that have been shown to bind to the let-7 family pre-microRNA and block production of the mature microRNAs (27). We found that 1,25-VD suppressed LIN28A and LIN28B expression (Fig. 3, D and E), and therefore could consequently result in miR-98 induction. Overall, these results suggest 1,25-VD regulates miR-98 via both direct and indirect mechanisms.

FIGURE 3.

FIGURE 3.

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The regulation of miR-98 by 1,25-VD is through both direct and indirect mechanism. A, position of four putative VDREs in miR-98 genome (VDRE-A∼D) was identified and shown. The sequence of putative VDREs corresponding to VDRE motif models is listed on the right. B, ChIP assay demonstrated VDR-bound VDREs in miR-98 5′ region. LNCaP cells were treated with EtOH or 100 nm 1,25-VD for 2 h then fixed by formaldehyde. ChIP assays were performed to pull-down VDR-DNA complex by antibody against VDR (αVDR) or control IgG. Primers amplifying DNA fragments containing potential VDREs were applied to detect VDREs bound by VDR. Primers amplifying CYP24 VDRE were served as positive controls. C, for each putative VDRE site, the relative intensity of PCR products amplified from ChIP complex compared with IgG control in each treatment group from three independent experiments was calculated and mean ± S.D. plotted. **, p < 0.01. D and E, LIN28 expression was suppressed by 1,25-VD. LNCaP cells were treated with EtOH or 100 nm 1,25-VD for 24 h then harvested for RNA and protein extraction. D, mRNA expression levels of LIN28A and LIN28B were detected by Q-PCR. The relative expression compared with EtOH-treated group was calculated then mean ± S.D. plotted. **, p < 0.01. E, protein expression levels of LIN28A and LIN28B were detected by Western blotting (upper panel). After being normalized by actin, the relative intensity of bends representing LIN28A or LIN28B in 1,25-VD-treated group compared with EtOH-treated group was calculated and plotted (lower chart).

MiR-98 Induces G2/M Arrest and Down-regulates CCNJ

The significant growth suppressive effect of miR-98 in LNCaP was further investigated by cell cycle analysis. LNCaP cells were stably transfected with miR-98 or control vector, then cells were treated with 1,25-VD and analyzed for cell cycle distribution. The result showed that 1,25-VD treatment alone increased G1 distribution while miR-98 overexpression induced G2/M arrest (Fig. 4, A and B). Interestingly, miR-98-overexpressed cells were further arrested in the G1 cell cycle by 1,25-VD. To investigate the miR-98's molecular target that controls G2/M cell cycle, we searched targets of miR-98 by TargetScan and found cyclin J (CCNJ), which has been linked with G2/M cell cycle in early Drosophila embryogenesis (28). We therefore examined the expression of CCNJ in 1,25-VD-treated and miR-98-overexpressed cells and found both 1,25-VD and miR-98 suppressed CCNJ expression but the combination did not have an additive effect (Fig. 4C). The molecular mechanism by which miR-98 regulates CCNJ was further investigated where we inserted 3′-UTR of CCNJ that contains the putative miR-98 target site into 3′ region of LUC cDNA in pGL3-p-LUC plasmid (Fig. 4D). Overexpression of miR-98 reduced LUC expression while knockdown miR-98 induced LUC expression (Fig. 4E). These results suggested that miR-98 down-regulates CCNJ, hence impairing mitosis progression.

FIGURE 4.

FIGURE 4.

