the microrna let-7a modulates interleukin-6 … · tushar patel from the division of...

THE MICRORNA LET-7A MODULATES INTERLEUKIN-6 DEPENDENT STAT-3 SURVIVAL SIGNALING IN MALIGNANT HUMAN CHOLANGIOCYTES

Fanyin Meng, Roger Henson, Hania Wehbe-Janek, Heather Smith, Yoshiyuki Ueno*, and Tushar Patel

From the Division of Gastroenterology, Scott and White Clinic, Texas A&M University System Health Science Center College of Medicine, Temple, Texas and the *Division of

Gastroenterology, Tohoku University, Sendai, Japan

Running title: let-7a modulates IL-6 survival signaling

Address correspondence to: Tushar Patel, Scott and White Clinic, Texas A&M University Health Science Center, 2401 South 31st Street, Temple, TX 76508, Tel: 254 724 4764 or 254 724 2489; Fax: 254 742 7181; e-mail: [email protected]

The inflammation-associated cytokine Interleukin-6 (IL-6) can contribute to tumor growth and resistance to therapy by the activation of survival mechanisms. In several human cancers, IL-6 activated survival signaling involves the signal transducers and activators of transcription (Stat) factors, or protein kinase cascades. microRNAs (miRNAs) are endogenous regulators of gene expression that are altered in expression in many cancers. However, the effect of inflammatory cytokines on miRNA expression, and the role of miRNA in modulating IL-6 mediated cell survival are unknown. We investigated the involvement of miRNA in malignant cholangiocytes stably transfected to over-express IL-6, which enhances tumor growth in vivo by inhibition of apoptosis. We provide evidence that (i) miRNA expression both in vitro and in vivo is altered by over-expression of IL-6; (ii) selective miRNA including let-7a are up-regulated and contribute to the survival effects of enforced IL-6 activity and (iii) let-7a contributes to the constitutively increased phosphorylation of Stat-3 by a mechanism involving the neurofibromatosis 2 (NF2) gene. These findings reveal a novel mechanism by which IL-6 mediates tumor cell survival that may be therapeutically targeted and emphasize the presence of complex interrelationships between

deregulated expression of miRNA and transcription factors in human cancers.

Increased expression of the inflammation-associated cytokine Interleukin-6 (IL-6) occurs in chronic inflammatory conditions and in several human cancers such as multiple myeloma, prostate cancer and cholangiocarcinoma. IL-6 has been implicated in tumor growth in many of these tumors, and elevated IL-6 expression has been associated with poor outcomes, and resistance to chemotherapy (1). Experimentally, growth of prostate cancer and cholangiocarcinoma xenografts in athymic mice has been shown to be increased by enforced expression of IL-6 by activation of cell survival signaling (2;3). The mechanisms by which IL-6 promotes cell survival in cancers are of considerable interest because they may be therapeutically targeted. IL-6 activated survival signaling has been shown to involve the signal transducers and activators of transcription (Stat) factors, or various protein kinase cascades (4;5). Although constitutive activation of Stat has been described in many cancers, the precise mechanisms involved are incompletely understood. Cholangio-carcinoma are highly resistant to chemotherapy. However, inhibition of IL-6 dependent pathways such as the Jak-Stat pathway, PI3-kinase or the p38 MAPK pathways can enhance chemotherapy-induced cell death. Thus, aberrant IL-6 dependent survival signaling may contribute to the

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http://www.jbc.org/cgi/doi/10.1074/jbc.M607712200The latest version is at JBC Papers in Press. Published on January 12, 2007 as Manuscript M607712200

Copyright 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

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refractoriness of cholangiocarcinoma to most chemotherapeutic agents. We sought to understand the role of microRNAs (miRNAs) in IL-6 mediated tumor cell survival. miRNAs are endogenous regulators of gene expression that are altered in expression in many cancers (6-8). The expression of several microRNAs (miRNAs) in cholangiocarcinoma xenografts in athymic mice is altered during in vivo treatment with gemcitabine (9). miRNAs are small endogenous molecules that can regulate gene expression in a sequence complementary manner. Several hundred miRNAs have been identified, and details of the mechanisms by which they regulate gene expression are being unraveled (10;11). Less is known about the mechanism by which miRNAs contribute to cellular behavior and function. Alterations in miRNA expression occur in many different cancers (8). Thus, individual miRNAs may play contributory or regulatory roles in tumor cell pathogenesis or behavior. Potential downstream targets of miRNA include oncogenes or tumor suppressor genes, but few miRNA regulated targets relevant to tumor biology have been described such as the Ras oncogene(12). We postulated that genetic reprogramming resulting from altered miRNA regulatory networks may contribute to tumor cell response and resistance to chemotherapy.

EXPERIMENTAL PROCEDURES

Cell Lines and Cultures – Mz-ChA-1 and KMCH-1 human malignant cholangiocytes and their respective IL-6 over-expressing stable transfectants, Mz-IL-6 and KM-IL-6 were obtained and cultured as previously described (2). Basal IL-6 expression was increased by ~1.5-fold in KM-IL-6 and ~ 3-fold in Mz-IL-6 cells relative to their respective controls. Transfections - 20 µl of 100 nM microRNA precursor, antisense inhibitor or controls were added to 1x106 cells suspended in 100 μl Nucleofector solution (Amaxa Biosystems) at room temperature. Electroporation was performed using the Nucleofector system (Amaxa Biosystems, Koln, Germany). Transfected cells were then re-suspended in regular culture media containing 10% serum for 48-72 hours prior to study.

