Cancer Cell Biology
MicroRNA regulates human vitamin D receptor
Version of Record online: 2 APR 2009
Copyright © 2009 UICC
International Journal of Cancer
Volume 125, Issue 6, pages 1328–1333, 15 September 2009
How to Cite
Mohri, T., Nakajima, M., Takagi, S., Komagata, S. and Yokoi, T. (2009), MicroRNA regulates human vitamin D receptor. Int. J. Cancer, 125: 1328–1333. doi: 10.1002/ijc.24459
- Issue online: 14 JUL 2009
- Version of Record online: 2 APR 2009
- Manuscript Accepted: 13 MAR 2009
- Manuscript Received: 17 DEC 2008
- Ministry of Health, Labor, and Welfare of Japan
- posttranscriptional regulation
Most of the biological effects of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) are elicited by the binding to vitamin D receptor (VDR), which regulates gene expression. Earlier studies reported no correlation between the VDR protein and mRNA levels, suggesting the involvement of posttranscriptional regulation. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression through translational repression or mRNA degradation. A potential miR-125b recognition element (MRE125b) was identified in the 3′-untranslated region of human VDR mRNA. We investigated whether VDR is regulated by miR-125b. In luciferase assays using a plasmid containing the MRE125b, the antisense oligonucleotide for miR-125b significantly increased (130% of control) the reporter activity in KGN cells, whereas the precursor for miR-125b significantly decreased (40% of control) the reporter activity in MCF-7 cells, suggesting that miR-125b functionally recognized the MRE125b. By electrophoretic mobility shift assays, it was demonstrated that the overexpression of miR-125b significantly decreased the endogenous VDR protein level in MCF-7 cells to 40% of control. 1,25(OH)2D3 drastically induced the CYP24 mRNA level in MCF-7 cells, but the induction was markedly attenuated by the overexpression of miR-125b. In addition, the antiproliferative effects of 1,25(OH)2D3 in MCF-7 cells were significantly abolished by the overexpression of miR-125b. These results suggest that the endogenous VDR level was repressed by miR-125b. In conclusion, we found that miR-125b posttranscriptionally regulated human VDR. Since the miR-125b level is known to be downregulated in cancer, such a decrease may result in the upregulation of VDR in cancer and augmentation of the antitumor effects of 1,25(OH)2D3. © 2009 UICC
1α,25-Dihydroxyvitamin D3 (1,25(OH)2D3 or calcitriol), a biologically active metabolite of vitamin D3, is known as a classical regulator of calcium and bone homeostasis.1, 2 Vitamin D deficiency is linked to rickets and osteoporosis.3 Over the last 25 years, additional roles have been found for vitamin D in the regulation of cell processes such as cell growth, differentiation and apoptosis. Accumulating evidence has revealed that vitamin D deficiency is also associated with the risk of cancer.4 Since the vitamin D system has relevance for both the prevention and treatment of cancer,3 the development of a number of novel synthetic vitamin D analogues as a therapeutic agent in cancer has been attempted.
Most of the biological effects of 1,25(OH)2D3 are elicited by the binding to vitamin D receptor (VDR; NR1I1),5 which belongs to the superfamily of nuclear steroid hormone receptors. After ligand binding, the VDR forms a heterodimer with retinoid X receptor (RXR; NR2B1) and binds to vitamin D responsive element (VDRE) in the regulatory region of the target genes.6 The VDR is expressed not only in the classical vitamin D responsive organs including the intestine, bone and kidney but also in many other nonclassical vitamin D responsive organs including the liver, suggesting a broader role of the receptor.7 It has been reported that, at the protein level, the VDR expression is higher in breast8 and thyroid9 cancers than in normal tissues, but no obvious difference was found in cancer and normal tissues at the mRNA level. In colon cancer, the VDR mRNA and protein expression levels are gradually increased in the early stages of cancerogenesis, but the VDR mRNA decreases subsequently to lower levels during advancement.10 Thus, the VDR expression is upregulated in cancers, although the expression levels seem to change during disease progression and in response to therapies. However, the mechanism of the upregulation of VDR protein in cancer has not been clarified. One clue is that there is no correlation between the VDR protein and mRNA levels, suggesting the involvement of posttranscriptional regulation.
