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miR-17-92 Cluster Promotes Cholangiocarcinoma Growth

Evidence for PTEN as Downstream Target and IL-6/Stat3 as Upstream Activator
      miR-17-92 is an oncogenic miRNA cluster implicated in the development of several cancers; however, it remains unknown whether the miR-17-92 cluster is able to regulate cholangiocarcinogenesis. This study was designed to investigate the biological functions and molecular mechanisms of the miR-17-92 cluster in cholangiocarcinoma. In situ hybridization and quantitative RT-PCR analysis showed that the miR-17-92 cluster is highly expressed in human cholangiocarcinoma cells compared with the nonneoplastic biliary epithelial cells. Forced overexpression of the miR-17-92 cluster or its members, miR-92a and miR-19a, in cultured human cholangiocarcinoma cells enhanced tumor cell proliferation, colony formation, and invasiveness, in vitro. Overexpression of the miR-17-92 cluster or miR-92a also enhanced cholangiocarcinoma growth in vivo in hairless outbred mice with severe combined immunodeficiency (SHO-PrkdcscidHrhr). The tumor-suppressor, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), was identified as a bona fide target of both miR-92a and miR-19a in cholangiocarcinoma cells via sequence prediction, 3′ untranslated region luciferase activity assay, and Western blot analysis. Accordingly, overexpression of the PTEN open reading frame protein (devoid of 3′ untranslated region) prevented miR-92a– or miR-19a–induced cholangiocarcinoma cell growth. Microarray analysis revealed additional targets of the miR-17-92 cluster in human cholangiocarcinoma cells, including APAF-1 and PRDM2. Moreover, we observed that the expression of the miR-17-92 cluster is regulated by IL-6/Stat3, a key oncogenic signaling pathway pivotal in cholangiocarcinogenesis. Taken together, our findings disclose a novel IL-6/Stat3–miR-17-92 cluster–PTEN signaling axis that is crucial for cholangiocarcinogenesis and tumor progression.
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      Whereas mutations of PTEN have not been reported in cholangiocarcinomas, loss of functional PTEN has been implicated in cholangiocarcinogenesis.
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      Consistent with the observations that loss of PTEN protein is a prognostic factor in other malignancies, loss of PTEN expression in patients with cholangiocarcinoma has been linked to more aggressive tumor growth parameters and worse survival outcome.
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      In the current study, we provide the first evidence that the miR-17-92 cluster is highly expressed in cholangiocarcinoma compared with nontumorous biliary epithelial cells. We show that miR-92a is the most abundant miRNA of the miR-17-92 cluster in cholangiocarcinoma. Overexpression of the miR-17-92 cluster or miR-92a enhanced human cholangiocarcinoma cell proliferation, colony formation, and invasiveness in vitro and enhanced tumor growth in mice. PTEN was identified as a bona fide target of both miR-92a and miR-19a in cholangiocarcinoma cells. Furthermore, we observed that IL-6/Stat3 enhances the expression of the miR-17-92 cluster in cholangiocarcinoma cells. Our findings suggest a novel interaction among IL-6/Stat3, miR-17-92 cluster, and PTEN signaling pathways in human cholangiocarcinoma cells.

      Materials and Methods

      Materials

      Dulbecco's modified minimum essential medium and heat-inactivated fetal bovine serum (FBS) were purchased from Sigma (St. Louis, MO). OPTI-MEM–reduced serum medium, RPMI 1640 medium, Lipofectamine 2000 reagent, and puromycin were purchased from Invitrogen (Carlsbad, CA). Bronchial Epithelial Cell Basal Medium, with supplemental growth factors in BEGM SingleQuot Kit, was purchased from Lonza (Walkersville, MD). The miR-19a, miR-92a, or miR-17-92 cluster expressed and scrambled control lentiviral particles with enhanced green fluorescent protein were obtained from GeneCopoeia (Rockville, MD). Dulbecco's Phosphate-Buffered Saline Solution was purchased from Thermo Scientific (Logan, UT). Protease inhibitor cocktail and protein phosphatase inhibitor were purchased from Roche (Mannheim, Germany). The Stat3 inhibitor V (Stattic) was purchased from Calbiochem (Darmstadt, Germany). The JAK inhibitors AZD1480 and INCB18424 were purchased from ChemieTek (Indianapolis, IN). Mouse monoclonal anti–β-actin antibody was from Sigma; rabbit anti-PTEN and anti–phospho-Stat3 antibodies were from Cell Signaling (Danvers, MA).

      Cell Culture

      Four human cholangiocarcinoma cell lines, including CCLP1, SG231, HuCCT1, and TFK1, and one immortalized noncancerous human cholangiocyte cell line (H69) were used. HuCCT1 and TFK1 cells were obtained from the Japanese Cancer Research Resources Bank (Ibaraki City, Japan); H69 cells were kindly provided by Dr. Gregory J. Gores (Mayo Clinic College of Medicine, Rochester, MN). The CCLP1 cells were cultured in Dulbecco's modified minimum essential medium containing 10% FBS, SG231 cells were cultured in OPTI-MEM medium containing 5% FBS, and TFK1 and HuCCT1 cells were cultured in RPMI 1640 medium containing 10% FBS. The H69 cells were cultured in BEBM Basal Medium supplemented with growth factors (BEGM SingleQuot Kit) and 10% FBS. All cells were cultured in a humidified atmosphere of 5% CO2 incubator at 37°C. The miR-19a, miR-92a, or miR-17-92 cluster–overexpressed and scramble control stable cell lines were established by transduction with the corresponding lentiviral vector or miRNA-scrambled control lentiviral vector, followed by selection with media containing puromycin.