MicroRNA isolation and expression profiling - miRNA was isolated by PAGE purification of total RNA and expression profiling was performed using a custom-generated microarray as previously described (9). Microarrays were scanned using a GenePix 4000A array scanner (Axon Instruments, Union City, CA). Normalization was performed by expressing each miRNA replicate relative to a control miRNA (Ambion, Austin, TX) added to each sample, thus allowing comparisons between chips. Data were analyzed using GeneSpring 7.0 Software (Silicon Genetics, Redwood City, CA), and an average value of the median intensity of each replicate in four groups was generated. MicroRNA expression levels were clustered using a self organizing tree algorithm using the Multi-experiment Viewer Version 3.1 from The Institute for Genomic Research (13). Quantitative Real-Time PCR - RNA was isolated using the ToTALLY RNA isolation kit (Ambion, Austin, TX), and cDNA generated by reverse transcription using 1 µg of total RNA and the reverse transcription kit (Invitrogen, Carlsbad, CA). Mature let-7a miRNA expression was assessed using a TaqMan® human microRNA assay kit (Applied Biosystems, Foster City, CA). Real-time PCR was performed using a MX 3000PTM PCR Instrument (Stratagene, San Diego, CA). Cytotoxicity Assay - Cell viability was assessed using a commercially available tetrazolium bio-reduction assay as previously described (14). 10,000 viable cells/well were seeded into 96-well plates and incubated with gemcitabine, 5-fluorouracil, camptothecin or appropriate diluent controls in a final volume of 200 μL medium containing 0.5% FBS. Caspase assay - Cells were plated in 96-well plates (20,000 cells/well) and incubated with different chemotherapeutic agents or diluent control. Caspase 3/7 activity was assayed using the fluorometric Apo-ONE homogenous caspase 3/7 assay (Promega, Madison, WI) and a Cytofluor microplate fluorescence plate reader. Stat-3 kinase assay - Stat-3 activity was assessed in cell lysates after immunoprecipitation using monoclonal P-Stat-3-Tyr 705 antibody (Cell Signaling Technology) and using a tyrosine Kinase Activity Assay Kit (Chemicon, Temecula, CA).

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Luciferase reporter vectors - The pMIR-NF2-luc and pMIR-NF2-MUT-luc firefly luciferase reporter vectors, which contain the intact or mutated putative let-7a recognition sequence from the 3’UTR of NF2 respectively cloned downstream of the firefly luciferase gene were constructed as follows. Synthetic oligonucleotides encompassing the intact or mutated let-7a recognition sequence, sticky ends for HindIII and SpeI to eliminate digestion of the insert, and a unique BlpI site to test for positive clones were synthesized and annealed. The oligonucleotides used were 5’–CTAGTGCTCAGC TACAAGAGA TTCTCCTGCCTCAA - 3’ (sense) and 5’ - AGCTTTGAGGCAGGAGAATCTCTTGTAGCTGAGCA - 3’ (antisense) for pMIR-NF2-luc, or 5’ CTAGTGCTCAGCTATAG CGCTTTGCTCGAGTGGAA-3’(sense) and 5’- AGCTTTCCATCGAG CAAAGCGCCTATAGCTGAGCA- 3’ (antisense) for pMIR-NF2-MUT-luc. The recognition sequence is underlined in the former and the random mutations introduced are italicized in the latter sequence. To anneal the oligonucleotides, 2 μg of each strand was added to 46 μl of DNA annealing buffer (30 mM HEPES, pH 7.4, 100 nM potassium acetate, 2 mM magnesium acetate) for a final volume of 50 μl and incubated at 90°C for 3 min and then at 37°C for 1 hour. The annealed insert was then directly ligated into the HindIII and SpeI cloning sites of the pMIR-REPORT luciferase expression vector (Ambion, Austin, TX). Clones were selected after screening by restriction digestion with BlpI. Mz-IL-6 cells were co-transfected with 1 μg pMIR-NF2-UTR or pMIR-NF2-MUT-UTR construct and 1 μg pRL-TK Renilla luciferase expression construct without (empty vector control) or with anti-let-7a inhibitor using TransIT-siQUEST transfection reagent (Mirus, Madison, WI). Luciferase assays were performed 48 hours after transfection using the Dual Luciferase Reporter Assay system (Promega, Madison, WI). For validation of the effect of anti-let-7a, we used the pRL-TK let-7a firefly luciferase expression construct in which two let-7a sites are inserted into the XbaI site in the 3' UTR. Cells were co-transfected with pRL-TK and luciferase assays were performed 48 hours after transfection. Firefly luciferase activity was normalized to Renilla luciferase activity for each sample

Western Blotting - For immunoblot analysis of cells in culture, cell lysates were obtained from cells grown in 100-mm dishes, whereas for analysis of xenograft tissue, lysates were obtained after tissue homogenization. Equivalent amounts of protein were resolved by electrophoresis in a 4-20% Tris-HCl gel (Bio-Rad, Hercules, CA), and then transferred to nitrocellulose membranes. After blocking, membranes were incubated with primary antibodies and infra-red dye-labeled secondary antibodies. The protein of interest was then detected using the LI-COR Odyssey Infrared Imaging System (LI-COR Bioscience, Lincoln, NE). Nuclear and cytoplasmic fractions were obtained using the NE-PER extraction kit (Pierce, Rockford, IL) according to the manufacturer's