To uncover the molecular mechanism of the posttranscriptional regulation, we sought to determine whether microRNA (miRNA) might be involved in the regulation of VDR. MiRNAs are an evolutionarily conserved class of endogenous ∼22-nucleotide noncoding RNAs, and play a key role in diverse biological processes, including development, cell proliferation, differentiation, apoptosis and cancer initiation and progression.11–13 MiRNAs recognize the 3′-untranslated region (3′-UTR) of the target mRNA and cause translational repression or mRNA degradation.14 To date, ∼700 miRNAs have been identified in human, and more than one-third of all human genes have been predicted to be miRNA targets.15 The expression of global miRNAs is deregulated in most types of human cancers.13 In this study, we investigated the potential involvement of miRNAs in the posttranscriptional regulation of human VDR expression.
Material and methods
Chemicals and reagents
1,25(OH)2D3 was purchased from Wako Pure Chemical Industries (Osaka, Japan). The pGL3-promoter vector, phRL-TK plasmid, pT7Blue T-Vector and a dual-luciferase reporter assay system were purchased from Promega (Madison, WI). LipofectAMINE2000 and LipofectAMINE RNAiMAX were from Invitrogen (Carlsbad, CA). Pre-miR miRNA Precursors for miR-125b-1 and negative control #2 were from Ambion (Austin, TX). Antisense LNA/DNA mixed oligonucleotides (AsO) for miR-125b (5′-TCACAAGTTAGGGTCTCAGGGA-3′, underlined letters are LNA) and for negative control (5′-AGAC TAGCGGTATCTTAAACC-3′) were from Greiner Japan (Tokyo, Japan). All primers and oligonucleotides were commercially synthesized at Hokkaido System Sciences (Sapporo, Japan). Antibodies to VDR (C-20) and RXRα(D-20) were from Santa Cruz Biotechnology (Santa Cruz, CA). Restriction enzymes were from Takara (Shiga, Japan), TOYOBO (Osaka, Japan) and New England Biolabs (Beverly, MA). All other chemicals and solvents were of the highest grade commercially available.
Cells and culture conditions
The human breast adenocarcinoma cell lines MCF-7 and MDA-MB-435, the human colon carcinoma cell lines LS180 and the human embryonic kidney cell line HEK293 were obtained from the American Type Culture Collection (Rockville, MD). The human ovarian granulosa-like tumor cell line KGN16 and the human hepatoma cell line HepG2 were obtained from Riken Gene Bank (Tsukuba, Japan). MCF-7 cells and LS180 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 0.1 mmol/L nonessential amino acid (Invitrogen) and 10% fetal bovine serum (FBS) (Invitrogen). MDA-MB-435 cells and HepG2 cells were cultured in DMEM supplemented with 10% FBS. HEK293 cells were cultured in DMEM supplemented with 4.5 g/L glucose, 10 mmol/L HEPES and 10% FBS. KGN cells were cultured in a 1:1 mixture of DMEM and Ham's F-12 medium (Nissui Pharmaceutical) supplemented with 10% FBS. These cells were maintained at 37°C under an atmosphere of 5% CO2-95% air.
Real-time RT-PCR for mature miR-125b
For the quantification of mature miR-125b, polyadenylation and reverse transcription were performed using an NCode miRNA First-Strand cDNA Synthesis Kit (Invitrogen) according to the manufacturer's protocol. The forward primer for miR-125b was 5′-TCC CTG AGA CCC TAA CTT GTG A-3′, and the reverse primer was the supplemented universal qPCR primer. The real-time PCR was performed using a Smart Cycler (Cepheid, Sunnyvale, CA) with Smart Cycler software (version 1.2b) as follows. After an initial denaturation at 95°C for 30 sec, the amplification was performed by denaturation at 95°C for 10 sec, annealing and extension at 60°C for 10 sec for 45 cycles.
