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MiR-125b, a MicroRNA Downregulated in Psoriasis, Modulates Keratinocyte Proliferation by Targeting FGFR2
ORIGINAL ARTICLE
MiR-125b, a MicroRNA Downregulated in Psoriasis, Modulates Keratinocyte Proliferation by Targeting FGFR2 Ning Xu1, Petter Brodin2, Tianling Wei1, Florian Meisgen1, Liv Eidsmo1, Nikoletta Nagy3, Lajos Kemeny3, Mona Sta˚hle1, Eniko¨ Sonkoly1,4 and Andor Pivarcsi1,4 MicroRNAs (miRNAs) are short, single-stranded, noncoding RNAs that play important roles in the regulation of gene expression. We previously identified a characteristic miRNA expression profile in psoriasis, distinct from that of healthy skin. One of the most downregulated miRNAs in psoriasis skin was microRNA-125b (miR-125b). In this study, we aimed to identify the potential role(s) of miR-125b in psoriasis pathogenesis. In situ hybridization results showed that the major cell type responsible for decreased miR-125b levels in psoriasis lesions was the keratinocyte. Overexpression of miR-125b in primary human keratinocytes suppressed proliferation and induced the expression of several known differentiation markers. Conversely, inhibition of endogenous miR-125b promoted cell proliferation and delayed differentiation. Fibroblast growth factor receptor 2 (FGFR2) was identified as one of the direct targets for suppression by miR-125b by luciferase reporter assay. The expression of miR-125b and FGFR2 was inversely correlated in both transfected keratinocytes and in psoriatic skin. Knocking down FGFR2 expression by siRNA suppressed keratinocyte proliferation, but did not enhance differentiation. Altogether, our results demonstrate a role for miR-125b in the regulation of keratinocyte proliferation and differentiation, partially through the regulation of FGFR2. Loss of miR-125b in psoriasis skin may contribute to hyperproliferation and aberrant differentiation of keratinocytes. Journal of Investigative Dermatology (2011) 131, 1521–1529; doi:10.1038/jid.2011.55; published online 17 March 2011
INTRODUCTION Psoriasis is a common chronic inflammatory skin disease, which affects nearly 3% of the world’s population and decreases patients’ quality of life considerably (Lebwohl, 2003). Psoriasis is thought to be initiated by complex interactions between environmental and genetic factors. Psoriasis skin lesions are characterized by hyperproliferation and aberrant differentiation of keratinocytes and infiltration of inflammatory cells into the dermis and epidermis (Tonel and
1
Molecular Dermatology Research Group, Unit of Dermatology and Venereology, Department of Medicine, Karolinska Institutet, Stockholm, Sweden; 2Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden; 3Dermatological Research Group of the Hungarian Academy of Sciences, University of Szeged, Szeged, Hungary and 4 Department of Dermatology and Allergology, University of Szeged, Szeged, Hungary Correspondence: Ning Xu, Molecular Dermatology Research Group, Centre for Molecular Medicine (CMM), L8:02, Department of Medicine, Karolinska Institutet, Stockholm SE-17176, Sweden. E-mail: [email protected] Abbreviations: 30 UTR, 30 untranslated region; Anti-miR-125b, miR-125b inhibitor oligonucleotide; EdU, 5-ethynyl-20 -deoxyuridine; FGFR2, fibroblast growth factor receptor 2; K10, cytokeratin 10; K5, cytokeratin 5; miR-125b, microRNA-125b; miRNA, microRNA; mRNA, messenger RNA; Pre-miR125b, miR-125b precursor RNA; qRT-PCR, real-time quantitative reverse transcription-PCR; siRNA, small interfering RNA Received 16 September 2010; revised 7 January 2011; accepted 5 February 2011; published online 17 March 2011
& 2011 The Society for Investigative Dermatology
Conrad, 2009). However, the underlying mechanisms regulating these epidermal defects and immunological dysfunction remain largely elusive. MicroRNAs (miRNAs) are B22 nucleotide-long singlestranded noncoding RNAs that can mediate post-transcriptional silencing by binding with partial complementarity to the 30 untranslated region (UTR) of the target messenger RNA (mRNA) (Ambros et al., 2003). Base-pairing at position 2–8 nucleotides relative to the 50 end of small RNA, termed the ‘‘seed’’ region, appears to be important for target recognition (Bartel, 2004). A unique miRNA can regulate the expression of hundreds of proteins, and the expression of a specific protein may be controlled by several miRNAs (Krek et al., 2005). The high sequence conservation of many miRNAs between distantly related organisms suggests strong evolutionary pressure (Farh et al., 2005) and they have been shown to participate in many fundamental life processes, e.g., development, differentiation, organogenesis, growth control, and apoptosis. Accordingly, deregulation of miRNA expression has been shown to contribute to cancer, heart diseases, infectious diseases, inflammatory diseases, and other medical conditions, making them potential targets for medical diagnosis and therapy (reviewed in Czech, 2006). The role of miRNAs in psoriasis remains largely unexplored. In 2007, we performed a genome-wide microarray analysis of miRNA expression in psoriasis and identified a www.jidonline.org 1521
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distinct miRNA expression profile in psoriatic lesions, compared with healthy human skin and/or with another chronic inflammatory skin disease, atopic eczema (Sonkoly et al., 2007). In particular, this study identified the first skinand keratinocyte-specific miRNA, miR-203, which is highly upregulated in psoriasis and regulates inflammatory pathways as well as keratinocyte proliferation and differentiation (Sonkoly et al., 2007, 2010; Lena et al., 2008; Yi et al., 2008). MicroRNA-125b (miR-125b) was one of the miRNAs found to be significantly decreased in psoriasis skin in comparison with healthy skin in our miRNA microarray analysis (Sonkoly et al., 2007). Expression profiling in healthy human organs/tissues showed that miR-125b is expressed in most organs. In normal skin, it is mainly expressed by resident cells such as fibroblasts, keratinocytes, and melanocytes (Sonkoly et al., 2007). To date, nothing is known about the function of miR-125b in healthy or inflamed skin. The aim of this study was to determine the potential role of miR-125b in psoriasis.
In order to identify which cell type(s) in the skin are responsible for the reduced expression of miR-125b in psoriasis, we performed in situ hybridization on skin sections from healthy individuals (n ¼ 6) and psoriasis patients (n ¼ 8) using miR-125b-specific locked nucleic acid-modified probes (Figure 1b). MiR-125b was expressed throughout the epidermis in healthy skin (Figure 1b), whereas its expression was decreased in all epidermal layers in psoriasis lesions. To quantify the expression of miR-125b specifically in the epidermis, we separated epidermis from dermis by dispase treatment. Measurement of miR-125b by qRT-PCR showed a 4.8-fold (Po0.05) decrease of miR-125b expression in epidermis from psoriasis lesional skin when compared with healthy epidermis (Figure 1c). These results indicate that the decrease of miR-125b expression in psoriasis skin results from its reduced expression in keratinocytes. The mature miR-125b is processed from two primary miR-125b transcripts in human: pri-miR-125b-1, encoded on chromosome 11, and pri-miR-125b-2, encoded on chromosome 21 (Griffiths-Jones et al., 2006). To explore which primary transcript of the miR-125b gene is dominant in skin, we quantified the expression of pri-miR-125b-1 and pri-miR-125b-2 in RNA samples from seven healthy skin samples and nine psoriasis lesional skin samples by qRT-PCR using primer sets designed to detect the specific primary transcripts (Figure 1d). The results showed that pri-miR125b-1 was the major miR-125b precursor in human skin. Compared with healthy skin, pri-miR-125b-1 was significantly decreased in psoriatic lesional skin (Po0.01), which suggested that the downregulation of miR-125b in psoriasis
RESULTS MiR-125b is suppressed in keratinocytes in psoriasis lesions
To confirm previous results from microarray profiling (Sonkoly et al., 2007), we performed real-time quantitative reverse transcription-PCR (qRT-PCR) analysis of the mature miR-125b expression on RNA samples from healthy skin (n ¼ 27) and psoriasis lesional skin (n ¼ 25; Figure 1a). The results showed that miR-125b was 3.7-fold (Po0.001) downregulated in psoriasis skin lesions compared with healthy skin, which supported our previous microarray data.