instructions. Xenograft model - Studies were performed under an Institutional Animal Care and Use Committee (IACUC) approved protocol. Eight week old male athymic nu/nu mice (Charles River Laboratories, Wilmington, MA), were maintained in accordance with IACUC procedures and guidelines, and 5 x 106 Mz-1 or Mz-IL-6 cells were suspended in 0.25 mL of extracellular matrix gel, and the mixture injected subcutaneously into the right and left flanks. Serial measurements of xenograft growth were performed and tumor volume estimated using the formula 4/3 π (L*W*H/8). Once tumor volume was 200-230 mm3, xenografts were excised, and the tissue homogenized for miRNA isolation or immunoblot studies or used for miRNA inhibition studies. For the latter, mice with Mz-IL-6 xenografts were injected intratumorally with 4 ng/mm tumor volume of either anti-let-7a or diluent. The following day, gemcitabine (150 mg/kg) was administered intraperitoneally every three days for three doses. The change in tumor size was assessed and tumors were excised after 10 days. Homogenates were obtained for western blot analysis. Sections of tumors were obtained for immunofluorescence studies using mouse anti-P-Stat3 (Tyr 705) (1:75 dilution) or rabbit anti-NF2 (1:75 dilution) primary antibodies, and with detection using FITC labeled donkey anti-mouse IgG (1:100 dilution) or Texas Red labeled donkey anti-rabbit IgG (1:100 dilution) respectively. Imaging was performed using a Axiovert 200 Motorized Fluorescent Microscope Imaging system (Carl Zeiss, Thornwood, NY).

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Reagents - All microRNA precursors, inhibitors and controls were obtained from Ambion Inc., (Austin, TX). Primary antibodies against NF2, Mcl-1, Survivin were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA); phospho-AKT [Ser473], phospho-STAT-3 [Tyr705], phospho-p38 [Thy180/Tyr182], PARP and active-caspase-3 were from Cell Signaling, Inc. (Beverly, MA), and α-tubulin was from Sigma (St. Louis, MO). TBP monoclonal antibody was from ABcam Inc. (Cambridge, MA). FITC or Texas Red labeled secondary antibodies were from Jackson Immunoresearch Laboratories (West Grove, PA), whereas IRDye700 & IRDye800-labelled secondary antibodies were obtained from Rockland (Gilbertsville, PA). Gemcitabine was provided by Lilly (Indianapolis, IN). 5-fluoro-uracil and camptothecin were from Sigma (St Louis, MO). pRL-TK let7a from Phil Sharp (Addgene plasmid 11324) and pcDNA3/5’F encoding NF2 from Vijaya Ramesh (Addgene plasmid 11623) were obtained from Addgene (Cambridge, MA) (15;16) . Statistical Analysis - In vivo responses were compared using the t-test. Data are expressed as the mean ± 95% confidence interval unless otherwise noted. The difference between two groups was analyzed using a double sided Student’s t test and the null hypothesis was rejected at the 0.05 level.

RESULTS IL-6 survival signaling involves alterations in miRNA expression. To identify miRNA that may contribute to survival signaling and chemoresistance, we first assessed the effect of IL-6 on miRNA expression. Mz-ChA-1 human cholangiocarcinoma cells were stably transfected to over-express IL-6 (Mz-IL-6 cells) and implanted as xenografts in athymic nude mice. Compared to Mz-1 control cell xenografts, the growth rate of Mz-IL-6 xenografts was increased (Fig 1A). Moreover, there was a loss of sensitivity of Mz-IL-6 tumor xenografts to the chemotherapeutic agent gemcitabine (Fig 1B), in conjunction with a decrease in the number of TUNEL positive (apoptotic) cells compared to controls (Fig 1C). We used a miRNA microarray to assess the expression of human miRNAs in tumor cell xenografts and in two different cholangiocarcinoma cell lines over-expressing IL-

6. The pattern of miRNA expression in IL-6 over-expressing Mz-IL-6 and KM-IL-6 cells differed from their controls (supplementary Fig 1). A cluster of miRNAs that were increased with enforced IL-6 expression both in vivo as well as in vitro was identified, and included several members of the let-7 family and miRNA that have been implicated in oncogenesis such as miR-21 (Fig 2A) (17). These data showing altered miRNA expression profiles in vivo suggest that some effects of IL-6 on tumor cell growth and apoptosis may be mediated by miRNA dependent regulation of gene expression. The relative expression of several members of the let-7 family was altered in vivo (Fig 2B). Of these, let-7a was chosen for further study based on consistency and level of expression. The expression of mature let-7a miRNA was confirmed to be increased by real-time PCR in IL-6 over-expressing cells in vitro, as well as in tumor cell xenografts in vivo compared to controls (Fig 2C). Let-7a contributes to survival signaling by IL-6. To determine the relevance of enhanced let-7a expression to survival signaling, we next evaluated the effect of let-7a inhibition on the response to chemotherapy in vitro. The effect of the anti-let-7a inhibitor was assessed by co-transfecting with the pRL-tk let-7a firefly luciferase construct which contains two let-7a sites in the 3’-UTR of the firefly luciferase reporter. An increase in luciferase activity confirmed the efficacy of anti-let-7a under the conditions used for our studies (Fig 3A). Cytotoxicity in response to diverse agents was enhanced by pre-incubation with antisense inhibitors to let-7a (Fig 3B). Moreover, there was an increase in caspase-3/7 activity in response to chemotherapy in cells pre-incubated with anti-let-7a compared to a control inhibitor (Fig 3C). Similarly, an increase in PARP cleavage and activated caspase-3 were noted in response to anti-let-7a in western blots from gemcitabine treated cells (Fig 3D). Thus, inhibition of let-7a increases chemotherapy induced apoptosis. Taken together, these data are consistent with an effect of let-7a on IL-6 mediated anti-apoptotic survival pathways. Let-7a regulates Stat-3 phosphorylation. Survival signaling by IL-6 can involve activation of the Stat family of transcription factors or protein kinases such as p38 MAPK and PI-3 kinase. Constitutive phosphorylation and