Construction of reporter plasmids
To construct luciferase reporter plasmids, various target fragments were inserted into the XbaI site, downstream of the luciferase gene in the pGL3-promoter vector. The sequence from +1786 to +1813 in the human VDR mRNA (5′-CAG GAG AAA TGC ATC CAT TCC TCA GGG A-3′) was termed the miR-125b recognition element (MRE125b). The region from +1748 to +1860 containing the MRE125b in the human VDR mRNA was amplified by PCR using the following primers adapted to the XbaI site: 5′-TTT TCT AGA CTG CCT AAG TGG CTG CTG AC-3′ and 5′-TTT TCT AGA CGC TGG ACA AGC GGG GCC-3′. The PCR product was digested with XbaI and the 119-bp fragment was cloned into pGL3-promoter vector, resulting in single (pGL3/F1) and reverse single (pGL3/R1) insertions. The fragment containing 3 copies of the MRE125b, 5′-CTA GAC AGG AGA AAT GCA TCC ATT CCT CAG GGA CAG AGC AGG AGA AAT GCA TCC ATT CCT CAG GGA CAG AGC AGG AGA AAT GCA TCC ATT CCT CAG GGA CAG AGT-3′ (MRE125b is italicized), was cloned into the pGL3-promoter vector (pGL3/3xMRE). The complementary sequence of 3 copies of the MRE125b was also cloned into the pGL3-promoter plasmid (pGL3/3xMRE-Rev). A fragment containing the perfect matching sequence with the mature miR-125b, 5′-CTA GAT CAC AAG TTA GGG TCT CAG GGA T-3′ (the matching sequence of miR-125b is italicized), was cloned into the pGL3-promoter vector (pGL3/c-125b). The nucleotide sequences of the constructed plasmids were confirmed by DNA sequencing analyses.
Various luciferase reporter plasmids (pGL3) were transiently transfected with phRL-TK plasmid into MCF-7 and KGN cells. Briefly, the day before transfection, the cells were seeded into 24 well plates. After 24 hr, 450 ng of pGL3 plasmid, 50 ng of phRL-TK plasmid and the precursors for miR-125b or control were cotransfected into MCF-7 cells using LipofectAMINE 2000. For KGN cells, 450 ng of pGL3 plasmid, 50 ng of phRL-TK plasmid and the AsOs for miR-125b or control were cotransfected using LipofectAMINE 2000. After incubation for 48 hr, the cells were resuspended in passive lysis buffer and then the luciferase activity was measured with a luminometer (Wallac, Turku, Finland) using the dual-luciferase reporter assay system.
Transfection of precursor for miR-125b into MCF-7 cells and preparation of nuclear extract and total RNA
To investigate the effects of miR-125b on the expression level of VDR protein, 50 nM precursors for miR-125b or control were transfected into MCF-7 cells using LipofectAMINE RNAiMAX. After 72 hr, nuclear extract was prepared using NE-PER Nuclear and Cytoplasmic extraction reagents (Pierce, Rockford, IL) and total RNA was prepared using ISOGEN according to the manufacturer's protocols. The protein concentration in the nuclear extract was determined using Bradford protein assay reagent (Bio-Rad, Hercules, CA) with γ-globulin as a standard.
Electrophoretic mobility shift assays
Human VDR cDNA was amplified by PCR using cDNA from human normal kidney with the forward primer 5′-TCC TTC AGG GAT GGA GGC AAT GGC-3′ and the reverse primer 5′-CTG TCC TAG TCA GGA GAT CTC ATT GCC-3′. The PCR fragment was cloned into the pT7Blue T-Vector. The nucleotide sequences of the constructed plasmids were confirmed by DNA sequencing analyses. Human RXRα expression vector (pGEM-3Z/hRXRα) was previously constructed.17 Using these plasmids and the TNT T7 Quick Coupled Transcription/Translation System (Promega), human VDR and RXRα proteins were synthesized in vitro. The oligonucleotide containing VDRE, 5′-aag CAC ACC cgg TGA ACT ccg-3′ (the hexamer half-sites are capitalized), was from the human CYP24 promoter.18 Double-stranded oligonucleotides were labeled with [γ-32P]ATP using T4 polynucleotide kinase (TOYOBO) and purified by Microspin G-50 columns (GE Healthcare Bio-Sciences, Piscataway, NJ). The labeled probe (40 fmol, ∼10,000 cpm) was applied to each binding reaction in 25 mM HEPES-KOH buffer (pH 7.9), 0.5 mM EDTA, 50 mM KCl, 10% glycerol, 0.5 mM dithiothreitol, 0.5 mM (p-amidinophenyl) methanesulfonyl fluoride, 2 μg of poly(dI-dC) and 2 μL of in vitro transcribed/translated proteins to a final reaction volume of 15 μL. For supershift experiments, 0.2 μg of anti-VDR antibodies or 2 μg of anti-RXRα antibodies were preincubated with in vitro transcribed/translated proteins or the nuclear extract at room temperature for 30 min. The mixtures were incubated on ice for 15 min and then loaded on 4% acrylamide gels in 0.5 × Tris-borate EDTA buffer. The gels were dried and then the DNA-protein complexes were detected with a Fuji Bio-Imaging Analyzer BAS 1000 (Fuji Film, Tokyo, Japan).