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Figure 1. The expression of microRNA-125b (miR-125b) in healthy and psoriasis skin. (a) MiR-125b expression was analyzed in healthy (n ¼ 27) and psoriasis lesional skin samples (n ¼ 25) using real-time quantitative reverse transcription-PCR (qRT-PCR). ***Po0.001. (b) In situ hybridization was performed on healthy (n ¼ 6, left panel) and psoriasis lesional skin sections (n ¼ 8, right panel) using miR-125b-specific locked nucleic acid (LNA) probe (upper panel) or scrambled probe (lower panel). The blue–purple color indicates miR-125b expression. Bar ¼ 50 mm. (c) MiR-125b expression was analyzed on RNA from epidermis of healthy (n ¼ 5) and psoriasis lesional skin (n ¼ 3) using qRT-PCR. *Po0.05. (d) Expression of primary miR-125b transcripts was analyzed in healthy (n ¼ 7) and psoriasis lesional skin samples (n ¼ 9) using qRT-PCR. Results for individual patients and mean are shown. **Po0.01. (e) qRT-PCR analysis of primary miR-125b transcripts in primary keratinocytes.
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skin occurred at the transcriptional level rather than during processing of the primary transcript. In addition, we showed that pri-miR-125b-1 was also the dominant form of miR-125b precursor in cultured primary keratinocytes (Figure 1e). MiR-125b suppresses the proliferation of keratinocytes
In order to determine the possible effects of aberrant miR-125b expression on keratinocyte proliferation, we transfected human primary keratinocytes with miR-125b precursor RNA (Pre-miR-125b, to overexpress miR-125b) or miR-125b inhibitor oligonucleotide (Anti-miR-125b, to inhibit endogenous miR-125b) and analyzed the cell proliferation rate by 5-ethynyl-20 -deoxyuridine (EdU) incorporation assay using flow cytometry and gating on live keratinocytes (Figure 2a–d). The EdU incorporation rate (percentage of cells that underwent cell division during the assay) was reduced Pre-miR-125b
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Terminal differentiation is severely altered in psoriasis. To determine whether suppressed expression of miR-125b in psoriasis keratinocytes may contribute to these alterations, we investigated the role of miR-125b on keratinocyte differentiation. We analyzed the differentiation status of keratinocytes at 24, 48, 72, and 96 hours after transfection with specific precursors or inhibitors for miR-125b as well as their respective control oligonucleotides and mock controls. We measured the expression of several keratinocyte differentiation markers including the early differentiation markers involucrin and cytokeratin 10 (K10), the late differentiation marker filaggrin, the differentiation-induced miRNA miR-203 (Sonkoly et al., 2010), as well as the basal cell marker cytokeratin 5 (K5), whose expression does not depend on cellular differentiation (Fuchs, 1990). The level of K5 remained constant as the confluence of the cultured cells increased with time, whereas the expression of the other four differentiation markers increased as expected (Figure 3 and Supplementary Figure S1 online). Overexpression of miR-125b significantly increased involucrin expression at both mRNA and protein levels, whereas inhibition of endogenous miR-125b decreased involucrin protein expression (Figure 3a). In addition, we observed a trend of reduced involucrin mRNA expression by inhibition of miR-125b in three independent experiments, although the change was not significant. Similar results were observed for the expression of K10 and filaggrin mRNA as well as miR-203 in the keratinocytes with overexpressed or inhibited miR-125b (Supplementary Figure S1a–d online). However, neither overexpression nor inhibition of miR-125b changed the mRNA level of K5 (Figure 3b). Together, these data indicate that miR-125b promotes keratinocyte differentiation. Fibroblast growth factor receptor 2 (FGFR2) is a direct target for miR-125b
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to 47% (Po0.001) in the cells overexpressing miR-125b compared with the cells overexpressing a control miRNA with a scrambled sequence, whereas inhibition of the endogenous miR-125b increased the EdU incorporation rate by 58% (Po0.01) compared with the cells transfected with scrambled miRNA inhibitor (Figure 2e). These results indicate that miR-125b functions as a negative regulator of keratinocyte proliferation.
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Figure 2. MicroRNA-125b (miR-125b) suppresses the proliferation of keratinocytes. Primary human keratinocytes were transfected with (a) miR-125b precursor RNAs (Pre-miR-125b), (b) miRNA precursor control (Pre-miR-ctrl), (c) miR-125b inhibitor oligonucleotides (Anti-miR-125b), or (d) miRNA inhibitor control (Anti-miR-ctrl). The percentage of cells that underwent cell division was assessed by anti-5-ethynyl-20 -deoxyuridine (anti-EdU) Alexa Fluor 647 staining 48 hours post-transfection. The representative flow cytometry histograms gated on live propidium iodide (PI)-negative cells are shown and the percentages of EdU-positive cells are indicated. (e) The data of one representative experiment with quintuplicate are shown. The experiment was repeated five times. **Po0.01, ***Po0.001.
Identifying the targets for miRNAs is essential for understanding their functions. In order to identify the direct target for miR-125b, we applied three standards for selection of the potential targets for further experimental validation. The target genes should be: (1) directly regulated by miR-125b binding; (2) coexpressed with miR-125b in keratinocytes; and (3) upregulated in psoriatic skin compared with healthy skin. To predict the potential targets for miR-125b, we performed an integrated prediction using three conventional bioinformatics methods: PicTar (http://pictar.mdc-berlin.de) (Krek et al., 2005), miRWalk (http://mirwalk.uni-hd.de), and RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/ submission.html) (Rehmsmeier et al., 2004) (Figure 4a and www.jidonline.org 1523
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Figure 3. MicroRNA-125b (miR-125b) promotes the differentiation of keratinocytes. Primary human keratinocytes were transfected with miR-125b precursor RNA (Pre-miR-125b) or miRNA precursor control (Pre-miR-ctrl; left panel); miR-125b inhibitor oligonucleotide (Anti-miR-125b) or miRNA inhibitor control (Anti-miR-ctrl; right panel). Both RNA and protein were collected at 24, 48, 72, and 96 hours after transfection. The involucrin messenger (m)RNA (a, upper panel) and cytokeratine 5 mRNA (b) were analyzed by real-time quantitative reverse transcription-PCR (qRT-PCR) and normalized to 18S RNA. Data are expressed as relative units (RU). The data of one representative experiment with triplicates are shown. The experiment was repeated three times. *Po0.05, **Po0.01. (a, lower panel) The expression of involucrin protein was analyzed by western blotting. b-Actin was detected on the same blots as loading control.
Supplementary Table S1 online). One of the potential target genes predicted by all three algorithms was FGFR2, a receptor expressed on keratinocytes and reported to be upregulated in lesional psoriasis skin (Finch et al., 1997). Bioinformatic tools predicted four potential miR-125b target sites in the 30 UTR of FGFR2 (target sites 1–4; Figure 4b and c). To verify whether FGFR2 is indeed a direct target gene for miR-125b, we performed 30 UTR luciferase-binding assays. To this end, the whole 30 UTR (1,594 nucleotides) of FGFR2 mRNA was cloned into a luciferase reporter plasmid and subsequently co-transfected with Pre-miR-125b or AntimiR-125b as well as their respective control oligonucleotides into HeLa cells (Figure 4d) and primary keratinocytes (Supplementary Figure S2 online). The endogenous miR125b expression levels in both cell types are shown in Supplementary Figure S3 online. Luciferase activity was analyzed 48 hours after transfection. In HeLa cells, overexpression of miR-125b reduced the luciferase activity to 46% (Po0.01), whereas inhibition of endogenous miR-125b increased the luciferase activity 2.5-fold (Po0.05) compared with scrambled pre-miR or anti-miR controls, respectively (Figure 4d). Similar results were observed in primary keratinocytes (Supplementary Figure S2 online). To determine whether the regulation of luciferase reporter gene by miR-125b is mediated by the predicted target sites in the 30 UTR of FGFR2, we deleted five to seven nucleotides within the seed-matching sequences of the predicted target sites 1, 2, 3, and 4 in plasmids named MUT1, MUT2, MUT3, 1524 Journal of Investigative Dermatology (2011), Volume 131
and MUT4, respectively (Figure 4c). Moreover, we created a construct named plasmid MUT 1–4 in which all four target sites were deleted (Figure 4c). Overexpression of miR-125b reduced the luciferase activity of the construct containing the wild-type 30 UTR of FGFR2 to 39% (Figure 4e, WT). Deletion of the seed-matching sequence in each potential target site led to partial restoration of luciferase activity to 67% for MUT1, 79% for MUT2, 64% for MUT3, and 67% for MUT4 (Figure 4e). Deletion of all four predicted miR-125b target sites led to a nearly complete restoration of luciferase activity (Figure 4e, MUT 1–4). Taken together, these results demonstrate that miR-125b directly regulates FGFR2 expression by binding to the four target sites in the 30 UTR of FGFR2 mRNA. MiR-125b regulates FGFR2 protein expression in keratinocytes
In the previous experiment we demonstrated that miR-125b can mediate post-transcriptional suppression through FGFR2 30 UTR. Next, we investigated whether miR-125b regulates FGFR2 expression at the mRNA or at the protein level in keratinocytes (Figure 5a and b). Although miR-125b overexpression or inhibition did not affect FGFR2 mRNA levels (Figure 5a), the results of western blot analysis showed that overexpression of miR-125b suppressed FGFR2 protein expression, and inhibition of miR-125b led to the upregulation of FGFR2 at the protein level (Figure 5b). To determine whether the interaction of miR-125b and FGFR2 is relevant in vivo, we performed immunohistochemical analysis of FGFR2 protein in healthy skin and active psoriatic plaques.