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Expression of miR-98 controls G2/M arrest via down-regulation of CCNJ. A, ectopic expression of miR-98 arrest cells in G2/M phase. LNCaP and LNCaPmiR98 cells (with miR-98 overexpression) were treated with EtOH or 1,25-VD for 48 h then harvested for cell cycle analysis. The representative histograms analyzed by FlowJo were shown. B, percentage of cells in G1, S and G2/M phase were calculated and mean ± S.D. of three independent experiments plotted. *, p < 0.05. C, expression of CCNJ was suppressed by miR-98 and 1,25-VD. LNCaP cells with or without ectopic expression of miR-98 were treated with EtOH or 1,25-VD (1, 10, or 100 nm). After 24 h, cells were harvested for RNA extraction. The relative mRNA expression level of CCNJ compared with EtOH treated LNCaP cells was detected by Q-PCR and mean ± S.D. plotted. *, p < 0.05 comparing with EtOH-treated LNCaP. D, MiR-98-regulated CCNJ via complementing its 3′ sequence. Software predicted miR-98 binding site in CCNJ mRNA is shown. E, expression of CCNJ was suppressed by miR-98 through 3′-UTR. LNCaP cells were transfected with control pre-miRNA (pre-C), miR-98 mimics (pre-miR98), control antagomirs (anti-C), or antagomirs of miR-98 (anti-miR98). After 24 h, cells were transfected with pGL3-p-LUC (p-LUC) or pGL3-p-LUC containing CCNJ 3′-UTR in the 3′ of LUC (p-LUC-CCNJ). After 24 h, cells were harvested for LUC activity assay. After being normalized by Renilla-LUC activity, the relative LUC activity of p-LUC-CCNJ compared with p-LUC in each group was calculated and mean ± S.D. plotted. *, p < 0.05; **, p < 0.01 compared with p-LUC of the same transfectants.

The Level of miR-98 in Blood Is Correlated with 1,25-VD Treatment in Mice

The potential of miRNA as a biomarker in body fluid has been reported. We expect miR-98 will be a useful functional biomarker predicting 1,25-VD response. The individual variation in response to 1,25-VD is contributed by a variety of factors, including metabolizing enzyme level, VDR polymorphism, cellular context of coregulators, etc. Functional biomarker measuring response to 1,25-VD will be useful in predicting individual response to vitamin D-based therapy. To prove the principle, mice including wild type and prostate cancer mice models, PTEN heterozygous deletion and TRAMP, were treated with 1,25-VD, and their blood sample was harvested for measuring miR-98 level. The result showed an induction of miR-98 in blood cells from mice treated with 1,25-VD regardless of genetic background (Fig. 5). These data indicated that miR-98 can be measured from the blood and 1,25-VD can induce its expression, thus, miR-98 level can potentially be developed as a biomarker to predict 1,25-VD anti-tumor activity.

FIGURE 5.

FIGURE 5.

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MiR-98 expression in blood is correlated with vitamin D treatment. Mice of different genotypes (wild type, PTEN−/+, and TRAMP) were intraperitoneally injected with corn oil containing EtOH or 25 ng of 1,25-VD per gram of the mouse body weight. After 24 h, mice were sacrificed and cardiac blood harvested for extraction of total RNA from cell fraction. The miR-98 expression was detected by Q-PCR then the relative expression compared with EtOH-injected mice was calculated and mean ± S.D. plotted. *, p < 0.05; **, p < 0.01 compared with EtOH-treated group.

DISCUSSION

Exploring miRNAs regulation as a novel mechanism through which vitamin D inhibits cell growth identifies the proliferation inhibitive effect of miR-98. Previous studies identify miR-22 and miR-32 induced by vitamin D in colon cancer cells and human myeloid leukemia cells, respectively (20, 29). The function of miR-22 contributes to the anti-growth and anti-migration effect of 1,25-VD in colon cancer cells (20). On the other hand, vitamin D induced miR-32 down-regulates the pro-apoptotic protein Bim and leads to anti-apoptotic effect of vitamin D. It is therefore suggested that down-regulation of miR-32 can enhance the therapeutic efficacy in leukemia patients (29).

Considerable preclinical and epidemiologic data suggest that vitamin D plays a critical role in pathogenesis, progression and therapy for PCa. However, the outcomes from the clinical trials that used 1,25-VD as a second line of therapy for advanced PCa patients are inconclusive. This is mainly due to lack of suitable pharmaceutical preparation that optimizes biologic dose and/or maximum-tolerated dose of 1,25-VD for clinical study. Thus, developing the vitamin D based therapy aiming to reduce hypercalcemic side effects has been one of the major efforts in the field. The discovery of oncomirs, tumor-suppressive, and oncogenic miRNAs, provides novel therapeutic targets for cancer therapy. There are promising results showing that locked-nucleic acid modified oligonucleotides knockdown miR-122, hence inhibiting HCV replication in liver; and intranasal administration of let-7 effectively reduces lung cancer in K-ras mutant mouse model (30). In addition to directly targeting oncomirs, miRNAs modulators can also sensitize cancer to radiation, chemotherapy, and hormone depletion therapy (31–33). Therefore, miRNAs modulators are suitable for developing combination therapy to sensitize cancer to vitamin D treatment. The findings of others and our study indicate that miR-22 and miR-98 mediate the growth inhibitory effect of 1,25-VD. These provide a therapy strategy where combining 1,25-VD and miRNAs modulators, such as ectopically introducing tumor suppressive miRNAs (miR-22 and miR-98) or knockdown VDR signal down-regulating miRNAs (miR-125b and miR-27), could reduce the dosage of 1,25-VD, hence avoiding the hypercalcemic side effect. Nonetheless, developing safe and tissue specific delivery methods to knockdown or replaced miRNAs for PCa therapy is necessary to prevent toxicity and off-target effects.