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activation of Stat-3 in cholangiocarcinoma cells has been shown to be IL-6 dependent (18). Consistent with these observations there was an increase in Tyr705-phosphorylated Stat-3 (p-Stat-3) in Mz-IL-6 cells compared to controls. An increase of Ser473 phosphorylated Akt was also noted in these cells. We next evaluated the effect of antisense inhibition of miR-21 and let-7a on these pathways (Fig 4A). Constitutive Stat-3 phosphorylation in Mz-IL-6 cells was markedly decreased by anti-let-7a, but not by anti-miR-21. Furthermore, incubation of Mz-IL-6 cells with anti-let-7a decreased basal Stat-3 kinase activity to 76.9 ± 2.5% of controls after 48 hours (n=8, p=0.002). Stat-3 has several well characterized targets that inhibit apoptosis and can modulate survival, such as survivin, Bcl-XL and Mcl-1 (19). Of these, survivin expression can predict prognosis in human cholangiocarcinoma, and Mcl-1 expression can modulate responses to chemotherapy (20). Consistent with a role for Stat-3 in mediating the survival effects of IL-6 in chemoresistance, the expression of both survivin and Mcl-1 was increased in Mz-IL-6 cells in a let-7a and Stat-3 dependent manner (Fig 4B). NF2 is a target for let-7a. To elucidate potential mediators of let-7a modulation of Stat-3 phosphorylation, we performed a bioinformatics analysis. The let-7a sequence was compared with proposed regulators of Stat-3, using the criteria of Doench and Sharp, but modified to include 85% sequence complementarity at the positions 2-9 of the miRNA (15). The tumor suppressor gene NF2, a known modulator of Stat-3 activation, was identified as a putative target for let-7a. Moreover, interrogation of various target prediction databases such as TargetScan, miRScan, miRanda and PicTar did not identify any other known regulators of Stat-3 as potential targets for let-7a (21). The location of the let-7a complementary site in the 3’-UTR of NF2 is shown in Fig 5. The site is conserved in human and rat homologs of NF2. NF2 has been previously shown to regulate Stat-3 phosphorylation by a mechanism involving the hepatocyte growth factor tyrosine kinase substrate HRS (22). We verified that NF2 was a target for let-7a using luciferase reporter constructs containing the let-7a recognition sequence from the 3’-UTR of NF2 inserted downstream of the luciferase gene (pMIR-NF2-

luc), along with a similar construct in which random mutations were introduced at sites involved in base-pairing (pMIR-NF2-MUT-luc) (Fig 5A). Transfection with anti-let-7a increased reporter activity in Mz-IL-6 cells, whereas let-7a precursor decreased reporter activity in Mz-1 cells However, these effects were ameliorated when the mutated reporter construct pMIR-NF2-MUT was used in place of pMIR-NF2-luc (Fig 5B, C). Constitutive expression of NF2 was decreased and p-Stat-3 increased in Mz-IL-6 cells compared to Mz-1 controls. Moreover, inhibition of let-7a increased NF2 expression and concomitantly decreased p-Stat-3 (Fig 6A). Conversely, p-Stat-3 was increased during incubation of Mz-1 cells with NF2 siRNA (Fig 6B). Incubation with the let-7a precursor miRNA increased nuclear Stat-3 expression suggesting that let-7a enhances activation of Stat-3 and nuclear translocation. Similar effects were also observed with NF2 siRNA (Fig 6C). These studies support a mechanism by which enhanced IL-6 production enhances constitutive Stat-3 phosphorylation and activation via a mechanism involving let-7a mediated inhibition of NF2.

Let-7a can mediate downstream effects of IL-6 over-expression on Stat-3 phosphporylation. We evaluated whether an increase in let-7a expression alone could recapitulate the effects of IL-6 on NF2 and Stat3. Mz-1 cells were transfected with either let-7a precursor or control precursors. Interestingly, NF2 expression was decreased and Stat-3 phosphorylation was increased in Mz-1 cells transfected with let-7a precursor. Furthermore, the effect of let-7a expression on Stat-3 phosphorylation was blocked by enforced expression of NF-2 (Fig. 7a). To assess the involvement of NF2 on IL-6 dependent signaling, we measured phospho-Stat3 and NF2 levels in Mz-1 and Mz-IL-6 cells after transfection with NF2 or LacZ. Aberrant expression of NF2 successfully blocked IL-6 dependent Stat3 activation (Fig. 7b). Thus, enforced-expression of NF2 is sufficient to overcome the effects of IL-6 on constitutive Stat-3 expression in malignant cholangiocytes. Let-7a modulates NF2 and Stat-3 in vivo. To explore the in vivo relevance of these observations, we assessed the expression of p-Stat-3, NF2 and the Stat-3 regulated anti-apoptotic proteins in homogenates from xenograft tumors.