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Figure 4. Fibroblast growth factor receptor 2 (FGFR2) is a direct target of microRNA-125b (miR-125b). (a) Venn diagram depicting the number of potential targets of miR-125b predicted by three bioinformatics methods. (b) Schematic representation of the 30 untranslated region (30 UTR) of FGFR2 mRNA (gray bar) with the predicted target sites for miR-125b (red lines). Mfe, the minimal free energies for miR-125b binding. (c) Nucleotide resolution of the predicted target sites: the seed sequence (green letters); the target sequence (red letters); the evolutionarily conserved regions (gray boxes); the deleted miR-125b-binding sites (black boxes). (d) HeLa cells were transfected with firefly luciferase (FL) reporter construct containing FGFR2 30 UTR together with miR-125b precursor RNA (Pre-miR-125b) or miR-125b inhibitor oligonucleotide (Anti-miR-125b). (e) The FL constructs containing the wild-type (WT) or mutant (MUT) 30 UTR or empty FL vector (Vector) were transfected to HeLa cells together with Pre-miR-125b or miRNA precursor control (Pre-miR-ctrl). Means±SD of three independent experiments are shown. *Po0.05, **Po0.01, ***Po0.001.
FGFR2 was predominantly detected in the basal keratinocytes in healthy skin, whereas in psoriasis the expression of FGFR2 was increased and presented in both basal and suprabasal keratinocytes (Figure 5c). The concurrent upregulation of FGFR2 and downregulation of miR-125b in psoriasis supports the regulation of FGFR2 by miR-125b in the skin. Silencing of FGFR2 suppresses keratinocyte proliferation
To determine whether the observed effects of miR-125b on keratinocyte proliferation and differentiation are at least partially mediated through FGFR2, we analyzed the effects of silencing of FGFR2 expression by small interfering RNA (siRNA) on keratinocyte proliferation and differentiation. FGFR2 expression in keratinocytes was significantly
decreased by siRNA at both mRNA and protein levels at 48 and 72 hours post-transfection (Figure 6a and b). Results from EdU assays showed that keratinocytes transfected with FGFR2-specific siRNA had significantly less EdU-positive cells at both 48 and 72 hours after the transfection, corresponding to a suppressed proliferation rate (Figure 6c). Hence, the suppression of keratinocyte proliferation by miR-125b may at least partially be mediated through the regulation of FGFR2. In contrast, reduced expression of FGFR2 had no effect on the expression of the differentiation marker involucrin, whereas it decreased the expression of K10 at 96 hours post-transfection (Figure 6d). This suggests that the regulation of keratinocyte differentiation by miR125b is mediated through targets other than FGFR2. www.jidonline.org 1525
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Figure 5. Fibroblast growth factor receptor 2 (FGFR2) is regulated by microRNA-125b (miR-125b) in keratinocytes and overexpressed in psoriasis. Keratinocytes were transfected with miR-125b precursor RNA (Pre-miR-125b) or miR-125b inhibitor oligonucleotide (Anti-miR-125b) and both RNA and protein were collected. (a) The expression of FGFR2 mRNA was analyzed by real-time quantitative reverse transcription-PCR (qRT-PCR). (b) FGFR2 protein was detected by western blotting and the results for the protein collected 48 hours after transfection are shown. b-Actin was detected on the same blots as loading control. (c) The expression of FGFR2 in healthy (n ¼ 3) and psoriasis skin (n ¼ 4) was detected by immunohistochemistry and the rectangular areas marked in upper figures are shown at higher magnification below. The red–brown color indicates FGFR2 expression. Bar ¼ 50 mm.
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DISCUSSION In this study, we show that miR-125b is significantly downregulated in psoriatic epidermis and that it modulates keratinocyte functions by directly repressing FGFR2 expression. MiR-125b is a multifunctional miRNA that plays important roles in many physiological and pathological processes in humans. In particular, miR-125b has been implicated in 1526 Journal of Investigative Dermatology (2011), Volume 131
carcinogenesis. It can function as a tumor suppressor, e.g., in medulloblastoma (Ferretti et al., 2008), oral squamous cell carcinoma (Henson et al., 2009), breast cancer (Scott et al., 2007), bladder cancer (Huang et al., 2010), and ovarian cancer (Guan et al., 2010). However, several reports showed that miR-125b may also function as an oncogene in prostate cancer cells (Shi et al., 2007) and human neuroblastoma cells and lung fibroblasts (Le et al., 2009). Hence, miR-125b plays different or even opposite roles in different tissue/cell types, which might be attributed to the varied expression context of miR-125b target genes in each tissue/cell type. In this study we show the significant downregulation of miR-125b in a nonmalignant hyperproliferative disease, psoriasis. Our study revealed the function of miR-125b in human keratinocytes: it inhibits keratinocyte proliferation, but promotes terminal differentiation. Importantly, inhibition of endogenous miR125b has the opposite effect, i.e., increased keratinocyte proliferation and suppressed differentiation. Downregulation of miR-125b in the psoriatic epidermis may thus contribute to hyperproliferation and altered differentiation of keratinocytes. This study identified FGFR2 as one of the experimentally verified target genes for miR-125b in keratinocytes. The major isoform of FGFR2 expressed in keratinocytes is FGFR2IIIb (also known as keratinocyte growth factor receptor (KGFR)). Importantly, the mRNA encoding this isoform contains all four miR-125b-binding sites in its 30 UTR (Thierry-Mieg and Thierry-Mieg, 2006). FGF1, FGF3, FGF7, FGF10, and FGF22 specifically bind to FGFR2-IIIb and activate the downstream signals (reviewed in Katoh, 2009). FGFR2 is highly conserved through evolution, and it plays important roles in controlling epithelial growth and differentiation (Farkas et al., 2003; Katoh, 2009). The increased expression of FGFR2-IIIb has been observed in many human tumors of epithelial origins, which could be associated with cell transformation (Finch and Rubin, 2006). In healthy skin, FGFR2 was expressed mainly in the basal keratinocyte layer (Figure 5c), in line with previous studies (Finch et al., 1997; Revest et al., 2001; Mailleux et al., 2002). Unlike FGFR2, miR-125b is expressed in all layers of the epidermis (Figure 1b), indicating that the roles of miR-125b in skin may be mediated by different target gene sets in basal and suprabasal cell layers. In psoriasis lesions, we observed increased FGFR2 expression that also extended to the suprabasal layers. In accordance with our results, FGF-7 and FGFR2 were previously reported to be upregulated in psoriatic lesional
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Figure 6. The effects of silencing fibroblast growth factor receptor 2 (FGFR2) expression on keratinocyte proliferation and differentiation. Keratinocytes were transfected with 30 nM small interfering RNA (siRNA) for FGFR2 (siFGFR2) or siRNA-negative control (siRNA-Ctrl) and both RNA and protein were collected 48 and 72 hours post-transfection (hpt) and analyzed by (a) real-time quantitative reverse transcription-PCR (qRT-PCR) and (b) western blotting. (c) The percentage of 5-ethynyl-20 -deoxyuridine (EdU)-positive cells was analyzed by flow cytometry at 48 and 72 hpt. (d) The mRNAs of involucrin and cytokeratine 10 were analyzed by qRT-PCR at 24–96 hpt. The expression of FGFR2, involucrin, and cytokeratine 10 was normalized to 18S RNA and data are expressed as relative units (RU). The data of one representative experiment with triplicates are shown. The experiment was repeated three times. *Po0.05, **Po0.01, ***Po0.001.