The vitamin D regulation of miR-98 is correlated with VDR transactivity. We therefore seek VDREs in the promoter region of miR-98, which is located at its host gene HUWE1. We confirmed VDR binding of putative VDREs in the promoter region. In addition to direct regulation, we also found 1,25-VD indirectly regulates miR-98 via down-regulation of LIN28, the miRNAs processing proteins. The mechanism of 1,25-VD regulation of LIN28 is possible through regulation of NFκB activity that needs to be further investigated. The balance between LIN28 and Let-7 family is found to control the stemness of stem cells (34, 35). A recent study identifies that miR-125b, the microRNA down-regulating VDR, can suppress the differentiation of hair follicles and maintains stemness of stem cell progenies. Treatment with 1,25-VD can rescue the hair growth defect in mice with doxycycline-induced expression of miR-125b in skin (36). We therefore suspect 1,25-VD treatment could control the balance between quiescence/self-renewal and differentiation status of stem cells through regulation of LIN28.

The known targets of miR-98 include an oncogene (HMGA2), a tumor suppressor (FUS1), and a immune-modulator, cytokine-inducible src homology 2-containing protein (CIS) (25, 37, 38). We observed the G2/M arrest in cells overexpressing miR-98 and identified a mitosis controlling cyclin, CCNJ, which is down-regulated by miR-98 via targeting its 3′-UTR. Interestingly, treatment of 1,25-VD in miR-98 overexpressed cells results in increasing G1 arrest as the effect of 1,25-VD alone. We therefore suspect other mechanisms through which miR-98 triggers can sensitize the 1,25-VD-induced G1 arrest in LNCaP. One possible mechanism is through reciprocal suppression of LIN28B, which is one of the predicted miR-98 targets in TargetScan. The down-regulation of LIN28B promotes differentiation of LNCaP cells, thereby enabling cells responding to the G1 arrest effect of 1,25-VD. Another interesting candidate target of miR-98 is enhancer of zeste homolog 2 (EZH2), which functions in protein complex to trimethylate histone H3 lysine 27 leading to epigenetic silencing (16). It is suspected that by down-regulation of EZH2, anti-proliferative genes, especially those involved in G1 arresting, is de-silenced, therefore enabling 1,25-VD to induce VDR up-regulating their expression. Whether CCNJ or other not-yet identified miR-98 targets contribute to the role of miR-98 in mediating the growth inhibitory effect of 1,25-VD requires further study.

Biomarker potential of miRNAs relies on their high stability and existence in formalin-fixed tissue, cell free serum/plasma, and urine (39). The biological response to vitamin D varied among individuals. Factors contributing to a broad range of sensitivity to vitamin D include genomic polymorphisms of VDR and metabolism enzymes and pathological conditions altering VDR activity. Biomarkers representing biological response of vitamin D treatment will be more meaningful than measurement of serum vitamin D level. A recent study investigates the serum level of miRNAs in subjects given high dose vitamin D3 (20,000 and 40,000 IU) weekly for a year (40). Although there is not yet a conclusive result, measuring miRNAs in body fluid to identify the response to vitamin D is worth pursuing. We therefore explore the biomarker potential of miR-98 representing vitamin D treatment in mice bearing prostate cancer. The cellular fraction of blood contains miR-98 whose expression correlates well with vitamin D treatment in both wild type and cancerous mice. Further investigation correlating expression levels of miR-98 and other vitamin D-regulated anti-tumor miRNAs (such as miR-22) in blood or other body fluid with anti-tumor effect of vitamin D is demanded.

Footnotes

2
The abbreviations used are:
PCa
prostate cancer
Q-PCR
quantitative PCR
EtOH
ethanol
1,25-VD
1α,25-dihydroxyvitamin D3
miRNA
microRNA
TSS
transcription start site
VDRE
vitamin D receptor response element.