      In Situ Hybridization for miRNA

      In situ hybridization for miR-92a was performed in the formalin-fixed, paraffin-embedded tissue specimens surgically resected from patients diagnosed with cholangiocarcinoma by using the MiRCURY LNA microRNA ISH Optimization Kit (Exiqon, Vedbaek, Denmark) with the approval of the Institutional Review Board. Briefly, paraffin sections (6-μm thick) were deparaffinized and treated with 15 μg/mL proteinase-K at 37°C for 10 minutes. After dehydration, slides were incubated with 100 nmol/L miR-92a locked nucleic acid probe (5′-DIG-ACAGGCCGGGACAAGTGCAATA-3′-DIG) at 50°C for 2 hours. This was followed by stringent washes with 5× standard saline citrate, 1× saline sodium citrate, and 0.2× saline sodium citrate buffers at 50°C; DIG blocking reagent (Roche, Mannheim, Germany) in maleic acid buffer containing 2% sheep serum at room temperature for 15 minutes; and alkaline phosphatase–conjugated anti-digoxigenin (diluted 1:500 in blocking reagent; Roche) at room temperature for 2 hours. Enzymatic development was performed by incubating the slides with 4-nitro-blue tetrazolium and 5-brom-4-chloro-3′-indolylphosphate substrate (Roche) at 30°C for 2 hours to allow formation of dark-blue 4-nitro-blue tetrazolium formazan precipitate, followed by nuclear fast red counterstain (Vector Laboratories, Burlingame, CA) at room temperature for 10 minutes. Slides were then dismantled in water, dehydrated in alcohol solutions, and mounted with mounting medium (Vector Laboratories). Scrambled probe and U6 small nuclear RNA–specific probe were used as system control. A standard 4-point scale method was used to evaluate the staining intensity under microscope and the results were scored as follows: 0 (negative), 1 (+), 2 (++), or 3 (+++), according to established criteria.
      • Adams E.J.
      • Green J.A.
      • Clark A.H.
      • Youngson J.H.
      Comparison of different scoring systems for immunohistochemical staining.
      Specifically, 3 (+++) indicates dark staining that is easily visible with a low-power objective and involves >50% of cells; 2 (++), moderate darkly staining areas and <50% of cells; 1 (+), weak or pale staining in any proportion of cells not easily seen under a low power; and 0 (negative), no staining (showing none of the above staining). The same procedure was used to analyze the cholangiocarcinoma tissue arrays (obtained from BioCat GmbH, Heidelberg, Germany; 44 cases of cholangiocarcinoma and 4 cases of nonneoplastic tissues).

      Quantitative RT-PCR

      Cellular total RNA was isolated using TRIzol reagent (Invitrogen). Reverse transcription was performed by using an miScript Reverse Transcription Kit (Qiagen, Valencia, CA). miScript SYBR Green PCR Kit (Qiagen) and miScript Primer Assays for six different miRNAs were used to amplify individual mature forms of miR-17/18/19a/19b/20/92a on a C1000 Thermal Cycler (Bio-Rad, Hercules, CA). U6 small nuclear 2 (U6b) was used as the internal control. For quantitation of PTEN mRNA levels, reverse transcription was performed by using the SuperscriptTM II Reverse Transcriptase Kit (Invitrogen). Then, the PTEN gene was amplified by using the QuantiFast SYBR Green PCR Kit (Qiagen). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control. The GAPDH (Hs_GAPDH_1_SG), PTEN (Hs_PTEN_1_SG), APAF1 (Hs_APAF1_1_SG), and PRDM2 (Hs_PRDM2_1_SG) QuantiTect primers were from Qiagen. The pre–miR-17-92 cluster forward primer is 5′-CAGTAAAGGTAAGGAGAGCTCAATCTG-3′, and the reverse primer is 5′-CATACAACCACTAAGCTAAAGAATAATCTGA-3′. miRNA and mRNA expression levels were normalized to their corresponding internal control genes, and relative change was calculated using the 2−ΔΔCT method.

      Cell Proliferation WST-1 Assay

      The growth of human cholangiocarcinoma cells was measured by using the WST-1 reagent from Roche (Indianapolis, IN). Briefly, 2 × 103 cells were seeded onto each well of 96-well plates and cultured for 24 hours to allow attachment. Then, the medium was replaced with fresh medium containing 1% FBS or specific reagents, as indicated, and the cultures were continued for the indicated days. For cell growth measurement, WST-1 reagent was added to each well and the cells were incubated at 37°C and 5% CO2 for 1 hour; absorbance at 450 nm was measured using an ELISA plate reader.

      Colony-Formation Assay

      Cells (1 × 103) were cultured in 10-cm dishes for 14 days to allow colony formation. Colonies were fixed in 100% methanol and stained with 0.1% crystal violet solution (Amersco, Solon, OH) for counting.

      Cell Invasion Assay

      The cell invasion assay was performed in Matrigel-coated transwell chambers (BD Biosciences Discovery Labware, Bedford, MA). A cell suspension (500 μL; 5 × 104 cells/mL) was added to each of the upper chambers. Cell culture medium containing 10% FBS was added to each of the lower chambers as chemoattractant. After incubating cells at 37°C for 24 hours, the cells on the upper surface of the membrane were removed with a cotton swab. The invading cells on the lower surface of the membrane were fixed in 100% methanol and stained with Mayer's hematoxylin solution (IHC World, Woodstock, MD). Then, the invading cells were counted under a microscope, and for each chamber, eight fields were randomly selected for counting.