Real-time RT-PCR for CYP24
To investigate the effects of miR-125b on the induction of CYP24 mRNA by 1,25(OH)2D3, 50 nM precursors for miR-125b or control were transfected into MCF-7 cells using LipofectAMINE RNAiMAX. After 72 hr, the cells were treated with 100 nM 1,25(OH)2D3 (or 0.1% ethanol for control) for 24 hr. Total RNA was prepared using ISOGEN. The forward and reverse primers for CYP24 mRNA were 5′-CAG CAA ACA GTC TAA TGT GG-3′ and 5′-AGC ATA TTC ACC CAG AAC TG-3′, respectively. The real-time PCR analysis was performed as follows: after an initial denaturation at 95°C for 30 sec, the amplification was performed by denaturation at 94°C for 4 sec, annealing and extension at 62°C for 20 sec for 45 cycles. The CYP24 mRNA levels were normalized with GAPDH mRNA as described previously.19
To investigate the effects of miR-125b on the antiproliferative effects of 1,25(OH)2D3, growth assay was conducted according to the method by McGaffin et al.20 with slight modifications. MCF-7 cells were plated on 96 well plates (3000 cells/well) and 20 nM precursors for miR-125b or control were transfected using LipofectAMINE RNAiMAX. After 24 hr, the cells were treated with 1 μM 1,25(OH)2D3 (or 0.1% ethanol) for 48–96 hr. The cells were rinsed with phosphate-buffered saline, fixed with 3.7% formaldehyde for 15 min and stained with 0.1% crystal violet for 10 min. The stained cells were washed with water and air dried. Crystal violet was extracted from the stained cells with 2% sodium dodecyl sulfate, and the intensities were quantified spectrophotometrically 620 nm. The percent cell viability was calculated by comparison with the absorbance of control cells.
Data are expressed as mean ± SD of triplicate determinations. Comparison of 2 groups was made with an unpaired, two-tailed student's t-test. Comparison of multiple groups was made with ANOVA followed by Dunnett or Tukey test. A value of p < 0.05 was considered statistically significant.
A miR-125b complementary sequence on the 3′-UTR of human VDR mRNA
By a computational search (http://www.targetscan.org/), several miRNAs are found to share complementarity with a sequence in the 3′-UTR of human VDR mRNA. Among them, we focused on miR-125b because its binding site is highly conserved among species (Fig. 1). The seed sequence of miR-125b was perfectly matching with the predicted binding site of the VDR mRNA. We investigated whether miR-125b might be involved in the regulation of human VDR expression through the MRE125b.
Expression levels of miR-125b in human cancer cell lines
For gain- and loss-of-function experiments, we need to know the expression level of endogenous miR-125b in cell lines. For this purpose, the expression levels of mature miR-125b in 6 kinds of human cancer cell lines were determined by real-time RT-PCR analysis. As shown in Figure 2a, the mature miR-125b level was highest in KGN followed by MDA-MB-435 cells, whereas it was extremely low in MCF-7, HepG2, HEK293 and LS180 cells.