REFERENCES

  • 1. Spina C. S., Ton L., Yao M., Maehr H., Wolfe M. M., Uskokovic M., Adorini L., Holick M. F. (2007) Selective vitamin D receptor modulators and their effects on colorectal tumor growth. J. Steroid Biochem. Mol. Biol. 103, 757–762 [DOI] [PubMed] [Google Scholar]
  • 2. Marchiani S., Bonaccorsi L., Ferruzzi P., Crescioli C., Muratori M., Adorini L., Forti G., Maggi M., Baldi E. (2006) The vitamin D analogue BXL-628 inhibits growth factor-stimulated proliferation and invasion of DU145 prostate cancer cells. J. Cancer Res. Clin. Oncol. 132, 408–416 [DOI] [PubMed] [Google Scholar]
  • 3. Hansen C. M., Hamberg K. J., Binderup E., Binderup L. (2000) Seocalcitol (EB 1089): a vitamin D analogue of anti-cancer potential. Background, design, synthesis, pre-clinical and clinical evaluation. Curr. Pharm. Des. 6, 803–828 [DOI] [PubMed] [Google Scholar]
  • 4. Trump D. L., Muindi J., Fakih M., Yu W. D., Johnson C. S. (2006) Vitamin D compounds: clinical development as cancer therapy and prevention agents. Anticancer Res. 26, 2551–2556 [PubMed] [Google Scholar]
  • 5. Beer T. M., Ryan C. W., Venner P. M., Petrylak D. P., Chatta G. S., Ruether J. D., Chi K. N., Young J., Henner W. D. (2008) Intermittent chemotherapy in patients with metastatic androgen-independent prostate cancer: results from ASCENT, a double-blinded, randomized comparison of high-dose calcitriol plus docetaxel with placebo plus docetaxel. Cancer 112, 326–330 [DOI] [PubMed] [Google Scholar]
  • 6. Jensen S. S., Madsen M. W., Lukas J., Binderup L., Bartek J. (2001) Inhibitory effects of 1α,25-dihydroxyvitamin D(3) on the G(1)-S phase-controlling machinery. Mol. Endocrinol. 15, 1370–1380 [DOI] [PubMed] [Google Scholar]
  • 7. Polek T. C., Stewart L. V., Ryu E. J., Cohen M. B., Allegretto E. A., Weigel N. L. (2003) p53 Is required for 1,25-dihydroxyvitamin D3-induced G0 arrest but is not required for G1 accumulation or apoptosis of LNCaP prostate cancer cells. Endocrinology 144, 50–60 [DOI] [PubMed] [Google Scholar]
  • 8. Wang Q. M., Jones J. B., Studzinski G. P. (1996) Cyclin-dependent kinase inhibitor p27 as a mediator of the G1-S phase block induced by 1,25-dihydroxyvitamin D3 in HL60 cells. Cancer Res. 56, 264–267 [PubMed] [Google Scholar]
  • 9. Jiang F., Li P., Fornace A. J., Jr., Nicosia S. V., Bai W. (2003) G2/M arrest by 1,25-dihydroxyvitamin D3 in ovarian cancer cells mediated through the induction of GADD45 via an exonic enhancer. J. Biol. Chem. 278, 48030–48040 [DOI] [PubMed] [Google Scholar]
  • 10. Guzey M., Kitada S., Reed J. C. (2002) Apoptosis induction by 1α,25-dihydroxyvitamin D3 in prostate cancer. Mol. Cancer Ther. 1, 667–677 [PubMed] [Google Scholar]
  • 11. Pepper C., Thomas A., Hoy T., Milligan D., Bentley P., Fegan C. (2003) The vitamin D3 analog EB1089 induces apoptosis via a p53-independent mechanism involving p38 MAP kinase activation and suppression of ERK activity in B-cell chronic lymphocytic leukemia cells in vitro. Blood 101, 2454–2460 [DOI] [PubMed] [Google Scholar]
  • 12. Bao B. Y., Yao J., Lee Y. F. (2006) 1α, 25-dihydroxyvitamin D3 suppresses interleukin-8-mediated prostate cancer cell angiogenesis. Carcinogenesis 27, 1883–1893 [DOI] [PubMed] [Google Scholar]
  • 13. Getzenberg R. H., Light B. W., Lapco P. E., Konety B. R., Nangia A. K., Acierno J. S., Dhir R., Shurin Z., Day R. S., Trump D. L., Johnson C. S. (1997) Vitamin D inhibition of prostate adenocarcinoma growth and metastasis in the Dunning rat prostate model system. Urology 50, 999–1006 [DOI] [PubMed] [Google Scholar]