      Western Blot Analysis

      The logarithmically growing cells were washed twice with ice-cold phosphate-buffered saline (PBS), scraped off the plates with 1 mL PBS with 100 nmol/L phenylmethylsulfonyl fluoride, and centrifuged. The cells were then incubated in a lysis buffer containing 50 mmol/L HEPES and 1 mmol/L EDTA (pH 8.0); one tablet of protease inhibitor cocktail (Roche) and one tablet of phosphatase inhibitors (Roche) were added per 10-mL buffer. After sonication on ice, the cell lysates were centrifuged at 12,000 × g for 10 minutes at 4°C, and the supernatants were collected for Western blot analysis. The protein concentration was measured with the Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA). After boiling for 5 minutes in the protein loading buffer with 2-mercaptoethanol, the samples were separated on Any kD Mini-PROTEAN TGX Gel (Bio-Rad) and then transferred onto the nitrocellulose membrane (Bio-Rad). The membranes were blocked in PBS with 0.1% Tween 20 (PBS-T) containing 5% nonfat milk for 1 hour at room temperature. Blots were incubated with different primary antibodies (at appropriate dilutions) in PBS-T containing 5% nonfat milk at 4°C overnight. After three washes with PBS-T, the membranes were incubated with IRDye secondary antibody (Licor, Lincoln, NE) at 1:10,000 dilutions in PBS-T containing 5% nonfat milk for 1 hour. After three washes with PBS-T, signals were visualized and the relative densities of protein bands were quantified and normalized to actin using the ODYSSEY Infrared Imaging System (Licor).

      miRNA, Anti-miRNA, siRNA, and Plasmid Transfection

      siRNA targeting Stat3, GP130, or scrambled control (Ambion, Grand Island, NY), miR-19a mimic or miR-19a–specific anti-miRNA (Qiagen), pcDNA3 control, or pcDNA3-PTEN orf plasmid (Addgene, Cambridge, MA) was transfected into cells by using Lipofectamine 2000 (Invitrogen), as per the manufacturer's instruction. After transfection at indicated time periods, cells were analyzed for proliferation and the cell lysates were obtained for Western blot analysis.

      IL-6 and Inhibitor Treatment

      Recombinant human IL-6 (R&D Systems, Minneapolis, MN) was prepared according to the product's manual, dissolved in sterile PBS containing 0.1% bovine serum albumin. Stattic, AZD1480, or INCB18424 was dissolved in dimethyl sulfoxide at 10 mmol/L. Serum was withdrawn 24 hours before IL-6 and/or inhibitor treatment to synchronize cells.

      DNA Pull-Down Assay

      Cells were lysed by sonication in HKMG buffer containing 10 mmol/L HEPES (pH 7.9), 100 mmol/L KCl, 5 mmol/L MgCl2, 10% glycerol, 1 mmol/L dithiothreitol, and 0.5% NP-40 with protease inhibitor cocktail and phosphatase inhibitors for the preparation of nuclear extract. The obtained nuclear extracts were precleared with streptavidin-agarose resin (Thermo Scientific) for 1 hour. The precleared samples were then incubated with 1 μg biotinylated double-stranded oligonucleotide STAT3-binding site (5′-biotin-GTTTCTGAGAATTCCGGAATTTCCTGAACCAC-3′ and 5′-biotin- GTGGTTCAGGAAATTCCGGAATTCTCAGAAAC-3′; synthesized by Integrated DNA Technologies, Coralville, IA) and 10 μg poly(dI-dC) at 4°C for overnight. DNA-protein complex was collected by incubation with streptavidin-agarose resin on a shaker to prevent precipitation at 4°C for 1 hour. The resin-bound complex was washed at least 5× with 1.0 mL HKMG buffer, followed by centrifugation at 2000 × g for 1 to 2 minutes to discard the supernatant. After the resin-bound complex was boiled in SDS-PAGE sample loading buffer, the released protein was loaded onto Any kD Mini-PROTEAN TGX Gel (Bio-Rad) and identified by using Western blot analysis with specific antibodies.

      Chromatin Immunoprecipitation

      The assay was performed by using a Simplechip Enzymatic Chromatin IP kit (Cell Signaling Technology, Billerica, MA), according to the manufacturer's instructions. Immunoprecipitation was performed by using 5 μg of anti–phospho-STAT3 antibody; rabbit IgG antibody was used as a negative control. Chromatin immunoprecipitation primers were designed to amplify the STAT3 binding region in the miR-17-92 cluster promoter. The primers are as follows: 5′-GGGCATTAAAGACATCCTTTGG-3′ and 5′-GAGCACCTCCTTCTTCACATT-3′.

      Luciferase Reporter Activity Assay

      Cells were seeded in 12-well plates and cultured overnight to allow attachment. The cells were then cotransfected with miRNA mimic or Allstar negative control siRNA (Qiagen) and 0.1 μg per well of pEZX-MT01-PTEN 3′UTR Renilla/firefly dual-luciferase reporter plasmid (GeneCopoeia). The cell lysates were obtained 24 hours after transfection, and luciferase activity was measured in a Centro XS3 LB 960 Microplate Fluorescence Reader (Berthold Technologies, Oak Ridge, TN) by using the Dual-Luciferase reporter assay system (Promega, Madison, WI). The Renilla luciferase activity was used as internal control, and the ratio of Firefly/Renilla activity was calculated for normalization.

      mRNA Microarray Analysis

      Total RNA from miR-17-92 cluster–overexpressed or control CCLP1 cells were used for Whole Human Genome Oligo Microarray (performed by Arraystar, Rockville, MD). Briefly, total RNA from each sample was amplified and transcribed into fluorescent cRNA using the manufacturer's Agilent's Quick Amp Labeling protocol, version 5.7 (Agilent Technologies, Santa Clara, CA). The labeled cRNAs were hybridized onto the Whole Human Genome Oligo Microarray (4 × 44K; Agilent Technologies). After having washed the slides, the arrays were scanned by the Agilent Scanner G2505B (Agilent Technologies). Agilent Feature Extraction software version 11.0.1.1 (Agilent Technologies) was used to analyze acquired array images. Quantile normalization and subsequent data processing were performed using the GeneSpring GX software version 11.5.1 (Agilent Technologies). Raw data of detected genes were normalized for further analysis. Differentially expressed genes were identified through fold change filtering. The data were deposited into Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo; accession number GSE58060).