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The results corresponded to those observed in vitro with an increase in basal expression of p-Stat-3 and decrease in NF2 in Mz-IL-6 xenografts compared to control cell xenografts (Fig 8A). Intra-tumoral administration of anti-let-7a increased NF2 and decreased p-Stat-3 expression in Mz-IL-6 xenografts in vivo (Fig 8B and C). Moreover, a decrease in tumor growth consistent with increased gemcitabine toxicity was observed in response to anti-let-7a compared to tumors that were untreated. Anti-let-7a (n=6) or diluent (n=4) was administered intra-tumorally into Mz-IL-6 xenografts. Animals subsequently received a course of three doses of gemcitabine 150 mg/kg i.p. given every three days. The mean change in tumor growth at the end of treatment was 0.6 ± 2.0% in tumors that received anti-let-7a compared to 18.4 ± 8.2% in controls (p=0.02). In combination, these findings identify a previously unrecognized mechanism that contributes to constitutive Stat-3 phosphorylation involving NF2, a target of regulation by the let-7a microRNA.

DISCUSSION Although the association between chronic inflammation and malignancy has been recognized for many decades, the role of miRNA in cancer cell biology has only recently been appreciated. The role of cytokines as stimulators of miRNA expression have not explored, and herein we demonstrate a role for persistent IL-6 stimulation on altered miRNA expression in a human cancer. The demonstration of an inflammation-associated cytokine-regulated miRNA mediated survival mechanism is highly relevant to both tumor biology and regulation of cytokine signaling. Moreover, these studies emphasize the emerging complexity of miRNA mediated cellular responses. There is compelling evidence of a critical role for activated Stat-3 in human cancers. Constitutively activated Stat-3 is observed in many cancers, and abrogation of Stat-3 activation results in the loss of the malignant phenotype (23). Moreover, cells expressing persistently activated Stat-3 are dependent on it for survival. Thus Stat-3 can act as an oncogene and may contribute to tumor growth (24-26). Although several cytokines including IL-6 can induce Stat-3 tyrosine phosphorylation, the mechanisms by which it is

constitutively activated in cancers are unknown. Although activating Stat-3 mutations have not been described, aberrant expression of modulators of Stat expression or phosphorylation such as PIAS-3, modulators of upstream Stat-3 activation such as SOCS-1, or as yet uncharacterized Stat-3 tyrosine phosphatases may all contribute (5). None of these mechanisms have been shown to predominate in tumor cells. The contribution of miRNA modulation of NF2 expression warrants further investigation as an alternative mechanism contributing to constitutive Stat-3 activation. We speculate that the NF-2 dependent mechanism may be more relevant to constitutively increased Stat-3 phosphorylation in the setting of chronic IL-6 stimulation, rather than transient, non-sustained activation of Stat-3 in response to acute stimulation of IL-6 signaling which has been well characterized and involves, among others, Jak-Stat interactions. NF2 is located on chromosome 22q12.2 and encodes for merlin, a putative tumor suppressor gene. Merlin has strong binding to HRS, a potent regulator of receptor tyrosine kinase trafficking, and the interaction of HRS and Merlin can result in inhibition of Stat activation (22). Merlin has been shown to act as growth regulator and its decreased expression could partly contribute to the increased growth rate observed in IL-6 over-expressing tumor cell xenografts. In response to IL-6 stimulation, activation of Stat3 is associated with the endocytotic pathway (27). Thus, a plausible mechanism by which decreased NF2 expression in response to persistent IL-6 stimulation results in activation of Stat-3 could involve facilitating its association with the endocytotic pathway through an HRS-mediated mechanism. The miRNA family of let-7 and its homologs have been implicated as cancer-associated miRNAs in recent studies (28;29). Although we focused our studies on let-7a, we note that the relative expression of other members of the let-7 family such as let-7d and let-7f-2 were also increased in vivo (Fig 2B). It is quite likely that these other members of the let-7 family that are differentially altered in response to increased IL-6 stimulation may also have cellular actions. NF2 is not a predicted target for either let-7d and let-7f-2. However, both these miRNA could potentially target SOCS-1, an established inhibitor

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of the Jak-Stat3 pathway, and thereby modulate IL-6 dependent Stat3 activation.

In reported series of lung cancers, let-7 expression is down-regulated in association with Ras expression in the setting of activating Ras mutations, and decreased expression of let-7a2 has been shown to correlate with a poor prognosis (30-32). However, these observations are likely to be cell-type specific since let-7 is only sporadically reduced in tumor types other than lung cancer. Our experimental model differs considerably from these studies in representing a state of persistent

cytokine stimulation, and it is unknown if let-7 expression can be modulated by IL-6 in a similar manner in lung cancer. Although augmenting let-7 expression is being touted as a potential therapeutic strategy, such approaches may be inappropriate for cancers that are associated with elevated IL-6 levels such as cholangiocarcinoma. In contrast, potential interventions to decrease survival signaling and Stat-3 activation by IL-6 may be a useful approach for these cancers.