skin compared with nonlesional skin (Finch et al., 1997). As FGFR2 is directly targeted by miR-125b, increased FGFR2 levels in psoriasis may partially be attributable to the reduced expression of miR-125b. Interestingly, two other miRNAs were shown to regulate FGFR2 signaling: miR-433 inhibits FGF20 expression in individuals with the C allele of the rs12720208 singlenucleotide polymorphism (Wang et al., 2008) and miR-21 represses the FGFR2 signaling inhibitor, sprouty 1 (Spry1), in cardiac fibroblasts (Thum et al., 2008). MiR-21 was also found to be upregulated in psoriatic skin compared with healthy skin (Sonkoly et al., 2007). Future studies will address whether the upregulation of miR-21 contributes to the increased FGFR2 signaling in psoriasis. Are the biological effects of ectopic miR-125b expression on keratinocyte proliferation and differentiation mediated by modulation of FGFR2 levels? Downregulation of FGFR2 expression by introducing FGFR2-specific siRNA repressed keratinocyte proliferation. This is in line with mouse studies showing that knocking out FGFR2 caused severe epidermal hypoplasia (Revest et al., 2001; Petiot et al., 2003) and which suggest that the antiproliferation effect of miR-125b in human keratinocyte is, at least partially, mediated by repressing FGFR2 expression. On the other hand, when we specifically knocked down FGFR2 expression by siRNA, the expression of differentiation marker K10 was slightly downregulated, in agreement with the previous findings that FGFR2 promotes early differentiation program (Marchese et al., 1990, 1997). Obviously, the prodifferentiation effects of miR-125b in keratinocytes cannot be explained by inhibiting FGFR2 expression. Instead, these are likely to be mediated by downregulation of other miR-125b target
genes, which might function as the suppressors of keratinocyte differentiation. This hypothesis will be examined in our future studies. According to our current understanding of miRNA function, each miRNA regulates dozens to hundreds of genes simultaneously. Thus, it is likely that additional targets contribute to the effect of miR-125b on keratinocyte proliferation and differentiation—in addition to FGFR2. Several target genes for miR-125b, which are involved in regulating cell proliferation and differentiation, have been identified in other cell types, e.g., Smoothened, activator of the Hedgehog pathway in medulloblastoma (Ferretti et al., 2008), oncogene ERBB2 and ERBB3 in breast cancer (Scott et al., 2007), proto-oncogene BCL3 in ovarian cancer (Guan et al., 2010), and E2F3 in bladder cancer (Huang et al., 2010). It would be interesting to study the expression of these proteins in keratinocyte and psoriasis skin, which may further explain the mechanisms of miR-125b regulating keratinocyte proliferation and differentiation. Taken together, our results demonstrate that miR-125b inhibits keratinocyte proliferation and promotes differentiation. Suppression of keratinocyte proliferation by miR-125b is partially mediated by the inhibition of its direct target, FGFR2. Downregulation of miR-125b in psoriasis skin lesions may contribute to hyperproliferation and aberrant differentiation of keratinocytes. This study implicates miR-125b as a potential therapeutic target for psoriasis. PATIENTS AND METHODS Patients Punch biopsies (6 mm) were taken, after informed consent, from lesional skin (n ¼ 25) of patients with moderate or severe chronic www.jidonline.org 1527
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plaque psoriasis, and from noninflamed, nonirritated skin of healthy individuals (n ¼ 27). The psoriasis patients had not received systemic immunosuppressive treatment or psoralen plus ultraviolet light A/solarium/UV for at least 1 month, and topical therapy for at least 2 weeks before skin biopsy. The study was approved by the Stockholm Regional Ethics Committee, and conducted according to the Declaration of Helsinki Principles.
Immunohistochemistry FGFR2 protein expression was analyzed in frozen skin sections (7 mm in thickness) using rabbit anti-human FGFR2 antibody (Bek (C-17), 1:100; Santa Cruz Biotechnology, Santa Cruz, CA) and the avidin-biotin-peroxidase complex staining system (Vector Laboratories, Burlingame, CA) following the manufacturer’s instructions. The omission of the primary antibody in the staining procedure was used as a negative control.
RNA extraction The details of RNA extraction are described in the Supplementary Materials and Methods online.
qRT-PCR The details of qRT-PCR are described in the Supplementary Materials and Methods online.
In situ hybridization In situ hybridizations were performed on formalin-fixed, paraffinembedded sections of skin biopsy specimens from eight psoriasis patients and six healthy individuals using digoxygenin-labeled miRCURY locked nucleic acid probes (Exiqon, Vedbaek, Denmark) as previously described (Sonkoly et al., 2007). The details are described in the Supplementary Materials and Methods online.
Cells and transfections The details of cell culture are described in the Supplementary Materials and Methods online. To study the biological effects of miR-125b on keratinocytes, third-passage keratinocytes at 30–50% confluence were transfected with 10 nM Pre-miR-125b miRNA precursor or Pre-miR miRNA precursor negative control 1 (Ambion, Foster City, CA); 25 nM miRIDIAN miR-125b hairpin inhibitor or microRNA hairpin inhibitor negative control 1 (ThermoFisher Scientific, Lafayette, CO); 30 nM Silencer select pre-designed siRNA for FGFR2 (siRNA ID: S5175) or siRNA negative control 1 (Ambion) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA), following the manufacturer’s instructions. The mock controls were included in each transfection experiment, in which only Lipofectamine 2000 reagent was added to the cell culture and there were no oligos present. Overexpression/downregulation of the mature, biologically active form of miR-125b was confirmed by qRT-PCR after transfection (data not shown).
Cell proliferation analysis EdU was added at a 10 mM final concentration to the transfected cells 2 hours before harvesting. Click-iT EdU Flow Cytometry Assay (Invitrogen) was carried out according to the manufacturer’s instructions and analyzed by flow cytometry on a FACScan (Becton Dickinson, Franklin Lakes, NJ) to determine EdU-positive cells and the cell-cycle distribution.
Western blotting Keratinocytes were lysed by RIPA buffer supplemented with Halt protease inhibitor cocktail and phosphatase inhibitor cocktail (Pierce, Rockford, IL) and the protein concentrations were determined by BCA protein assay kit (ThermoFisher Scientific). The details of western blotting are described in the Supplementary Materials and Methods online. 1528 Journal of Investigative Dermatology (2011), Volume 131
Plasmids mutagenesis and 30 UTR luciferase-binding assays Firefly luciferase reporter plasmids containing 30 UTR of the FGFR2 gene and empty luciferase vector were obtained from SwitchGear Genomics (Menlo Park, CA). The deletions were generated with the predicted target sites of FGFR2 30 UTR using the QuickChange XL site-directed mutagenesis kit (Stratagene, Lo Jolla, CA) according to the manufacturer’s instructions. The details of 30 UTR luciferasebinding assays are described in the Supplementary Materials and Methods online. CONFLICT OF INTEREST The authors state no conflict of interest.
ACKNOWLEDGMENTS We thank Anna-Lena Kastman, Clara Ibel Chamorro Jimenez, Jakob Love´n, Per Johnsson, and Martin Corcoran for the excellent assistance and technical support. This work was supported by the Swedish Research Council (Vetenskapsra˚det), Medical Research Council, the Swedish Psoriasis Association (Psoriasisfo¨rbundet), the Welander and Finsens Foundation, the Tore Nilssons Foundation, the Lars Hierta Memorial Foundation, the Sigurd and Elsa Goljes Foundation, TAMOP-4.2.2/08/1, Centre of Excellence for Research on Inflammation and Cardiovascular disease (CERIC), Karolinska Institutet, and the Stockholm County Council. A.P. was supported by LEO Pharma Research Foundation.
SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper at http:// www.nature.com/jid
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Rehmsmeier M, Steffen P, Hochsmann M et al. (2004) Fast and effective prediction of microRNA/target duplexes. RNA 10:1507–17
Henson BJ, Bhattacharjee S, O’Dee DM et al. (2009) Decreased expression of miR-125b and miR-100 in oral cancer cells contributes to malignancy. Genes Chromosomes Cancer 48:569–82
Revest JM, Spencer-Dene B, Kerr K et al. (2001) Fibroblast growth factor receptor 2-IIIb acts upstream of Shh and Fgf4 and is required for limb bud maintenance but not for the induction of Fgf8, Fgf10, Msx1, or Bmp4. Dev Biol 231:47–62
Huang L, Luo J, Cai Q et al. (2010) MicroRNA-125b suppresses the development of bladder cancer by targeting E2F3. Int J Cancer 128:1758–69 Katoh M (2009) FGFR2 abnormalities underlie a spectrum of bone, skin, and cancer pathologies. J Invest Dermatol 129:1861–7 Krek A, Grun D, Poy MN et al. (2005) Combinatorial microRNA target predictions. Nat Genet 37:495–500 Le MT, Teh C, Shyh-Chang N et al. (2009) MicroRNA-125b is a novel negative regulator of p53. Genes Dev 23:862–76 Lebwohl M (2003) Psoriasis. Lancet 361:1197–204 Lena AM, Shalom-Feuerstein R, Rivetti di Val Cervo P et al. (2008) miR-203 represses ‘stemness’ by repressing DeltaNp63. Cell Death Differ 15:1187–95 Mailleux AA, Spencer-Dene B, Dillon C et al. (2002) Role of FGF10/FGFR2b signaling during mammary gland development in the mouse embryo. Development 129:53–60 Marchese C, Rubin J, Ron D et al. (1990) Human keratinocyte growth factor activity on proliferation and differentiation of human keratinocytes: differentiation response distinguishes KGF from EGF family. J Cell Physiol 144:326–32 Marchese C, Sorice M, De Stefano C et al. (1997) Modulation of keratinocyte growth factor receptor expression in human cultured keratinocytes. Cell Growth Differ 8:989–97 Petiot A, Conti FJ, Grose R et al. (2003) A crucial role for Fgfr2-IIIb signalling in epidermal development and hair follicle patterning. Development 130:5493–501
Scott GK, Goga A, Bhaumik D et al. (2007) Coordinate suppression of ERBB2 and ERBB3 by enforced expression of micro-RNA miR-125a or miR-125b. J Biol Chem 282:1479–86 Shi XB, Xue L, Yang J et al. (2007) An androgen-regulated miRNA suppresses Bak1 expression and induces androgen-independent growth of prostate cancer cells. Proc Natl Acad Sci USA 104:19983–8 Sonkoly E, Wei T, Janson PC et al. (2007) MicroRNAs: novel regulators involved in the pathogenesis of Psoriasis? PLoS One 2:e610 Sonkoly E, Wei T, Pavez Lorie E et al. (2010) Protein kinase C-dependent upregulation of miR-203 induces the differentiation of human keratinocytes. J Invest Dermatol 130:124–34 Thierry-Mieg D, Thierry-Mieg J (2006) AceView: a comprehensive cDNA-supported gene and transcripts annotation. Genome Biol 7 (Suppl 1):S12.1–4 Thum T, Gross C, Fiedler J et al. (2008) MicroRNA-21 contributes to myocardial disease by stimulating MAP kinase signalling in fibroblasts. Nature 456:980–4 Tonel G, Conrad C (2009) Interplay between keratinocytes and immune cells–recent insights into psoriasis pathogenesis. Int J Biochem Cell Biol 41:963–8 Wang G, van der Walt JM, Mayhew G et al. (2008) Variation in the miRNA-433 binding site of FGF20 confers risk for Parkinson disease by overexpression of alpha-synuclein. Am J Hum Genet 82:283–9 Yi R, Poy MN, Stoffel M et al. (2008) A skin microRNA promotes differentiation by repressing ‘stemness’. Nature 452:225–9
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MiR-125b, a MicroRNA Downregulated in Psoriasis, Modulates Keratinocyte Proliferation by Targeting FGFR2 Ning Xu1, Petter Brodin2, Tianling Wei1, Florian Meisgen1, Liv Eidsmo1, Nikoletta Nagy3, Lajos Kemeny3, Mona Sta˚hle1, Eniko¨ Sonkoly1,4 and Andor Pivarcsi1,4 MicroRNAs (miRNAs) are short, single-stranded, noncoding RNAs that play important roles in the regulation of gene expression. We previously identified a characteristic miRNA expression profile in psoriasis, distinct from that of healthy skin. One of the most downregulated miRNAs in psoriasis skin was microRNA-125b (miR-125b). In this study, we aimed to identify the potential role(s) of miR-125b in psoriasis pathogenesis. In situ hybridization results showed that the major cell type responsible for decreased miR-125b levels in psoriasis lesions was the keratinocyte. Overexpression of miR-125b in primary human keratinocytes suppressed proliferation and induced the expression of several known differentiation markers. Conversely, inhibition of endogenous miR-125b promoted cell proliferation and delayed differentiation. Fibroblast growth factor receptor 2 (FGFR2) was identified as one of the direct targets for suppression by miR-125b by luciferase reporter assay. The expression of miR-125b and FGFR2 was inversely correlated in both transfected keratinocytes and in psoriatic skin. Knocking down FGFR2 expression by siRNA suppressed keratinocyte proliferation, but did not enhance differentiation. Altogether, our results demonstrate a role for miR-125b in the regulation of keratinocyte proliferation and differentiation, partially through the regulation of FGFR2. Loss of miR-125b in psoriasis skin may contribute to hyperproliferation and aberrant differentiation of keratinocytes. Journal of Investigative Dermatology (2011) 131, 1521–1529; doi:10.1038/jid.2011.55; published online 17 March 2011
INTRODUCTION Psoriasis is a common chronic inflammatory skin disease, which affects nearly 3% of the world’s population and decreases patients’ quality of life considerably (Lebwohl, 2003). Psoriasis is thought to be initiated by complex interactions between environmental and genetic factors. Psoriasis skin lesions are characterized by hyperproliferation and aberrant differentiation of keratinocytes and infiltration of inflammatory cells into the dermis and epidermis (Tonel and
1
Molecular Dermatology Research Group, Unit of Dermatology and Venereology, Department of Medicine, Karolinska Institutet, Stockholm, Sweden; 2Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden; 3Dermatological Research Group of the Hungarian Academy of Sciences, University of Szeged, Szeged, Hungary and 4 Department of Dermatology and Allergology, University of Szeged, Szeged, Hungary Correspondence: Ning Xu, Molecular Dermatology Research Group, Centre for Molecular Medicine (CMM), L8:02, Department of Medicine, Karolinska Institutet, Stockholm SE-17176, Sweden. E-mail: [email protected] Abbreviations: 30 UTR, 30 untranslated region; Anti-miR-125b, miR-125b inhibitor oligonucleotide; EdU, 5-ethynyl-20 -deoxyuridine; FGFR2, fibroblast growth factor receptor 2; K10, cytokeratin 10; K5, cytokeratin 5; miR-125b, microRNA-125b; miRNA, microRNA; mRNA, messenger RNA; Pre-miR125b, miR-125b precursor RNA; qRT-PCR, real-time quantitative reverse transcription-PCR; siRNA, small interfering RNA Received 16 September 2010; revised 7 January 2011; accepted 5 February 2011; published online 17 March 2011
& 2011 The Society for Investigative Dermatology
Conrad, 2009). However, the underlying mechanisms regulating these epidermal defects and immunological dysfunction remain largely elusive. MicroRNAs (miRNAs) are B22 nucleotide-long singlestranded noncoding RNAs that can mediate post-transcriptional silencing by binding with partial complementarity to the 30 untranslated region (UTR) of the target messenger RNA (mRNA) (Ambros et al., 2003). Base-pairing at position 2–8 nucleotides relative to the 50 end of small RNA, termed the ‘‘seed’’ region, appears to be important for target recognition (Bartel, 2004). A unique miRNA can regulate the expression of hundreds of proteins, and the expression of a specific protein may be controlled by several miRNAs (Krek et al., 2005). The high sequence conservation of many miRNAs between distantly related organisms suggests strong evolutionary pressure (Farh et al., 2005) and they have been shown to participate in many fundamental life processes, e.g., development, differentiation, organogenesis, growth control, and apoptosis. Accordingly, deregulation of miRNA expression has been shown to contribute to cancer, heart diseases, infectious diseases, inflammatory diseases, and other medical conditions, making them potential targets for medical diagnosis and therapy (reviewed in Czech, 2006). The role of miRNAs in psoriasis remains largely unexplored. In 2007, we performed a genome-wide microarray analysis of miRNA expression in psoriasis and identified a www.jidonline.org 1521
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distinct miRNA expression profile in psoriatic lesions, compared with healthy human skin and/or with another chronic inflammatory skin disease, atopic eczema (Sonkoly et al., 2007). In particular, this study identified the first skinand keratinocyte-specific miRNA, miR-203, which is highly upregulated in psoriasis and regulates inflammatory pathways as well as keratinocyte proliferation and differentiation (Sonkoly et al., 2007, 2010; Lena et al., 2008; Yi et al., 2008). MicroRNA-125b (miR-125b) was one of the miRNAs found to be significantly decreased in psoriasis skin in comparison with healthy skin in our miRNA microarray analysis (Sonkoly et al., 2007). Expression profiling in healthy human organs/tissues showed that miR-125b is expressed in most organs. In normal skin, it is mainly expressed by resident cells such as fibroblasts, keratinocytes, and melanocytes (Sonkoly et al., 2007). To date, nothing is known about the function of miR-125b in healthy or inflamed skin. The aim of this study was to determine the potential role of miR-125b in psoriasis.