      SCID Mice Tumor Xenograft Studies

      Four-week-old male severe combined immunodeficiency (SCID) hairless outbred (SHO) mice (Crl:SHO-PrkdcscidHrhr, strain 474) were purchased from Charles River (Wilmington, MA). Two complementary xenograft models (s.c. and orthotopic) were used. For the s.c. xenograft model, 3 × 106 miR-92a– or miR-17-92 cluster–overexpressed or control CCLP1 cells were inoculated into the flanks of mice (six mice per group), and the mice were observed over 4 weeks for tumor formation. The tumor volume was measured weekly with a caliper and calculated by using the following formula: largerdiameter×(smallerdiameter)2/2. On sacrifice, the tumors were recovered and the wet weights of each tumor were determined. A portion of each tumor was selected for H&E staining, Western blot analysis, and quantitative RT-PCR (RT-qPCR) analysis. For the orthotopic transplant model, 3 × 107/mL miR-17-92 cluster–overexpressed or control CCLP1 cell suspension was mixed with Corning Matrigel Basement Membrane Matrix High Concentration (Corning, NY) in a 1:1 ratio; a 50-μL final volume (containing 7.5 × 105 cells) was injected directly into the liver of mice in surgery under local anesthesia. The mice were sacrificed 6 weeks after intrahepatic inoculation to document tumor growth.

      Statistical Analysis

      Results are presented as means ± SD or SEM from a minimum of three replicates. Differences between groups were compared using either Student's t-test or one-way analysis of variance. P < 0.05 was considered statistically significant. The analysis was performed using SPSS statistical software version 19.0 (IBM, Armonk, NY). For data from human samples, statistical significance between means was determined by the nonparametric Kruskal-Wallis test.

      Results

      Expression of miR-92a Is Increased in Human Cholangiocarcinoma Tissues and Cell Lines

      The miR-17-92 cluster is a polycistronic primary transcript and yields six mature miRNAs: miR-17, miR-18a, miR-19a, miR-20a, miR-19b, and miR-92a (Figure 1A). To determine the expression patterns of these six miRNAs, we measured their levels by RT-qPCR analyses in human cholangiocarcinoma cells and noncancerous human biliary epithelial cells. Higher levels of miR-92a, miR-20, and miR-17 expression were observed in all four human cholangiocarcinoma cell lines (CCLP1, HuCCT1, SG231, and TFK1) when compared with the noncancerous human biliary epithelial cell line (H69) (Figure 1B). The expression levels of miR-18, miR-19a, and miR-19b were also increased in cholangiocarcinoma cells (noticeably increased in SG231 cells and slightly increased in HuCCT1, CCLP1, and TFK1 cells). miR-92a is the highest among all of the six miRNAs of the cluster in all four human cholangiocarcinoma cell lines.
      Figure thumbnail gr1
      Figure 1High expression of the miR-17-92 cluster in human cholangiocarcinoma tissues and cell lines. A: Genomic structure of the miR-17-92 cluster. The miR-17-92 cluster is polycistronic, and located at C13orf25. The pre–miR-17-92 cluster is cleaved, generating six mature miRNAs (miR-17, miR-18, miR-19a, miR-20, miR-19b, and miR-92). B: Expression of the miR-17-92 cluster in cholangiocarcinoma cell lines. RT-qPCR for mature miRNA in the miR-17-92 cluster in a human transformed cholangiocyte cell line (H69) and four human cholangiocarcinoma cell lines (CCLP1, HuCCT1, SG231, and TFK1). Total cellular RNA was extracted for RT-qPCR (n = 3). The expression level of each individual miRNA is normalized to RNU6B (internal control). All of the six miRNAs (miR-17/18/19a/19b/20/92) are highly expressed in four cholangiocarcinoma cell lines (CCLP-1, HuCCT1, SG231, and TFK-1 cells) compared with the noncancerous control cell line H69. Among the six mature miRNAs, miR-92a is the most abundant, followed by miR-17 and miR-20. C: In situ hybridization for mature miR-92a in human cholangiocarcinoma tissues. Representative in situ hybridization images depict the expression of miR-92a in human cholangiocarcinoma tissues. Note positive miR-92a staining in cholangiocarcinoma cells and negative staining in normal bile duct epithelial cells (positive signals are in dark blue; nuclei were counterstained in red). D: Distribution of miR-92a staining intensity scores in human cholangiocarcinoma tissue arrays (N = 44). P < 0.05 (Kruskal-Wallis nonparameter test).
      Given that miR-92a is the most abundant miRNA among the miR-17-92 cluster in human cholangiocarcinoma cell lines, we performed in situ hybridization using locked nucleic acid–modified probe against miR-92a in human cholangiocarcinoma tissue samples and nonneoplastic bile duct epithelia. Formalin-fixed, paraffin-embedded tumor and liver tissues from 12 patients who underwent surgical resections for cholangiocarcinoma and 41 cases of cholangiocarcinoma tissue arrays were analyzed. We observed that miR-92a is highly expressed in 12 (100%) of 12 cases of archived cholangiocarcinoma tissues, which is in contrast to the weak or absent staining in nonneoplastic bile duct and peribiliary glands (Figure 1C). For cholangiocarcinoma tissue array analysis, the staining frequency and intensity of miR-92a are also significantly higher in cholangiocarcinoma cells (63.41% +++, 19.51% ++, 12.20% +, and 4.88% −) compared with the nonneoplastic bile duct epithelial cells (0.0% +++, 0.0% ++, 66.67% +, and 33.33% −; P = 0.04) (Figure 1D and Supplemental Table S1). These findings document increased expression of the miR-17-92 cluster, particularly miR-92a, in human cholangiocarcinoma tissues and cell lines.