FOOTNOTES

This study was supported by grant DK069370 from the National Institutes of Health and the Scott & White Hospital Foundation. We thank Dr. Reza Forough, Dr. Robert Jamroz and Ms. Usha Chowdhury of the Microarray Core Facility in the Department of Medical Physiology, Texas A & M University System Health Science Center for assistance with the microRNA microarray studies. The abbreviations used are: IL-6, Interleukin-6; miRNA, micro-RNA; NF2, neurofibromatosis 2; Stat-3, Signal transducers and activators of transcription -3

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Ciliberto, G., Moscinski, L., Fernandez-Luna, J. L., Nunez, G., Dalton, W. S., and Jove, R. (1999) Immunity. 10, 105-115

27. Shah, M., Patel, K., Mukhopadhyay, S., Xu, F., Guo, G., and Sehgal, P. B. (2006) J. Biol. Chem. 281, 7302-7308

28. Esquela-Kerscher, A. and Slack, F. J. (2006) Nat. Rev. Cancer 6, 259-269 29. Akao, Y., Nakagawa, Y., and Naoe, T. (2006) Biol. Pharm. Bull. 29, 903-906 30. Takamizawa, J., Konishi, H., Yanagisawa, K., Tomida, S., Osada, H., Endoh, H., Harano, T.,

Yatabe, Y., Nagino, M., Nimura, Y., Mitsudomi, T., and Takahashi, T. (2004) Cancer Res 64, 3753-3756

31. Yanaihara, N., Caplen, N., Bowman, E., Seike, M., Kumamoto, K., Yi, M., Stephens, R. M., Okamoto, A., Yokota, J., Tanaka, T., Calin, G. A., Liu, C. G., Croce, C. M., and Harris, C. C. (2006) Cancer Cell 9, 189-198

32. Johnson, S. M., Grosshans, H., Shingara, J., Byrom, M., Jarvis, R., Cheng, A., Labourier, E., Reinert, K. L., Brown, D., and Slack, F. J. (2005) Cell 120, 635-647

FIGURE LEGENDS

Figure 1. Enforced expression of IL-6 decreases apoptosis in vivo. (a) IL-6 over-expressing Mz-IL-6 cell xenografts in athymic nude mice grow faster than control Mz-1 tumor cell xenografts. (b) Mz-1 and Mz-IL-6 tumor cell xenografts (n=4 each) were treated with gemcitabine (120 mg/kg i.p) every three days for a total of five doses. Tumor volume was assessed at baseline and at the end-of-treatment. The starting size of tumors was similar in both groups, and the studies were performed with tumors ranging from 200-230 mm3. The data represents the mean and 95% confidence intervals from four tumors. P <0.05 for differences between change in volume from baseline for Mz-IL-6 compared to Mz-1 xenografts. (c) Xenograft sections were obtained and TUNEL staining for apoptotic cells performed. The data represents mean and standard deviation of the number of TUNEL positive cells from eight random high power fields

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(HPF). There was a decrease in chemosensitivity in vivo and in the numbers of TUNEL positive cells in Mz-IL6 xenografts. Thus, enhanced IL-6 expression decreases chemotherapy induced apoptosis in vivo. Figure 2. IL-6 over-expression alters miRNA expression. (a) miRNA was isolated from IL-6 over-expressing Mz-IL-6 and KM-IL-6 cells and their respective control Mz-1 and KMCH-1 cells in vitro, and from Mz-IL-6 and Mz-1 tumor cell xenografts in nude mice. Cluster analysis of miRNA expression profiles identified a group of miRNA that was consistently altered with enforced expression of IL-6 both in vivo and in vitro. This cluster contained the listed members of the let-7 family, and miR-21. (b) Quantitative analysis of let-7 family of miRNA in vivo representing the average and 95% confidence intervals from eight samples. (c) Real-time PCR confirmed increased expression of mature let-7a in Mz-IL-6 and KM-IL-6 cells as well as tumor xenografts compared to expression in their respective controls. Data represents the mean and standard error of four determinations performed in triplicate. * p< 0.05 compared to respective controls. Figure 3. Let-7a decreases chemotherapy-induced apoptosis in vitro. (a) To validate the efficacy of the anti-let-7a inhibitor, Mz-1 cells plated (2 × 106 cells/well) in 6-well plates were transfected with 1 ug pRL-TK let-7a (let-7a firefly luciferase construct), 1 ug pRL-TK (renilla luciferase construct) and either anti-let-7a or control inhibitor. Luciferase assays were performed 48 hours after transfection. Firefly luciferase activity was normalized to Renilla luciferase activity for each sample. The results represent the mean and SE of 10 separate determinations. The anti-let-7a inhibitor directly inhibited the effect of endogenous let-7a on the luciferase reporter. (b) Tumor cells were transfected with anti-let-7a or negative control miRNA inhibitors. After 48 hours, 104 live cells were plated in each well of a 96-well plate and incubated with 100 μM gemcitabine, 100 μM 5-fluorouracil, 10 μM camptothecin or their respective diluent controls, and cell viability was assessed after 48 hours. (c) Cells were transfected with either control or anti-let-7a inhibitors for 48 hours as above, then incubated with the indicated chemotherapeutic agent. Caspase-3/7 activation was assessed using a fluorometric assay after 24 hours. Data represent mean ± 95% confidence interval from 3 separate studies done in triplicate. (d) Transfected cells were incubated with 100 μM gemcitabine for 24 hours, and lysates obtained for western blot analysis for activated caspase-3 or PARP expression. Inhibition of let-7a increased caspase activity and PARP cleavage in response to gemcitabine.

Figure 4. Over-expression of IL-6 increases constitutive STAT-3 activation. (a) IL-6 over-expressing Mz-IL-6 or control Mz-1 cells were transfected with negative control miRNA inhibitors or with miR-21 or let-7a miRNA specific inhibitors for 48 hours. The expression of active Stat-3, Akt and p38 MAPK was assessed by western blot analysis using active-site phosphorylation specific antibodies to evaluate IL-6 activated survival signaling pathways (b) Let-7a modulates expression of activated Stat-3 and its downstream anti-apoptotic targets Mcl-1 and survivin in vitro. Cells were transfected with anti-let-7a or negative control miRNA inhibitors in vitro, and cell lysates obtained after 48 hours. The expression of survivin and Mcl-1 is increased in Mz-IL-6 cells compared to Mz-1 cells, and moreover is decreased by either anti-let-7a or AG490, an inhibitor of Jak mediated STAT-3 activation.