In order to identify which cell type(s) in the skin are responsible for the reduced expression of miR-125b in psoriasis, we performed in situ hybridization on skin sections from healthy individuals (n ¼ 6) and psoriasis patients (n ¼ 8) using miR-125b-specific locked nucleic acid-modified probes (Figure 1b). MiR-125b was expressed throughout the epidermis in healthy skin (Figure 1b), whereas its expression was decreased in all epidermal layers in psoriasis lesions. To quantify the expression of miR-125b specifically in the epidermis, we separated epidermis from dermis by dispase treatment. Measurement of miR-125b by qRT-PCR showed a 4.8-fold (Po0.05) decrease of miR-125b expression in epidermis from psoriasis lesional skin when compared with healthy epidermis (Figure 1c). These results indicate that the decrease of miR-125b expression in psoriasis skin results from its reduced expression in keratinocytes. The mature miR-125b is processed from two primary miR-125b transcripts in human: pri-miR-125b-1, encoded on chromosome 11, and pri-miR-125b-2, encoded on chromosome 21 (Griffiths-Jones et al., 2006). To explore which primary transcript of the miR-125b gene is dominant in skin, we quantified the expression of pri-miR-125b-1 and pri-miR-125b-2 in RNA samples from seven healthy skin samples and nine psoriasis lesional skin samples by qRT-PCR using primer sets designed to detect the specific primary transcripts (Figure 1d). The results showed that pri-miR125b-1 was the major miR-125b precursor in human skin. Compared with healthy skin, pri-miR-125b-1 was significantly decreased in psoriatic lesional skin (Po0.01), which suggested that the downregulation of miR-125b in psoriasis
RESULTS MiR-125b is suppressed in keratinocytes in psoriasis lesions
To confirm previous results from microarray profiling (Sonkoly et al., 2007), we performed real-time quantitative reverse transcription-PCR (qRT-PCR) analysis of the mature miR-125b expression on RNA samples from healthy skin (n ¼ 27) and psoriasis lesional skin (n ¼ 25; Figure 1a). The results showed that miR-125b was 3.7-fold (Po0.001) downregulated in psoriasis skin lesions compared with healthy skin, which supported our previous microarray data.
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Figure 1. The expression of microRNA-125b (miR-125b) in healthy and psoriasis skin. (a) MiR-125b expression was analyzed in healthy (n ¼ 27) and psoriasis lesional skin samples (n ¼ 25) using real-time quantitative reverse transcription-PCR (qRT-PCR). ***Po0.001. (b) In situ hybridization was performed on healthy (n ¼ 6, left panel) and psoriasis lesional skin sections (n ¼ 8, right panel) using miR-125b-specific locked nucleic acid (LNA) probe (upper panel) or scrambled probe (lower panel). The blue–purple color indicates miR-125b expression. Bar ¼ 50 mm. (c) MiR-125b expression was analyzed on RNA from epidermis of healthy (n ¼ 5) and psoriasis lesional skin (n ¼ 3) using qRT-PCR. *Po0.05. (d) Expression of primary miR-125b transcripts was analyzed in healthy (n ¼ 7) and psoriasis lesional skin samples (n ¼ 9) using qRT-PCR. Results for individual patients and mean are shown. **Po0.01. (e) qRT-PCR analysis of primary miR-125b transcripts in primary keratinocytes.
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skin occurred at the transcriptional level rather than during processing of the primary transcript. In addition, we showed that pri-miR-125b-1 was also the dominant form of miR-125b precursor in cultured primary keratinocytes (Figure 1e). MiR-125b suppresses the proliferation of keratinocytes
In order to determine the possible effects of aberrant miR-125b expression on keratinocyte proliferation, we transfected human primary keratinocytes with miR-125b precursor RNA (Pre-miR-125b, to overexpress miR-125b) or miR-125b inhibitor oligonucleotide (Anti-miR-125b, to inhibit endogenous miR-125b) and analyzed the cell proliferation rate by 5-ethynyl-20 -deoxyuridine (EdU) incorporation assay using flow cytometry and gating on live keratinocytes (Figure 2a–d). The EdU incorporation rate (percentage of cells that underwent cell division during the assay) was reduced Pre-miR-125b
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Terminal differentiation is severely altered in psoriasis. To determine whether suppressed expression of miR-125b in psoriasis keratinocytes may contribute to these alterations, we investigated the role of miR-125b on keratinocyte differentiation. We analyzed the differentiation status of keratinocytes at 24, 48, 72, and 96 hours after transfection with specific precursors or inhibitors for miR-125b as well as their respective control oligonucleotides and mock controls. We measured the expression of several keratinocyte differentiation markers including the early differentiation markers involucrin and cytokeratin 10 (K10), the late differentiation marker filaggrin, the differentiation-induced miRNA miR-203 (Sonkoly et al., 2010), as well as the basal cell marker cytokeratin 5 (K5), whose expression does not depend on cellular differentiation (Fuchs, 1990). The level of K5 remained constant as the confluence of the cultured cells increased with time, whereas the expression of the other four differentiation markers increased as expected (Figure 3 and Supplementary Figure S1 online). Overexpression of miR-125b significantly increased involucrin expression at both mRNA and protein levels, whereas inhibition of endogenous miR-125b decreased involucrin protein expression (Figure 3a). In addition, we observed a trend of reduced involucrin mRNA expression by inhibition of miR-125b in three independent experiments, although the change was not significant. Similar results were observed for the expression of K10 and filaggrin mRNA as well as miR-203 in the keratinocytes with overexpressed or inhibited miR-125b (Supplementary Figure S1a–d online). However, neither overexpression nor inhibition of miR-125b changed the mRNA level of K5 (Figure 3b). Together, these data indicate that miR-125b promotes keratinocyte differentiation. Fibroblast growth factor receptor 2 (FGFR2) is a direct target for miR-125b
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to 47% (Po0.001) in the cells overexpressing miR-125b compared with the cells overexpressing a control miRNA with a scrambled sequence, whereas inhibition of the endogenous miR-125b increased the EdU incorporation rate by 58% (Po0.01) compared with the cells transfected with scrambled miRNA inhibitor (Figure 2e). These results indicate that miR-125b functions as a negative regulator of keratinocyte proliferation.
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Figure 2. MicroRNA-125b (miR-125b) suppresses the proliferation of keratinocytes. Primary human keratinocytes were transfected with (a) miR-125b precursor RNAs (Pre-miR-125b), (b) miRNA precursor control (Pre-miR-ctrl), (c) miR-125b inhibitor oligonucleotides (Anti-miR-125b), or (d) miRNA inhibitor control (Anti-miR-ctrl). The percentage of cells that underwent cell division was assessed by anti-5-ethynyl-20 -deoxyuridine (anti-EdU) Alexa Fluor 647 staining 48 hours post-transfection. The representative flow cytometry histograms gated on live propidium iodide (PI)-negative cells are shown and the percentages of EdU-positive cells are indicated. (e) The data of one representative experiment with quintuplicate are shown. The experiment was repeated five times. **Po0.01, ***Po0.001.
Identifying the targets for miRNAs is essential for understanding their functions. In order to identify the direct target for miR-125b, we applied three standards for selection of the potential targets for further experimental validation. The target genes should be: (1) directly regulated by miR-125b binding; (2) coexpressed with miR-125b in keratinocytes; and (3) upregulated in psoriatic skin compared with healthy skin. To predict the potential targets for miR-125b, we performed an integrated prediction using three conventional bioinformatics methods: PicTar (http://pictar.mdc-berlin.de) (Krek et al., 2005), miRWalk (http://mirwalk.uni-hd.de), and RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/ submission.html) (Rehmsmeier et al., 2004) (Figure 4a and www.jidonline.org 1523
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Figure 3. MicroRNA-125b (miR-125b) promotes the differentiation of keratinocytes. Primary human keratinocytes were transfected with miR-125b precursor RNA (Pre-miR-125b) or miRNA precursor control (Pre-miR-ctrl; left panel); miR-125b inhibitor oligonucleotide (Anti-miR-125b) or miRNA inhibitor control (Anti-miR-ctrl; right panel). Both RNA and protein were collected at 24, 48, 72, and 96 hours after transfection. The involucrin messenger (m)RNA (a, upper panel) and cytokeratine 5 mRNA (b) were analyzed by real-time quantitative reverse transcription-PCR (qRT-PCR) and normalized to 18S RNA. Data are expressed as relative units (RU). The data of one representative experiment with triplicates are shown. The experiment was repeated three times. *Po0.05, **Po0.01. (a, lower panel) The expression of involucrin protein was analyzed by western blotting. b-Actin was detected on the same blots as loading control.