      miR-92a Enhances Cholangiocarcinoma Cell Growth, in Vitro and in Vivo

      To investigate the role of miR-92a in cholangiocarcinoma cell growth, we constructed human cholangiocarcinoma cell lines with stable overexpression of miR-92a by infecting the parental cell line (CCLP1) with lentivirus particles carrying the miR-92a gene (this vector also carries the enhanced green fluorescent protein gene under the control of the same promoter). High infection efficiency was confirmed by the expression of enhanced green fluorescent protein in nearly all transduced cells. Successful overexpression of miR-92a in miR-92a lentivirus stably transduced cells was verified by RT-qPCR (cells transduced with lentivirus carrying scrambled control miRNA were used as control). Overexpression of miR-92a significantly increased the growth of cholangiocarcinoma cells in vitro (Figure 2A). miR-92a overexpression also increased colony-formation efficiency and enhanced cell invasion (Figure 2, B and C). These findings demonstrate that miR-92a increases the malignant potential of cholangiocarcinoma cells in vitro.
      Figure thumbnail gr2
      Figure 2miR-92a overexpression promotes cholangiocarcinoma cell growth in vitro and in vivo. AC: miR-92a overexpression promotes cholangiocarcinoma cell growth, colony formation, and cell invasion in vitro. Human cholangiocarcinoma cells (CCLP1) were infected with miR-92a lentivirus and miRNA scrambled control lentivirus, respectively. The stably transduced cells were analyzed for proliferation, colonogenic potential, and invasion ability, as described in . A: miR-92a overexpression promotes cholangiocarcinoma cell growth. Cell growth curves of human cholangiocarcinoma cells (CCLP1) stably transduced with miR-92a lentivirus (indicated as LV-miR-92a) or scrambled control (indicated as LV-control). miR-92a levels were measured by RT-qPCR. B: miR-92a overexpression enhances cholangiocarcinoma cell colony formation. The numbers of colonies were counted after 14 days. Representative images showing colonies formed in cell culture dishes. Average colony formation efficiency is shown. C: miR-92a overexpression promotes cholangiocarcinoma cell invasion. Representative images showing migrated cell staining by hematoxylin. Average numbers of the invaded cells are shown. DG: miR-92a promotes cholangiocarcinoma growth in vivo. miR-92a–overexpressed or scrambled control CCLP1 cells (1.0 × 108) were inoculated s.c. into flank areas of SCID hairless mice (n = 6), and the mice were closely monitored for tumor growth. Thirty days after inoculation, the mice were sacrificed and the tumors were recovered. D: Photographs of xenograft tumor masses from SCID mice highlight that miR-92a promotes tumor growth. Images are of the mice and the xenograft tumors that were recovered. E: miR-92a overexpression significantly increases xenograft tumor volume. F: miR-92a significantly increases the weight of the xenograft tumors. G: Western blot analysis reveals decreased PTEN protein levels in xenograft tumor tissues by miR-92a. Data are presented as means ± SEM (AC) or means ± SD (E and F). N = 3 (AC); N = 6 (E and F). P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.
      To determine the effect of miR-92a on cholangiocarcinoma growth in vivo, we inoculated miR-92a–overexpressed and scrambled control CCLP1 cells into the flanks of SCID hairless outbred mice (SHO-PrkdcscidHrhr). Although only three of six mice inoculated with scrambled control cells showed visible tumors, six of six mice inoculated with miR-92a overexpression cells developed visible tumors 30 days after inoculation (Figure 2D). The scrambled control tumors were smaller (P < 0.05) and lower in weight compared with the miR-92a–overexpressed tumors (Figure 2, D and E). An approximately sixfold increase in tumor weight was observed in miR-92a–overexpressed tumors compared with the controls (0.19 ± 0.04 g versus 0.02 ± 0.01 g; P = 0.014) (Figure 2F). Western blot analysis of the tumor tissues showed reduction of PTEN protein in miR-92a–overexpressed tumors (Figure 2G).

      miR-17-92 Cluster Increases Cholangiocarcinoma Cell Growth, in Vitro and in Vivo

      Given that miR-92a is able to increase cholangiocarcinoma cell growth, as shown above, we sought to further evaluate the effect of the miR-17-92 cluster by stably transducing cholangiocarcinoma cells with lentivirus particles expressing miR-17-92 cluster or the scrambled control miRNA. Overexpression of the miR-17-92 cluster significantly increased the cholangiocarcinoma cell growth in vitro (Figure 3A). miR-17-92 overexpression also increased colony-formation efficiency and enhanced cell invasion (Figure 3, B and C). When these cells were inoculated s.c. into SCID hairless mice (SHO-PrkdcscidHrhr), the miR-17-92 cluster–overexpressed cells produced visible tumors 30 days after inoculation; in contrast, the scrambled control cells failed to produce any tumor under the same condition (Figure 3, D–F). When these cells were transplanted orthotopically into the livers of SCID hairless mice, the miR-17-92 cluster–overexpressed tumors exhibited larger tumor volume compared with the scrambled control tumors (Supplemental Figure S1). These findings provide in vitro and in vivo evidences for a tumor-promoting effect of the miR-17-92 cluster in cholangiocarcinoma.
      Figure thumbnail gr3
      Figure 3Overexpression of miR-17-92 cluster promotes cholangiocarcinoma cell growth in vitro and in vivo. The miR-17-92 cluster promotes human cholangiocarcinoma cell proliferation (A), colony formation (B), and cell invasion (C) in vitro. Human cholangiocarcinoma cells (CCLP1) were infected with miR-17-92 cluster lentivirus and miRNA scrambled control lentivirus, respectively, and the stable cells were analyzed for proliferation, colonogenic potential, and invasion ability, as described in . A: Cell proliferation assay (WST-1). Cell growth curves of human cholangiocarcinoma cells (CCLP1) stably transduced with miR-17-92 cluster lentivirus (indicated as LV-miR-17-92) or scrambled control LV-control. The miR-17-92 cluster promotes cell growth in cholangiocarcinoma cell lines. Pre–miR-17-92 cluster levels were measured by RT-qPCR. B: miR-17-92 cluster overexpression enhances cholangiocarcinoma cell colony formation. The numbers of colonies were counted after 14 days. Representative images show colonies formed in cell culture dishes. Average colony-forming efficiency is shown. C: miR-17-92 cluster overexpression promotes cholangiocarcinoma cell invasion. Representative images show migrated cell staining by hematoxylin. Average numbers of invaded cells are shown. DF: The miR-17-92 cluster promotes cholangiocarcinoma growth in vivo. miR-17-92 cluster–overexpressed or scrambled control CCLP1 cells (1.0 × 108) were inoculated s.c. into flank areas of SCID hairless mice (n = 6), and the mice were closely monitored for tumor growth. Thirty days after inoculation, the mice were sacrificed and the tumors were recovered. D: Photographs of xenograft tumor masses from SCID mice show that the miR-17-92 cluster promotes tumor growth. E: The volume of xenograft tumors is significantly increased by miR-17-92 cluster overexpression. F: The weight of xenograft tumors is significantly increased by miR-17-92 cluster overexpression. Data are from four SCID mice (two mice died before 30 days). Data represent means ± SEM (AC) or means ± SD (E and F). N = 3 (AC); N = 4 to 6 (E); N = 4 (F). P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.
      Because miR-19a is another member of the miR-17-92 cluster implicated in the growth regulation of other cancer cells,
      • Olive V.
      • Bennett M.J.
      • Walker J.C.
      • Ma C.
      • Jiang I.
      • Cordon-Cardo C.
      • Li Q.J.
      • Lowe S.W.
      • Hannon G.J.
      • He L.
      miR-19 is a key oncogenic component of mir-17-92.
      we performed further experiments to determine whether miR-19a might play a role in human cholangiocarcinoma cells. Overexpression of miR-19a enhanced cholangiocarcinoma cell proliferation, colony formation, and cell invasion (Figure 4A). Collectively, our findings document an important oncogenic role of the miR-17-92 cluster and its members (including miR-92a and miR-19a) in cholangiocarcinoma cell growth.
      Figure thumbnail gr4
      Figure 4miR-19a overexpression promotes cholangiocarcinoma cell growth and colony formation in vitro. A: miR-19a overexpression promotes cholangiocarcinoma cell growth. Cell growth curves of human cholangiocarcinoma cells (CCLP1) stably transduced with miR-19a lentivirus (indicated as LV-miR-19a) or scrambled control (indicated as LV-control). miR-19a levels were measured by RT-qPCR. B: miR-19a overexpression promotes cholangiocarcinoma cell colony formation. The numbers of colonies were counted after 14 days. Representative images show colonies formed in cell culture dishes. Average colony formation efficiency is shown. C: miR-19a overexpression promotes cholangiocarcinoma cell invasion. Representative images show migrated cell staining by hematoxylin. Average numbers of invaded cells are shown. Data are presented as means ± SEM. N = 3. P < 0.05, ∗∗∗P < 0.001.