Figure 5. NF2 is a target of let-7a. (a) The location of the putative let-7a target site in the NF2 3’-UTR is shown. A comparison of base pairs between mature human let-7a (hsa-let-7a), human NF-2, rat rno-let-7a and rat NF-2 shows sequence conservation between species. The sequence of the mutated target site with mutations to disrupt base pairing between let-7a binding sites and NF2 is also shown. (b) Mz-IL-6 cells were transfected with the renilla luciferase expression construct pRL-tk and either the luciferase construct pMIR-NF2-luc or pMIR-NF2-MUT-luc (in which mutations were introduced in the let-7a target site) with either anti-let-7a or control inhibitor. After 48 hours, dual luciferase assays were performed. An increase in relative firefly luciferase with pMIR-NF2-luc (solid bars) but not with the pMIR-NF2-MUT-luc construct (hatched bars) confirms that the let-7a complementary sequence in the 3’UTR of NF2

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is a target of modulation by let-7a. The data represents the mean and standard deviations from six determinations from three independent transfections. (c) Mz-1 cells were co-transfected with either let-7a or control precursor, pRL-TK and either pMIR-NF2-luc or pMIR-NF-2-MUT-luc constructs. After 48 hours, dual luciferase assays were performed. A decrease in relative firefly luciferase was observed with pMIR-NF2-luc but not with p-MIR-NF2-MUT-luc. The data represents the mean and standard errors from six determinations from three independent transfections. Figure 6. NF2 modulates Stat-3 phosphorylation. (a) NF-2, a regulator of STAT-3 activation is decreased in Mz-IL-6 in a let-7a dependent manner. Anti-let-7a increases NF2 expression in Mz-IL-6 cells concomitant with a decrease in Tyr705 phospho-Stat-3. Quantitative data showing the mean and 95% confidence interval from four separate studies are shown. (b) Mz-1 cells were transfected with siRNA to NF2 or scrambled control siRNA. Compared to control siRNA transfected cells, the phosphorylation of Stat-3 was increased by NF2 siRNA. (c) Mz-1 cells were incubated with either let-7a precursor or siRNA to NF2 for 48 hours. Nuclear and cytoplasmic fractions were obtained, and immunoblots performed for Stat-3 expression. TBP and β-actin were used as nuclear and cytoplasmic

markers respectively and as loading controls. An increase in nuclear Stat-3 occurs with either let-7a precursor or with siRNA to NF2. Figure 7. Let-7a and NF2 expression are mediators of the effects of IL-6 on Stat-3 phosphorylation. (a) Mz-1 cells were transfected with 30 nM specific let-7a or control precursors, along with 6 μg pcDNA3/5’F encoding NF2 or Lac Z control plasmids for 48 hrs. Representative immunoblots are shown along with quantitative data representing the mean ± standard error from four separate blots. Let-7a over-expression increases Stat-3 phosphorylation, which is blocked by over-expression of NF2. *p < 0.05 relative to expression in control precursor group. (b) Mz-1 or Mz-IL-6 cells were transfected with 6 μg NF2 or Lac Z control plasmids. Immunoblot analysis for NF2 and phosphorylated Stat3 was performed 48 hours after transfection. A decrease in NF2 expression along with an increase in Stat3 phosphorylation is observed in Mz-IL-6 cells compared to Mz-1 controls. Enforced expression of NF2 is sufficient to overcome the effect of IL-6 over-expression on constitutive Stat-3 phosphorylation. Representative immunoblots and quantitative data (mean ± SE) from four separate blots are shown. *p < 0.05 relative to expression in control Lac Z group. Figure 8. STAT-3 phosphorylation is increased by enforced expression of IL-6 in vivo. (a) Tumor cell xenografts in nude mice (3 for each cell type) were excised once they grew to a volume of ~200 mm3, and tissue homogenized. The expression of 705Tyr phosphorylated Stat-3, its negative upstream modulator NF2, and the downstream anti-apoptotic Stat-3 targets survivin and Mcl-1 were assessed by western blot analysis. Quantitative data of mean and 95% confidence intervals from three separate blots are shown. (b) Mz-IL-6 tumor xenografts were treated with anti-let-7a or control miRNA inhibitor, and homogenates obtained for immunoblot analysis. Representative blots and quantitative data showing the average and 95% confidence interval of four separate blots are shown. (c) Immunohistochemistry for phosphorylated Stat-3 and NF-2 was performed on xenograft sections, showing a decrease in phospho-Stat-3 expression and an increase in NF2 in xenografts injected with anti-let-7a compared to controls.