Effects of overexpression or inhibition of miR-125b on luciferase activity
To investigate whether MRE125b is functional in the regulation by miR-125b, luciferase assays were performed. First, we transfected the precursor for miR-125b into MCF-7 cells in which the mature miR-125b level was low (Fig. 2b). Using the pGL3/c-125b plasmid containing the miR-125b complementary sequence, it was demonstrated that the luciferase activity was significantly (p < 0.001) decreased by the transfection of precursor for miR-125b. The luciferase activity of the pGL3/F1 plasmid was significantly (p < 0.001) decreased (60% of control) by the overexpression of miR-125b, but that of the pGL3/R1 plasmid was not. When the pGL3/3xMRE plasmid containing 3 copies of the MRE125b was used, a prominent suppression was observed (40% of control, p < 0.001) by the overexpression of miR-125b. Next, we transfected the AsO for miR-125b into KGN cells in which the mature miR-125b was highly expressed (Fig. 2b). The luciferase activity of the pGL3/c-125b plasmid was significantly (p < 0.01) lower than that of the control pGL3-p plasmid. The luciferase activity of the pGL3/c-125b plasmid was significantly (p < 0.01) restored by the transfection of AsO for miR-125b (3.1-fold of control). The luciferase activity of the pGL3/F1 plasmid was increased by the transfection of AsO for miR-125b, although the effects were statistically insignificant. The luciferase activity of the pGL3/3xMRE plasmid was significantly (p < 0.01) lower than that of the control pGL3-p plasmid. The luciferase activity of the pGL3/3xMRE plasmid was significantly (1.3-fold of control, p < 0.05) restored by the inhibition of miR-125b by AsO. These results suggest that miR-125b functionally recognized the MRE125b on the human VDR mRNA.
Effects of overexpression of miR-125b on the endogenous VDR protein level
We sought to examine the effects of miR-125b on the endogenous VDR protein level. When we first attempted to determine the endogenous VDR protein level in human cancer cell lines by Western blot analysis using commercially available antibodies, we could not identify the VDR protein because of multiple nonspecific bands. Therefore, we utilized electrophoretic mobility shift assays to evaluate the endogenous VDR level. The VDRE of human CYP24 gene, which is known to be a target of VDR,18 was used as a probe. It was confirmed that in vitro-synthesized VDR/RXRα heterodimers bound to the VDRE (Fig. 3a). With the anti-VDR or anti-RXRα antibodies, the band density of the VDR/RXRα heterodimer was decreased and the supershifted band was observed. When the probe was incubated with the nuclear extracts prepared from MCF-7 cells, the band representing the VDR/RXRα heterodimer was observed and the band density was diminished with the anti-VDR or anti-RXRα antibodies. When the precursor for miR-125b was transfected, the mature miR-125b level was prominently increased, and the band density of the VDR/RXRα heterodimer was significantly (p < 0.001) decreased compared with that of control (40% of control). We confirmed by Western blot analysis that the expression level of RXRα was not affected by the overexpression of miR-125b (data not shown). These results suggest that the endogenous VDR level was repressed by miR-125b.
MiR-125b-dependent VDR regulation affects the target gene expression
We investigated whether the miR-125b-dependent regulation of VDR affects the expression of target genes. When the MCF-7 cells were treated with 100 nM 1,25(OH)2D3, the CYP24 mRNA level was significantly (p < 0.001) increased (588-fold) (Fig. 4). However, this induction was markedly attenuated by the overexpression of miR-125b. In addition, the basal CYP24 mRNA level was also decreased by the overexpression of miR-125b, although it was statistically insignificant. These results support that the endogenous VDR level was repressed by miR-125b, and this regulation mechanism affects the expression of target genes.
Effects of overexpression of miR-125b on the antiproliferative effects of 1,25(OH)2D3
We investigated the effects of miR-125b on the antiproliferative effects of 1,25(OH)2D3 (Fig. 5). The cells transfected with the precursor for control were grown during incubation for 48–96 hr, but the growth was significantly (p < 0.01 or p < 0.001) reduced in the presence of 1 μM 1,25(OH)2D3. Interestingly, the overexpression of miR-125b prominently (p < 0.05, p < 0.01 or p < 0.001) abolished the antiproliferative effects of 1,25(OH)2D3. In addition, the overexpression of miR-125b could significantly (p < 0.05, at 96 hr) increase the cell growth in the absence of 1,25(OH)2D3. These results suggest that miR-125b regulating VDR has a great impact on antiproliferative effects of 1,25(OH)2D3.