      PTEN Is a Direct Target of miR-92a and miR-19a in Cholangiocarcinoma Cells

      In an effort to identify the potential targets that mediate miR-17-92 actions in cholangiocarcinoma cells, we used the online prediction tool Miranda (http://www.microrna.org/microrna, last accessed October 12, 2013). This approach led to identification of PTEN as a key downstream target. Complementary sequences of miR-92a and miR-19a were identified in the 3′UTR of PTEN mRNA (Figure 5A). RT-qPCR and Western blot analyses showed that overexpression of the miR-17-92 cluster or its members (miR-92a or miR-19a) led to reduction of PTEN mRNA and protein levels (Figure 5, B and C). Treatment of human cholangiocarcinoma cells with miR-92a or miR-19a mimic also reduced PTEN expression (Figure 5C). Accordingly, miR-92a or miR-19a mimic treatment decreased the PTEN 3′UTR luciferase reporter activity (Figure 5D). The role of PTEN in miR-92a– and miR-19a–induced cholangiocarcinoma cell growth was further supported by the observation that forced overexpression of PTEN open reading frame protein (without the 3′UTR) partially prevented miR-92a– and miR-19a–induced tumor cell growth (Figure 5E). These results demonstrate that PTEN is a direct target of the miR-17-92 cluster through two of its members, miR-92a and miR-19a.
      Figure thumbnail gr5
      Figure 5miR-17-92 targets PTEN via miR-92a and miR-19a in cholangiocarcinoma cells. A: Predicted 8-mer and 7-mer-m8 binding sequence for miR-92a and miR-19a in the 3′UTR of PTEN mRNA. B: PTEN mRNA levels are decreased in miR-19a, miR-92a, or miR-17-92 cluster–overexpressed cells, as determined by RT-qPCR analysis. C: PTEN protein levels are down-regulated in miR-19a, miR-92a, and miR-17-92 cluster–overexpressed stable cell lines, as determined by using Western blot analysis. PTEN protein levels are also down-regulated by miR-19a mimic or miR-92a mimic in CCLP-1 cells. D: miR-92a or miR-19a mimic significantly reduces PTEN 3′UTR luciferase activity. CCLP1 cells were transfected with miRNA mimic or scrambled control, together with PTEN 3′UTR dual-luciferase reporter plasmid. The luciferase activity was analyzed 24 hours after transfection. E: PTEN (ORF) overexpression inhibits the cell growth induced by miR-19a or miR-92a. Data are presented as means ± SEM (B and E) or means ± SD (D). N = 3 (B and D); N = 6 (E). ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.

      Transcriptome and Sequence Analyses for Additional Targets of miR-17-92 Cluster

      To predict other targets of the miR-17-92 cluster, we performed sequence analysis by using TargetScan (http://www.targetscan.org, last accessed October 12, 2013), PicTar (http://pictar.mdc-berlin.de, last accessed October 12, 2013), and Miranda. Such sequence analysis predicted >30,000 targets that have at least one binding site for one or multiple miRNAs in the miR-17-92 cluster. As a parallel approach, we performed transcriptome analysis using mRNA microarray and found that 2861 genes were down-regulated in miR-17-92 cluster–overexpressed cells compared with scrambled control cells (Supplemental Figure S2A). There were 960 common genes identified by both sequence prediction and transcription analysis. PANTHER pathway analysis (http://www.pantherdb.org, last accessed October 12, 2013) indicates that these common genes are most enriched in Wnt, cadherin, and inflammation pathways (Supplemental Table S2), which is consistent with the molecular features of cholangiocarcinoma.
      • Malhi H.
      • Gores G.J.
      Cholangiocarcinoma: modern advances in understanding a deadly old disease.
      By comparing the miR-17-92 cluster–down-regulated genes with the online database of human tumor-suppressor gene,
      • Zhao M.
      • Sun J.
      • Zhao Z.
      TSGene: a web resource for tumor suppressor genes.
      we found several highly possible targets, including APAF-1 and PRDM2, in addition to PTEN, which are significantly down-regulated in miR-17-92 cluster–overexpressed cholangiocarcinoma cells (Supplemental Table S3 and Supplemental Figure S2B). These findings suggest that miR-17-92–regulated cholangiocarcinoma growth may also involve additional targets (including APAF-1 and PRDM2, among others).