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Days

20 40 60 80 100 1200

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Perc

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Days

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Perc

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Perc

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0

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Mz-1 Mz-IL-6

Cha

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%

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Mz-1 Mz-IL-6

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Fig 1

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Let-7aLet-7cLet-7dLet-7f-1Let-7f-2miR 16-1miR 21

Mz-

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1 352 253 304 45 86 287 268 29 310 711 29

Numberof miRNA

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1 352 253 304 45 86 287 268 29 310 711 29

Numberof miRNA

1 352 253 304 45 86 287 268 29 310 711 29

Numberof miRNA

1 352 253 304 45 86 287 268 29 310 711 29

Numberof miRNA

0

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3

Con

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Let-7

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Let-7

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Relative in vivoExpression

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*

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Let-7

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Relative in vivoExpression

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Fig 2

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*


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Gemcitabine 5-FU Camptothecin

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Active Caspase 3

PARP

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Mz-1 Mz-IL-6

+--Anti-let-7a

-++control anti-miR+++Gemcitabine

Active Caspase 3

PARP

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AFig 5

HSA-Let-7a Binding Site

NF2 Coding Sequence An5’ 3’

421 2199 4368 4390

3' UUGAUAUGUUGGAU..GAUGGAGU 5'Human hsa-let-7a|| | | | :|| ||:|||||UACAAGAGAUUCUC..CUGCCUCA 3'Human NF2 | |||||| | | ||||||||

CA-AAGAGAAAC-C--CUGCCUCA 3'Rat NF2| : : ||| | ||:|||||

3' UU.GAUAUGUUG.GAUGAUGGAGU 5'Rat rno-let-7a

UAUAGGCGCUUUGC..UCGAUGGANF2-MUT: 3'5'

3' UUGAUAUGUUGGAU..GAUGGAGU 5'-let-7a|| | | | :|| ||:|||||UACAAGAGAUUCUC..CUGCCUCA 3'Human NF2 | |||||| | | ||||||||




HSA-Let-7a Binding Site

NF2 Coding Sequence An5’ 3’

421 2199 4368 4390









3' UUGAUAUGUUGGAU..GAUGGAGU 5'- -7a|| | | | :|| ||:|||||

5' UACAAGAGAUUCUC..CUGCCUCA 3'| |||||| | | ||||||||




3' UUGAUAUGUUGGAU..GAUGGAGU 5'- -7a|| | | | :|| ||:|||||UACAAGAGAUUCUC..CUGCCUCA 3'| |||||| | | ||||||||

5' CA-AAGAGAAAC-C--CUGCCUCA 3'Rat NF2| : : ||| | ||:|||||




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Fig 5

Ratio of Firefly / Renilla

luciferase activity

Control Precursor miRNA

let-7a precursor

*

*

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let-7a precursor

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-- -

---

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Negative Control anti-miRNA

Anti-let-7a

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++

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P -Stat3

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NF2

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Control siRNA

NF2 siRNA

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NF2

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Mz-1

P -Stat3

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NF2

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TBP

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NF2

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Control precursor

Let-7a precursor

Lac Z

NF2 - ++-+ --++ +--- -++

NF2

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NF2 - ++-+ --++ +--- -++

NF2

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Mz-IL-6 + NF2

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Mz-1 + NF2

Mz-IL-6 + Lac Z

Mz-IL-6 + NF2

Fig 7A

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n

0

0.3

0.6

0.9

1.2

P-Stat3 0

0.3

0.6

0.9

1.2

NF2

Rel

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n

0

0.3

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P-Stat3 0

0.3

0.6

0.9

1.2

NF2

Rel

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n

0

0.3

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Mcl-1 Survivin0

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Rel

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0

0.3

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Mcl-1 Survivin0

0.3

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Rel

ativ

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n

P-Stat3

NF2

Mcl-1

Survivin

Tubulin

Mz-1

Mz-IL-6

* P<0.05

* *

*

*

0

0.3

0.6

0.9

1.2

P-Stat3 0

0.3

0.6

0.9

1.2

NF2

Rel

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n

0

0.3

0.6

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Mcl-1 Survivin0

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Rel

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0

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P-Stat3 0

0.3

0.6

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NF2

Rel

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0

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P-Stat3 0

0.3

0.6

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NF2

Rel

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0

0.3

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Mcl-1 Survivin0

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Rel

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Mcl-1 Survivin0

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P-Stat3 0

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NF2

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Mcl-1 Survivin0

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P-Stat3 0

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Mcl-1 Survivin0

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Mcl-1 Survivin0

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Rel

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Survivin

Tubulin

Mz-1

Mz-IL-6

* P<0.05

Mz-1

Mz-IL-6

* P<0.05

* *

*

*

0

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P-Stat3 NF2

Rel

ativ

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n

0

0.3

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Mcl-1 Survivin

Rel

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e E

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n

Mz-IL-6 Xenografts

Con

trol

Inhi

bito

r

Ant

i-let

-7a

P-Stat3

NF2

Mcl-1

Survivin

Tubulin

Control Inhibitor

Anti-let-7a

* P<0.05

**

**

0

0.3

0.6

0.9

P-Stat3 NF2

Rel

ativ

e E

xpre

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n

0

0.3

0.6

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Mcl-1 Survivin

Rel

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P-Stat3 NF2

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ativ

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P-Stat3 P-Stat3 NF2 NF2

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Mcl-1 Mcl-1 Survivin Survivin

Rel

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Con

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Inhi

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Ant

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-7a

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Control Inhibitor

Anti-let-7a

* P<0.05

Control Inhibitor

Anti-let-7a

* P<0.05

**

**

A B Fig 8


http://www.jbc.org/

Control anti-miRNA

Anti-let-7a

miRNA

p-Stat3 NF2 Merged

Control anti-miRNA

Anti-let-7a

miRNA

p-Stat3 NF2 Merged

Fig 8

C


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Tushar PatelFanyin Meng, Roger Henson, Hania Wehbe-Janek, Heather Smith, Yoshiyuki Ueno and

malignant human cholangiocytesThe microRNA let-7A modulates Interleukin-6 dependent Stat-3 survival signaling in

published online January 12, 2007J. Biol. Chem.

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