Supplementary Table S1 online). One of the potential target genes predicted by all three algorithms was FGFR2, a receptor expressed on keratinocytes and reported to be upregulated in lesional psoriasis skin (Finch et al., 1997). Bioinformatic tools predicted four potential miR-125b target sites in the 30 UTR of FGFR2 (target sites 1–4; Figure 4b and c). To verify whether FGFR2 is indeed a direct target gene for miR-125b, we performed 30 UTR luciferase-binding assays. To this end, the whole 30 UTR (1,594 nucleotides) of FGFR2 mRNA was cloned into a luciferase reporter plasmid and subsequently co-transfected with Pre-miR-125b or AntimiR-125b as well as their respective control oligonucleotides into HeLa cells (Figure 4d) and primary keratinocytes (Supplementary Figure S2 online). The endogenous miR125b expression levels in both cell types are shown in Supplementary Figure S3 online. Luciferase activity was analyzed 48 hours after transfection. In HeLa cells, overexpression of miR-125b reduced the luciferase activity to 46% (Po0.01), whereas inhibition of endogenous miR-125b increased the luciferase activity 2.5-fold (Po0.05) compared with scrambled pre-miR or anti-miR controls, respectively (Figure 4d). Similar results were observed in primary keratinocytes (Supplementary Figure S2 online). To determine whether the regulation of luciferase reporter gene by miR-125b is mediated by the predicted target sites in the 30 UTR of FGFR2, we deleted five to seven nucleotides within the seed-matching sequences of the predicted target sites 1, 2, 3, and 4 in plasmids named MUT1, MUT2, MUT3, 1524 Journal of Investigative Dermatology (2011), Volume 131
and MUT4, respectively (Figure 4c). Moreover, we created a construct named plasmid MUT 1–4 in which all four target sites were deleted (Figure 4c). Overexpression of miR-125b reduced the luciferase activity of the construct containing the wild-type 30 UTR of FGFR2 to 39% (Figure 4e, WT). Deletion of the seed-matching sequence in each potential target site led to partial restoration of luciferase activity to 67% for MUT1, 79% for MUT2, 64% for MUT3, and 67% for MUT4 (Figure 4e). Deletion of all four predicted miR-125b target sites led to a nearly complete restoration of luciferase activity (Figure 4e, MUT 1–4). Taken together, these results demonstrate that miR-125b directly regulates FGFR2 expression by binding to the four target sites in the 30 UTR of FGFR2 mRNA. MiR-125b regulates FGFR2 protein expression in keratinocytes
In the previous experiment we demonstrated that miR-125b can mediate post-transcriptional suppression through FGFR2 30 UTR. Next, we investigated whether miR-125b regulates FGFR2 expression at the mRNA or at the protein level in keratinocytes (Figure 5a and b). Although miR-125b overexpression or inhibition did not affect FGFR2 mRNA levels (Figure 5a), the results of western blot analysis showed that overexpression of miR-125b suppressed FGFR2 protein expression, and inhibition of miR-125b led to the upregulation of FGFR2 at the protein level (Figure 5b). To determine whether the interaction of miR-125b and FGFR2 is relevant in vivo, we performed immunohistochemical analysis of FGFR2 protein in healthy skin and active psoriatic plaques.
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Figure 4. Fibroblast growth factor receptor 2 (FGFR2) is a direct target of microRNA-125b (miR-125b). (a) Venn diagram depicting the number of potential targets of miR-125b predicted by three bioinformatics methods. (b) Schematic representation of the 30 untranslated region (30 UTR) of FGFR2 mRNA (gray bar) with the predicted target sites for miR-125b (red lines). Mfe, the minimal free energies for miR-125b binding. (c) Nucleotide resolution of the predicted target sites: the seed sequence (green letters); the target sequence (red letters); the evolutionarily conserved regions (gray boxes); the deleted miR-125b-binding sites (black boxes). (d) HeLa cells were transfected with firefly luciferase (FL) reporter construct containing FGFR2 30 UTR together with miR-125b precursor RNA (Pre-miR-125b) or miR-125b inhibitor oligonucleotide (Anti-miR-125b). (e) The FL constructs containing the wild-type (WT) or mutant (MUT) 30 UTR or empty FL vector (Vector) were transfected to HeLa cells together with Pre-miR-125b or miRNA precursor control (Pre-miR-ctrl). Means±SD of three independent experiments are shown. *Po0.05, **Po0.01, ***Po0.001.
FGFR2 was predominantly detected in the basal keratinocytes in healthy skin, whereas in psoriasis the expression of FGFR2 was increased and presented in both basal and suprabasal keratinocytes (Figure 5c). The concurrent upregulation of FGFR2 and downregulation of miR-125b in psoriasis supports the regulation of FGFR2 by miR-125b in the skin. Silencing of FGFR2 suppresses keratinocyte proliferation
To determine whether the observed effects of miR-125b on keratinocyte proliferation and differentiation are at least partially mediated through FGFR2, we analyzed the effects of silencing of FGFR2 expression by small interfering RNA (siRNA) on keratinocyte proliferation and differentiation. FGFR2 expression in keratinocytes was significantly
decreased by siRNA at both mRNA and protein levels at 48 and 72 hours post-transfection (Figure 6a and b). Results from EdU assays showed that keratinocytes transfected with FGFR2-specific siRNA had significantly less EdU-positive cells at both 48 and 72 hours after the transfection, corresponding to a suppressed proliferation rate (Figure 6c). Hence, the suppression of keratinocyte proliferation by miR-125b may at least partially be mediated through the regulation of FGFR2. In contrast, reduced expression of FGFR2 had no effect on the expression of the differentiation marker involucrin, whereas it decreased the expression of K10 at 96 hours post-transfection (Figure 6d). This suggests that the regulation of keratinocyte differentiation by miR125b is mediated through targets other than FGFR2. www.jidonline.org 1525
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Figure 5. Fibroblast growth factor receptor 2 (FGFR2) is regulated by microRNA-125b (miR-125b) in keratinocytes and overexpressed in psoriasis. Keratinocytes were transfected with miR-125b precursor RNA (Pre-miR-125b) or miR-125b inhibitor oligonucleotide (Anti-miR-125b) and both RNA and protein were collected. (a) The expression of FGFR2 mRNA was analyzed by real-time quantitative reverse transcription-PCR (qRT-PCR). (b) FGFR2 protein was detected by western blotting and the results for the protein collected 48 hours after transfection are shown. b-Actin was detected on the same blots as loading control. (c) The expression of FGFR2 in healthy (n ¼ 3) and psoriasis skin (n ¼ 4) was detected by immunohistochemistry and the rectangular areas marked in upper figures are shown at higher magnification below. The red–brown color indicates FGFR2 expression. Bar ¼ 50 mm.
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DISCUSSION In this study, we show that miR-125b is significantly downregulated in psoriatic epidermis and that it modulates keratinocyte functions by directly repressing FGFR2 expression. MiR-125b is a multifunctional miRNA that plays important roles in many physiological and pathological processes in humans. In particular, miR-125b has been implicated in 1526 Journal of Investigative Dermatology (2011), Volume 131
carcinogenesis. It can function as a tumor suppressor, e.g., in medulloblastoma (Ferretti et al., 2008), oral squamous cell carcinoma (Henson et al., 2009), breast cancer (Scott et al., 2007), bladder cancer (Huang et al., 2010), and ovarian cancer (Guan et al., 2010). However, several reports showed that miR-125b may also function as an oncogene in prostate cancer cells (Shi et al., 2007) and human neuroblastoma cells and lung fibroblasts (Le et al., 2009). Hence, miR-125b plays different or even opposite roles in different tissue/cell types, which might be attributed to the varied expression context of miR-125b target genes in each tissue/cell type. In this study we show the significant downregulation of miR-125b in a nonmalignant hyperproliferative disease, psoriasis. Our study revealed the function of miR-125b in human keratinocytes: it inhibits keratinocyte proliferation, but promotes terminal differentiation. Importantly, inhibition of endogenous miR125b has the opposite effect, i.e., increased keratinocyte proliferation and suppressed differentiation. Downregulation of miR-125b in the psoriatic epidermis may thus contribute to hyperproliferation and altered differentiation of keratinocytes. This study identified FGFR2 as one of the experimentally verified target genes for miR-125b in keratinocytes. The major isoform of FGFR2 expressed in keratinocytes is FGFR2IIIb (also known as keratinocyte growth factor receptor (KGFR)). Importantly, the mRNA encoding this isoform contains all four miR-125b-binding sites in its 30 UTR (Thierry-Mieg and Thierry-Mieg, 2006). FGF1, FGF3, FGF7, FGF10, and FGF22 specifically bind to FGFR2-IIIb and activate the downstream signals (reviewed in Katoh, 2009). FGFR2 is highly conserved through evolution, and it plays important roles in controlling epithelial growth and differentiation (Farkas et al., 2003; Katoh, 2009). The increased expression of FGFR2-IIIb has been observed in many human tumors of epithelial origins, which could be associated with cell transformation (Finch and Rubin, 2006). In healthy skin, FGFR2 was expressed mainly in the basal keratinocyte layer (Figure 5c), in line with previous studies (Finch et al., 1997; Revest et al., 2001; Mailleux et al., 2002). Unlike FGFR2, miR-125b is expressed in all layers of the epidermis (Figure 1b), indicating that the roles of miR-125b in skin may be mediated by different target gene sets in basal and suprabasal cell layers. In psoriasis lesions, we observed increased FGFR2 expression that also extended to the suprabasal layers. In accordance with our results, FGF-7 and FGFR2 were previously reported to be upregulated in psoriatic lesional
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Figure 6. The effects of silencing fibroblast growth factor receptor 2 (FGFR2) expression on keratinocyte proliferation and differentiation. Keratinocytes were transfected with 30 nM small interfering RNA (siRNA) for FGFR2 (siFGFR2) or siRNA-negative control (siRNA-Ctrl) and both RNA and protein were collected 48 and 72 hours post-transfection (hpt) and analyzed by (a) real-time quantitative reverse transcription-PCR (qRT-PCR) and (b) western blotting. (c) The percentage of 5-ethynyl-20 -deoxyuridine (EdU)-positive cells was analyzed by flow cytometry at 48 and 72 hpt. (d) The mRNAs of involucrin and cytokeratine 10 were analyzed by qRT-PCR at 24–96 hpt. The expression of FGFR2, involucrin, and cytokeratine 10 was normalized to 18S RNA and data are expressed as relative units (RU). The data of one representative experiment with triplicates are shown. The experiment was repeated three times. *Po0.05, **Po0.01, ***Po0.001.