      The Expression of miR-17-92 Cluster Is Up-Regulated by IL-6/Stat3 Signaling

      Given the noticeable up-regulation of miR-17-92 cluster in cholangiocarcinoma cells, we sought to further explore the mechanism that regulates miR-17-92 expression in cholangiocarcinoma cells. Because the promoter region of the miR-17-92 cluster gene contains a conserved binding site for Stat3
      • Brock M.
      • Trenkmann M.
      • Gay R.E.
      • Gay S.
      • Speich R.
      • Huber L.C.
      MicroRNA-18a enhances the interleukin-6-mediated production of the acute-phase proteins fibrinogen and haptoglobin in human hepatocytes.
      • Brock M.
      • Trenkmann M.
      • Gay R.E.
      • Michel B.A.
      • Gay S.
      • Fischler M.
      • Ulrich S.
      • Speich R.
      • Huber L.C.
      Interleukin-6 modulates the expression of the bone morphogenic protein receptor type II through a novel STAT3-microRNA cluster 17/92 pathway.
      (Figure 6D), we postulated that activation of IL-6/Stat3 signaling might be a potential mechanism for miR-17-92 cluster up-regulation during cholangiocarcinogenesis. Indeed, treatment of human cholangiocarcinoma cells with IL-6 significantly increased the expression of pre–miR-17-92 cluster mRNA (Figure 6A). IL-6 treatment also significantly increased the levels of each individual mature miRNA of the miR-17-92 cluster (by 77- to 250-folds) (Supplemental Figure S3). IL-6–mediated induction of pre–miR-17-92 cluster mRNA was almost completely abolished by siRNA knockdown of Stat3 or GP130 (P < 0.01) (Figure 6B). Similarly, pretreatment of human cholangiocarcinoma cells with 20 μmol/L Stat3 phosphorylation inhibitor Stattic
      • Schust J.
      • Sperl B.
      • Hollis A.
      • Mayer T.U.
      • Berg T.
      Stattic: a small-molecule inhibitor of STAT3 activation and dimerization.
      or 20 μmol/L JAK inhibitor (AZD1480 or INCB18424) also abolished IL-6–induced up-regulation of the miR-17-92 cluster (Figure 6, C and D). The DNA pull-down assay and the chromatin immunoprecipitation assay showed that IL-6 treatment increased the association of phosphorylated Stat3 to its DNA binding site in miR-17-92 cluster gene promoter (Figure 6, E and F). These findings suggest an important role of IL-6/Stat3 signaling for up-regulation of the miR-17-92 cluster in human cholangiocarcinoma cells.
      Figure thumbnail gr6
      Figure 6IL-6 up-regulates miR-17-92 expression in cholangiocarcinoma cells through Stat3 activation. A: Stimulation of CCLP-1 cells with 20 ng/mL IL-6 increases the expression of the pre–miR-17-92 cluster, as determined by RT-qPCR. B: IL-6–mediated induction of the pre–miR-17-92 cluster is abolished by siRNA knockdown of Stat3 or the IL-6 receptor, GP130. RT-qPCR data are presented. C: IL-6–mediated induction of the pre–miR-17-92 cluster is abolished by 20 μmol/L Stat3 phosphorylation inhibitor Stattic. RT-qPCR data are presented. D: IL-6–mediated induction of the pre–miR-17-92 cluster is abrogated by 20 μmol/L JAK inhibitor, AZD1480 or INCB18424. RT-qPCR data are presented. E: IL-6 treatment increases binding of activated Stat3 to the promoter region of the miR-17-92 cluster, as determined by biotinylated oligonucleotide precipitation assay. Stat3 binding sequence at promoter region of pre–miR-17-92 was biotinylated-linked and used to precipitate proteins after 20 ng/mL IL-6 treatment for 1 hour. Phospho-Stat3 protein was detected by using Western blot analysis. F: Chromatin immunoprecipitation assay. The chromatin extracted from CCLP1 cells, treated with IL-6 or control vehicle, was subjected to immunoprecipitation with phospho-STAT3 antibody, and the precipitates were subjected to RT-qPCR analysis using primers to amplify the STAT3 binding region. Normal rabbit IgG was used as the negative control. RT-qPCR data are presented. Data are presented as means ± SEM (AD and F). N = 3 (AD and F). P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