  • 14. Bao B. Y., Yeh S. D., Lee Y. F. (2006) 1α,25-dihydroxyvitamin D3 inhibits prostate cancer cell invasion via modulation of selective proteases. Carcinogenesis 27, 32–42 [DOI] [PubMed] [Google Scholar]
  • 15. Bonci D., Coppola V., Musumeci M., Addario A., Giuffrida R., Memeo L., D'Urso L., Pagliuca A., Biffoni M., Labbaye C., Bartucci M., Muto G., Peschle C., De Maria R. (2008) The miR-15a-miR-16–1 cluster controls prostate cancer by targeting multiple oncogenic activities. Nat. Med. 14, 1271–1277 [DOI] [PubMed] [Google Scholar]
  • 16. Varambally S., Cao Q., Mani R. S., Shankar S., Wang X., Ateeq B., Laxman B., Cao X., Jing X., Ramnarayanan K., Brenner J. C., Yu J., Kim J. H., Han B., Tan P., Kumar-Sinha C., Lonigro R. J., Palanisamy N., Maher C. A., Chinnaiyan A. M. (2008) Genomic loss of microRNA-101 leads to overexpression of histone methyltransferase EZH2 in cancer. Science 322, 1695–1699 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. Galardi S., Mercatelli N., Giorda E., Massalini S., Frajese G. V., Ciafrè S. A., Farace M. G. (2007) miR-221 and miR-222 expression affects the proliferation potential of human prostate carcinoma cell lines by targeting p27Kip1. J. Biol. Chem. 282, 23716–23724 [DOI] [PubMed] [Google Scholar]
  • 18. Mohri T., Nakajima M., Takagi S., Komagata S., Yokoi T. (2009) MicroRNA regulates human vitamin D receptor. Int. J. Cancer J. Int. Cancer 125, 1328–1333 [DOI] [PubMed] [Google Scholar]
  • 19. Wang W. L., Chatterjee N., Chittur S. V., Welsh J., Tenniswood M. P. (2011) Effects of 1α,25 dihydroxyvitamin D3 and testosterone on miRNA and mRNA expression in LNCaP cells. Mol. Cancer 10, 58. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20. Alvarez-Díaz S., Valle N., Ferrer-Mayorga G., Lombardía L., Herrera M., Domínguez O., Segura M. F., Bonilla F., Hernando E., Muñoz A. (2012) MicroRNA-22 is induced by vitamin D and contributes to its antiproliferative, antimigratory and gene regulatory effects in colon cancer cells. Human Mol. Genet. 21, 2157–2165 [DOI] [PubMed] [Google Scholar]
  • 21. Ting H. J., Yasmin-Karim S., Yan S. J., Hsu J. W., Lin T. H., Zeng W., Messing J., Sheu T. J., Bao B. Y., Li W. X., Messing E., Lee Y. F. (2012) A positive feedback signaling loop between ATM and the vitamin D receptor is critical for cancer chemoprevention by vitamin D. Cancer Res. 72, 958–968 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22. Ting H. J., Bao B. Y., Reeder J. E., Messing E. M., Lee Y. F. (2007) Increased expression of corepressors in aggressive androgen-independent prostate cancer cells results in loss of 1α,25-dihydroxyvitamin D3 responsiveness. Mol. Cancer Res. 5, 967–980 [DOI] [PubMed] [Google Scholar]
  • 23. Bao B. Y., Ting H. J., Hsu J. W., Lee Y. F. (2008) Protective role of 1α,25-dihydroxyvitamin D3 against oxidative stress in nonmalignant human prostate epithelial cells. Int. J. Cancer 122, 2699–2706 [DOI] [PubMed] [Google Scholar]
  • 24. Bao B. Y., Ting H. J., Hsu J. W., Yasmin-Karim S., Messing E., Lee Y. F. (2010) Down-regulation of NF-κB signals is involved in loss of 1α,25-dihydroxyvitamin D3 responsiveness. J. Steroid Biochem. Mol. Biol. 120, 11–21 [DOI] [PubMed] [Google Scholar]