skin compared with nonlesional skin (Finch et al., 1997). As FGFR2 is directly targeted by miR-125b, increased FGFR2 levels in psoriasis may partially be attributable to the reduced expression of miR-125b. Interestingly, two other miRNAs were shown to regulate FGFR2 signaling: miR-433 inhibits FGF20 expression in individuals with the C allele of the rs12720208 singlenucleotide polymorphism (Wang et al., 2008) and miR-21 represses the FGFR2 signaling inhibitor, sprouty 1 (Spry1), in cardiac fibroblasts (Thum et al., 2008). MiR-21 was also found to be upregulated in psoriatic skin compared with healthy skin (Sonkoly et al., 2007). Future studies will address whether the upregulation of miR-21 contributes to the increased FGFR2 signaling in psoriasis. Are the biological effects of ectopic miR-125b expression on keratinocyte proliferation and differentiation mediated by modulation of FGFR2 levels? Downregulation of FGFR2 expression by introducing FGFR2-specific siRNA repressed keratinocyte proliferation. This is in line with mouse studies showing that knocking out FGFR2 caused severe epidermal hypoplasia (Revest et al., 2001; Petiot et al., 2003) and which suggest that the antiproliferation effect of miR-125b in human keratinocyte is, at least partially, mediated by repressing FGFR2 expression. On the other hand, when we specifically knocked down FGFR2 expression by siRNA, the expression of differentiation marker K10 was slightly downregulated, in agreement with the previous findings that FGFR2 promotes early differentiation program (Marchese et al., 1990, 1997). Obviously, the prodifferentiation effects of miR-125b in keratinocytes cannot be explained by inhibiting FGFR2 expression. Instead, these are likely to be mediated by downregulation of other miR-125b target
genes, which might function as the suppressors of keratinocyte differentiation. This hypothesis will be examined in our future studies. According to our current understanding of miRNA function, each miRNA regulates dozens to hundreds of genes simultaneously. Thus, it is likely that additional targets contribute to the effect of miR-125b on keratinocyte proliferation and differentiation—in addition to FGFR2. Several target genes for miR-125b, which are involved in regulating cell proliferation and differentiation, have been identified in other cell types, e.g., Smoothened, activator of the Hedgehog pathway in medulloblastoma (Ferretti et al., 2008), oncogene ERBB2 and ERBB3 in breast cancer (Scott et al., 2007), proto-oncogene BCL3 in ovarian cancer (Guan et al., 2010), and E2F3 in bladder cancer (Huang et al., 2010). It would be interesting to study the expression of these proteins in keratinocyte and psoriasis skin, which may further explain the mechanisms of miR-125b regulating keratinocyte proliferation and differentiation. Taken together, our results demonstrate that miR-125b inhibits keratinocyte proliferation and promotes differentiation. Suppression of keratinocyte proliferation by miR-125b is partially mediated by the inhibition of its direct target, FGFR2. Downregulation of miR-125b in psoriasis skin lesions may contribute to hyperproliferation and aberrant differentiation of keratinocytes. This study implicates miR-125b as a potential therapeutic target for psoriasis. PATIENTS AND METHODS Patients Punch biopsies (6 mm) were taken, after informed consent, from lesional skin (n ¼ 25) of patients with moderate or severe chronic www.jidonline.org 1527
N Xu et al. MiR-125b in Psoriasis
plaque psoriasis, and from noninflamed, nonirritated skin of healthy individuals (n ¼ 27). The psoriasis patients had not received systemic immunosuppressive treatment or psoralen plus ultraviolet light A/solarium/UV for at least 1 month, and topical therapy for at least 2 weeks before skin biopsy. The study was approved by the Stockholm Regional Ethics Committee, and conducted according to the Declaration of Helsinki Principles.
Immunohistochemistry FGFR2 protein expression was analyzed in frozen skin sections (7 mm in thickness) using rabbit anti-human FGFR2 antibody (Bek (C-17), 1:100; Santa Cruz Biotechnology, Santa Cruz, CA) and the avidin-biotin-peroxidase complex staining system (Vector Laboratories, Burlingame, CA) following the manufacturer’s instructions. The omission of the primary antibody in the staining procedure was used as a negative control.
RNA extraction The details of RNA extraction are described in the Supplementary Materials and Methods online.
qRT-PCR The details of qRT-PCR are described in the Supplementary Materials and Methods online.
In situ hybridization In situ hybridizations were performed on formalin-fixed, paraffinembedded sections of skin biopsy specimens from eight psoriasis patients and six healthy individuals using digoxygenin-labeled miRCURY locked nucleic acid probes (Exiqon, Vedbaek, Denmark) as previously described (Sonkoly et al., 2007). The details are described in the Supplementary Materials and Methods online.
Cells and transfections The details of cell culture are described in the Supplementary Materials and Methods online. To study the biological effects of miR-125b on keratinocytes, third-passage keratinocytes at 30–50% confluence were transfected with 10 nM Pre-miR-125b miRNA precursor or Pre-miR miRNA precursor negative control 1 (Ambion, Foster City, CA); 25 nM miRIDIAN miR-125b hairpin inhibitor or microRNA hairpin inhibitor negative control 1 (ThermoFisher Scientific, Lafayette, CO); 30 nM Silencer select pre-designed siRNA for FGFR2 (siRNA ID: S5175) or siRNA negative control 1 (Ambion) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA), following the manufacturer’s instructions. The mock controls were included in each transfection experiment, in which only Lipofectamine 2000 reagent was added to the cell culture and there were no oligos present. Overexpression/downregulation of the mature, biologically active form of miR-125b was confirmed by qRT-PCR after transfection (data not shown).
Cell proliferation analysis EdU was added at a 10 mM final concentration to the transfected cells 2 hours before harvesting. Click-iT EdU Flow Cytometry Assay (Invitrogen) was carried out according to the manufacturer’s instructions and analyzed by flow cytometry on a FACScan (Becton Dickinson, Franklin Lakes, NJ) to determine EdU-positive cells and the cell-cycle distribution.
Western blotting Keratinocytes were lysed by RIPA buffer supplemented with Halt protease inhibitor cocktail and phosphatase inhibitor cocktail (Pierce, Rockford, IL) and the protein concentrations were determined by BCA protein assay kit (ThermoFisher Scientific). The details of western blotting are described in the Supplementary Materials and Methods online. 1528 Journal of Investigative Dermatology (2011), Volume 131
Plasmids mutagenesis and 30 UTR luciferase-binding assays Firefly luciferase reporter plasmids containing 30 UTR of the FGFR2 gene and empty luciferase vector were obtained from SwitchGear Genomics (Menlo Park, CA). The deletions were generated with the predicted target sites of FGFR2 30 UTR using the QuickChange XL site-directed mutagenesis kit (Stratagene, Lo Jolla, CA) according to the manufacturer’s instructions. The details of 30 UTR luciferasebinding assays are described in the Supplementary Materials and Methods online. CONFLICT OF INTEREST The authors state no conflict of interest.
ACKNOWLEDGMENTS We thank Anna-Lena Kastman, Clara Ibel Chamorro Jimenez, Jakob Love´n, Per Johnsson, and Martin Corcoran for the excellent assistance and technical support. This work was supported by the Swedish Research Council (Vetenskapsra˚det), Medical Research Council, the Swedish Psoriasis Association (Psoriasisfo¨rbundet), the Welander and Finsens Foundation, the Tore Nilssons Foundation, the Lars Hierta Memorial Foundation, the Sigurd and Elsa Goljes Foundation, TAMOP-4.2.2/08/1, Centre of Excellence for Research on Inflammation and Cardiovascular disease (CERIC), Karolinska Institutet, and the Stockholm County Council. A.P. was supported by LEO Pharma Research Foundation.
SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper at http:// www.nature.com/jid
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