      Discussion

      The current study provides the first evidence that the miR-17-92 cluster is highly expressed in human cholangiocarcinoma cells compared with the noncancerous human biliary epithelial cells. Our data show that miR-92a is the most abundant miRNA among the six mature miRNAs of the miR-17-92 cluster in human cholangiocarcinoma. The role of the miR-17-92 cluster in cholangiocarcinoma growth is established by complementary in vitro and in vivo studies. We observed that forced overexpression of the miR-17-92 cluster or its members, miR-92a and miR-19a, in cultured human cholangiocarcinoma cells enhanced tumor cell proliferation, colony formation, and invasiveness in vitro. Overexpression of the miR-17-92 cluster or miR-92a was also found to enhance xenograft cholangiocarcinoma growth in mice. These findings establish a key tumor-promoting role of the miR-17-92 cluster in cholangiocarcinoma (Figure 7).
      Figure thumbnail gr7
      Figure 7Schematic illustration of the proposed mechanism for miR-17-92 cluster–induced cholangiocarcinoma growth. The miR-17-92 cluster can directly target PTEN via miR-19a and miR-92, which associate with 3′UTR of PTEN. Reduction of PTEN enhances cholangiocarcinoma cell proliferation and invasion in vitro and tumor formation in vivo. Furthermore, IL-6 can up-regulate the expression of miR-17-92 cluster through Stat3-mediated transcription of the pre–miR-17-92 cluster. CCA, cholangiocarcinoma.
      Our data show that PTEN is a direct target of the miR-17-92 cluster through two of its members, miR-92a and miR-19a. This assertion is based on the following observations: i) complementary sequences of miR-92a and miR-19a are present in the 3′UTR of PTEN mRNA, ii) overexpression of the miR-17-92 cluster or its members (miR-92a or miR-19a) decreased PTEN mRNA and protein levels in human cholangiocarcinoma cells, iii) treatment of human cholangiocarcinoma cells with miR-92a or miR-19a reduced PTEN protein expression, and iv) miR-92a or miR-19a mimic treatment decreased PTEN 3′UTR luciferase reporter activity in human cholangiocarcinoma cells. Therefore, inhibition of PTEN may represent an important mechanism for miR-17-92 cluster–mediated cholangiocarcinoma growth. The involvement of PTEN in miR-17-92–induced cholangiocarcinoma cell growth is further supported by the observation that overexpression of PTEN open reading frame protein prevented miR-92a– or miR-19a–induced tumor cell growth.
      In addition to PTEN, we observe that miR-17-92 cluster also targets other proteins, including APAF-1 and PRDM2, in human cholangiocarcinoma cells. It is possible that these additional targets may also be implicated in miR-17-92–mediated cholangiocarcinoma growth.
      Herein, we show that IL-6/Stat3 signaling transcriptionally regulates the expression of the miR-17-92 cluster in cholangiocarcinoma cells. This assertion is based on the following observations: i) a conserved Stat3 binding site is present in the miR-17-92 cluster gene promoter, ii) IL-6 treatment increased the levels of pre–miR-17-92 and mature miRNAs of the miR-17-92 cluster, iii) siRNA knockdown of Stat3 or GP130 or inhibition of Stat3 phosphorylation abolished IL-6–induced induction of the pre–miR-17-92 cluster, and iv) IL-6 treatment increased the association of phosphorylated Stat3 to its DNA binding site in the miR-17-92 cluster gene promoter. Thus, activation of IL-6/Stat3 signaling may be an important factor for induction of miR-17-92 expression in cholangiocarcinoma. In this context, it is notable that recent studies have shown that miR-17-92 targets JAK-STAT pathway inhibitor SOCS3 (suppressors of cytokine signaling)
      • Collins A.S.
      • McCoy C.E.
      • Lloyd A.T.
      • O'Farrelly C.
      • Stevenson N.J.
      miR-19a: an effective regulator of SOCS3 and enhancer of JAK-STAT signalling.
      and PIAS3 (protein inhibitor of activated Stat3),
      • Wu W.
      • Takanashi M.
      • Borjigin N.
      • Ohno S.I.
      • Fujita K.
      • Hoshino S.
      • Osaka Y.
      • Tsuchida A.
      • Kuroda M.
      MicroRNA-18a modulates STAT3 activity through negative regulation of PIAS3 during gastric adenocarcinogenesis.
      two key negative regulators of the JAK-STAT pathway. Thus, it is possible that the IL-6/Stat3–induced miR-17-92 cluster may further amplify IL-6/Stat3 signaling through targeting SOCS3 and PIAS3, forming a positive feed-forward loop that drives carcinogenesis and tumor progression.
      Cholangiocarcinoma is often difficult to diagnose at an early stage; accurate diagnosis frequently cannot be established until the disease is advanced and often in the metastatic stage, resulting in a poor prognosis. Given that the miR-17-92 cluster, particularly miR-92a, is highly expressed in cholangiocarcinoma, as shown in this study, further studies are needed to validate whether the miR-17-92 cluster or miR-92a can be used as a marker for early diagnosis of cholangiocarcinoma. Moreover, in light of the key role of miR-17-92 in cholangiocarcinogenesis and tumor progression, as described in the current study, further investigations are warranted to determine whether miR-17-92 can be targeted for the treatment of human cholangiocarcinoma.

      Supplemental Data

      • Supplemental Figure S1

        miR-17-92 cluster promotes cholangiocarcinoma growth in vivo. miR-17-92 cluster–overexpressed or scrambled control CCLP1 cells were mixed with Matrigel, and 50 μL was injected directly into the liver of SCID hairless mice (n = 6 for each group). Six weeks after inoculation, the mice were sacrificed and the livers were recovered. A: Photographs of liver from SCID mice show that the miR-17-92 cluster promotes tumor growth compared with control; one mouse died shortly after tumor cell inoculation surgery. B: Overexpression of the miR-17-92 cluster significantly increases tumor volume. Data represent means ± SD. P < 0.05 (Student's t-test).

      • Supplemental Figure S2

        Microarray analysis of miR-17-92 cluster targets. A: Transcriptome microarray analysis in miR-17-92–overexpressed and scrambled control cell lines. B: RT-qPCR analysis shows down-regulation of PTEN, APAF-1, and PRDM2 mRNAs by miR-17-92 cluster in CCLP-1 cells. Data are presented as means ± SEM (N = 3). P < 0.05, ∗∗P < 0.01 (Student's t-test).

      • Supplemental Figure S3

        IL-6 up-regulates miR-17-92 expression in cholangiocarcinoma cells through Stat3 activation. Stimulation of CCLP-1 cells with 20 ng/mL IL-6 increases the expression of each individual mature miRNA of the miR-17-92 cluster (miR-17/18/19a/19b/20/92). RT-qPCR data are presented as means ± SEM (N = 3). ∗∗∗P < 0.001 (Student's t-test).

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