  • 25. Hebert C., Norris K., Scheper M. A., Nikitakis N., Sauk J. J. (2007) High mobility group A2 is a target for miRNA-98 in head and neck squamous cell carcinoma. Mol. Cancer 6, 5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26. Monteys A. M., Spengler R. M., Wan J., Tecedor L., Lennox K. A., Xing Y., Davidson B. L. (2010) Structure and activity of putative intronic miRNA promoters. RNA 16, 495–505 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27. Kawahara H., Okada Y., Imai T., Iwanami A., Mischel P. S., Okano H. (2011) Musashi1 cooperates in abnormal cell lineage protein 28 (Lin28)-mediated let-7 family microRNA biogenesis in early neural differentiation. J. Biol. Chem. 286, 16121–16130 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28. Kolonin M. G., Finley R. L., Jr. (2000) A role for cyclin J in the rapid nuclear division cycles of early Drosophila embryogenesis. Dev. Biol. 227, 661–672 [DOI] [PubMed] [Google Scholar]
  • 29. Gocek E., Wang X., Liu X., Liu C. G., Studzinski G. P. (2011) MicroRNA-32 upregulation by 1,25-dihydroxyvitamin D3 in human myeloid leukemia cells leads to Bim targeting and inhibition of AraC-induced apoptosis. Cancer Res. 71, 6230–6239 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30. Heneghan H. M., Miller N., Kerin M. J. (2010) MiRNAs as biomarkers and therapeutic targets in cancer. Current Opin. Pharmacol. 10, 543–550 [DOI] [PubMed] [Google Scholar]
  • 31. Wouters M. D., van Gent D. C., Hoeijmakers J. H., Pothof J. (2011) MicroRNAs, the DNA damage response and cancer. Mutation Res. 717, 54–66 [DOI] [PubMed] [Google Scholar]
  • 32. Ward A., Balwierz A., Zhang J. D., Kublbeck M., Pawitan Y., Hielscher T., Wiemann S., Sahin O. (2012) Re-expression of microRNA-375 reverses both tamoxifen resistance and accompanying EMT-like properties in breast cancer. Oncogene, in press [DOI] [PubMed] [Google Scholar]
  • 33. Zhao Y., Deng C., Lu W., Xiao J., Ma D., Guo M., Recker R. R., Gatalica Z., Wang Z., Xiao G. G. (2011) let-7 microRNAs induce tamoxifen sensitivity by down-regulation of estrogen receptor α signaling in breast cancer. Mol. Med 17, 1233–1241 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34. Viswanathan S. R., Daley G. Q. (2010) Lin28: A microRNA regulator with a macro role. Cell 140, 445–449 [DOI] [PubMed] [Google Scholar]
  • 35. Viswanathan S. R., Daley G. Q., Gregory R. I. (2008) Selective blockade of microRNA processing by Lin28. Science 320, 97–100 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36. Zhang L., Stokes N., Polak L., Fuchs E. (2011) Specific microRNAs are preferentially expressed by skin stem cells to balance self-renewal and early lineage commitment. Cell Stem Cell 8, 294–308 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37. Du L., Schageman J. J., Subauste M. C., Saber B., Hammond S. M., Prudkin L., Wistuba, Ji L., Roth J. A., Minna J. D., Pertsemlidis A. (2009) miR-93, miR-98, and miR-197 regulate expression of tumor suppressor gene FUS1. Mol. Cancer Res. 7, 1234–1243 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38. Hu G., Zhou R., Liu J., Gong A. Y., Eischeid A. N., Dittman J. W., Chen X. M. (2009) MicroRNA-98 and let-7 confer cholangiocyte expression of cytokine-inducible Src homology 2-containing protein in response to microbial challenge. J. Immunol. 183, 1617–1624 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39. Coppola V., De Maria R., Bonci D. (2010) MicroRNAs and prostate cancer. Endocrine-related Cancer 17, F1–F17 [DOI] [PubMed] [Google Scholar]
  • 40. Jorde R., Svartberg J., Joakimsen R. M., Coucheron D. H. (2012) Plasma profile of microRNA after supplementation with high doses of vitamin D3 for 12 months. BMC Res. Notes 5, 245. [DOI] [PMC free article] [PubMed] [Google Scholar]

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