In this study, we investigated whether human VDR might be regulated by miRNA. In the 3′-UTR of human VDR mRNA, a potential miR-125b recognition element (MRE125b) was identified. Luciferase assays clearly revealed that the miR-125b negatively regulated the reporter activity through MRE125b. By electrophoretic mobility shift assays and evaluation of the induction potencies of CYP24 mRNA, it was demonstrated that the endogenous VDR level was repressed by the overexpression of miR-125b. These results clearly suggest that the human VDR is posttranscriptionally regulated by miR-125b. Because the sequences of VDR mRNA around MRE125b are highly conserved among species (Fig. 1), the regulation by miR-125b may also occur in other species.
The global expression of miRNAs is deregulated in most cancer types.21 Some studies have suggested that miRNA expression would be widely downregulated in human tumors relative to normal tissues, and other studies reported a tumor-specific mixed pattern of downregulation and upregulation of miRNA genes. Recent findings revealed that the miRNA deregulation in human cancers occurs by multiple mechanisms, including transcriptional deregulation, epigenetic alterations, mutation, DNA copy number abnormalities and dysfunction of key proteins in the miRNA biogenesis pathway.21 Among them, alterations in DNA copy numbers would be a major mechanism because over 50% of miRNAs are in genomic fragile sites or regions associated with cancers.12 It has been reported that miR-125b was downregulated in breast12, 22 and prostate23 cancers. Mature miR-125b is formed by 2 precursors, miR-125b-1 and miR-125b-2. The genes for miR-125b-1 and miR-125b-2 are located in chromosome 11q24.1 and 21q11.2, respectively (http://microrna.sanger.ac.uk/sequences/). Interestingly, it has been reported that the chromosome region 11q23-24 is most frequently deleted in breast, ovarian and lung cancers24, 25 and the chromosome region 21q11-21 is frequently deleted in breast, esophagus, stomach, ovary and lung cancers.26 This could be one of the mechanisms of the downregulation of miR-125b in cancers. Meanwhile, it is known that VDR is upregulated in several cancers,8, 9 and the upregulation appears to be associated with a good prognosis.27 As this study demonstrated that miR-125b negatively regulated the expression of VDR, it was directly proven that the upregulation of VDR in cancers would be due to the downregulation of miR-125b.
Previously, the role of miR-125b in cell proliferation and differentiation has been reported in human prostate cancer cell lines,28 thyroid carcinoma cells,29 a bone marrow stroma cell line30 and hepatocellular carcinoma.31 Scott et al.32 reported that the miR-125b suppressed ERBB2 and ERBB3 oncogenes. Li et al.31 reported that high expression of miR-125b was correlated with good survival in hepatocellular carcinoma patients. These previous studies suggest that miR-125b acts as a type of tumor suppressor gene. In contrast, our study demonstrated that miR-125b repressed the antiproliferative effects of 1,25(OH)2D3. Thus, this study provides new information concerning the role of miR-125b in cell proliferation. In cancer cells, the downregulation of miR-125b would result in an augmentation of the antitumor effects of 1,25(OH)2D3.
As regards other nuclear receptors, there are a few reports. Estrogen receptor (ER) α, which is an important marker for the prognosis and is predictive of the response to endocrine therapy in breast cancer patients, has been found to be regulated by miR-20633 and miR-221/222.34 These studies suggested that these miRNAs could serve as potential therapeutic targets for a subset of ERα-negative breast cancers. Previously, we found that pregnane X receptor (PXR), which is a key regulator of the expression of drug-metabolizing enzymes and transporters involved in the responses to steroids and xenobiotics, is regulated by miR-148a.35 Thus, accumulating evidence has revealed that nuclear receptors, to which steroid hormones bind as a ligand, are regulated by miRNAs. The regulation of nuclear receptors by miRNA would result in changes in the expression of a variety of target genes, constructing complex regulatory networks.
In conclusion, we clarified that human VDR is posttranscriptionally regulated by miR-125b. This study could provide new insights into the regulatory mechanism of VDR expression.
The authors acknowledge Mr. Brent Bell for reviewing the manuscript.
- 6Retinoid X receptor (RXR) ligands activate the human 25-hydroxyvitamin D3-24-hydroxylase promoter via RXR heterodimer binding to two vitamin D-responsive elements and elicit additive effects with 1,25-dihydroxyvitamin D3. J Biol Chem 1997; 272: 19027–34., , .