miR-24 Inhibition Increases Menin Expression and Decreases Cholangiocarcinoma Proliferation
2017; Elsevier BV; Volume: 187; Issue: 3 Linguagem: Inglês
10.1016/j.ajpath.2016.10.021
ISSN1525-2191
AutoresLaurent Ehrlich, Chad Hall, Julie Venter, David E. Dostal, Francesca Bernuzzi, Pietro Invernizzi, Fanyin Meng, Jerome P. Trzeciakowski, Tianhao Zhou, Holly Standeford, Gianfranco Alpini, Terry C. Lairmore, Shannon Glaser,
Tópico(s)Pediatric Hepatobiliary Diseases and Treatments
ResumoMenin (MEN1) is a tumor-suppressor protein in neuroendocrine tissue. Therefore, we tested the novel hypothesis that menin regulates cholangiocarcinoma proliferation. Menin and miR-24 expression levels were measured in the following intrahepatic and extrahepatic cholangiocarcinoma (CCA) cell lines, Mz-ChA-1, TFK-1, SG231, CCLP, HuCCT-1, and HuH-28, as well as the nonmalignant human intrahepatic biliary line, H69. miR-24 miRNA and menin protein levels were manipulated in vitro in Mz-ChA-1 cell lines. Markers of proliferation and angiogenesis (Ki-67, vascular endothelial growth factors A/C, vascular endothelial growth factor receptors 2/3, angiopoietin 1/2, and angiopoietin receptors 1/2) were evaluated. Mz-ChA-1 cells were injected into the flanks of nude mice and treated with miR-24 inhibitor or inhibitor scramble. Menin expression was decreased in advanced CCA specimens, whereas miR-24 expression was increased in CCA. Menin overexpression decreased proliferation, angiogenesis, migration, and invasion. Inhibition of miR-24 increased menin protein expression while decreasing proliferation, angiogenesis, migration, and invasion. miR-24 was shown to negatively regulate menin expression by luciferase assay. Tumor burden and expression of proliferative and angiogenic markers was decreased in the miR-24 inhibited tumor group compared to controls. Interestingly, treated tumors were more fibrotic than the control group. miR-24–dependent expression of menin may be important in the regulation of nonmalignant and CCA proliferation and may be an additional therapeutic tool for managing CCA progression. Menin (MEN1) is a tumor-suppressor protein in neuroendocrine tissue. Therefore, we tested the novel hypothesis that menin regulates cholangiocarcinoma proliferation. Menin and miR-24 expression levels were measured in the following intrahepatic and extrahepatic cholangiocarcinoma (CCA) cell lines, Mz-ChA-1, TFK-1, SG231, CCLP, HuCCT-1, and HuH-28, as well as the nonmalignant human intrahepatic biliary line, H69. miR-24 miRNA and menin protein levels were manipulated in vitro in Mz-ChA-1 cell lines. Markers of proliferation and angiogenesis (Ki-67, vascular endothelial growth factors A/C, vascular endothelial growth factor receptors 2/3, angiopoietin 1/2, and angiopoietin receptors 1/2) were evaluated. Mz-ChA-1 cells were injected into the flanks of nude mice and treated with miR-24 inhibitor or inhibitor scramble. Menin expression was decreased in advanced CCA specimens, whereas miR-24 expression was increased in CCA. Menin overexpression decreased proliferation, angiogenesis, migration, and invasion. Inhibition of miR-24 increased menin protein expression while decreasing proliferation, angiogenesis, migration, and invasion. miR-24 was shown to negatively regulate menin expression by luciferase assay. Tumor burden and expression of proliferative and angiogenic markers was decreased in the miR-24 inhibited tumor group compared to controls. Interestingly, treated tumors were more fibrotic than the control group. miR-24–dependent expression of menin may be important in the regulation of nonmalignant and CCA proliferation and may be an additional therapeutic tool for managing CCA progression. Cholangiocarcinoma (CCA) is a biliary epithelial adenocarcinoma associated with late diagnosis, poor long-term survival, and limited responsiveness to current therapies.1Razumilava N. Gores G.J. 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Histological diversity in cholangiocellular carcinoma reflects the different cholangiocyte phenotypes.Hepatology. 2012; 55: 1876-1888Crossref PubMed Scopus (232) Google Scholar Clearly, there is a need for advanced diagnostic strategies and improved targeted therapies for CCA. Menin, encoded by the MEN1 (multiple endocrine neoplasia type I) tumor-suppressor gene, is a 610–amino acid, 67-kDa nuclear protein that is ubiquitously expressed in all tissues and evolutionarily conserved, but shares little sequence homology with other proteins.5Chandrasekharappa S.C. Guru S.C. Manickam P. Olufemi S.-E. Collins F.S. Emmert-Buck M.R. Debelenko L.V. Zhuang Z. Lubensky I.A. Liotta L.A. Crabtree J.S. Wang Y. Roe B.A. Weisemann J. Boguski M.S. Agarwal S.K. Kester M.B. Kim Y.S. Heppner C. Dong Q. Spiegel A.M. Burns A.L. Marx S.J. 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One study has shown that menin expression is down-regulated in hepatocellular carcinoma (HCC), and that overexpressing menin in vitro decreased cell proliferation and gene expression of inflammatory cytokines.14Gang D. Hongwei H. Hedai L. Ming Z. Qian H. Zhijun L. The tumor suppressor protein menin inhibits NF-kappaB-mediated transactivation through recruitment of Sirt1 in hepatocellular carcinoma.Mol Biol Rep. 2013; 40: 2461-2466Crossref PubMed Scopus (22) Google Scholar However, another study has shown that menin is up-regulated in HCC samples from patients with underlying cirrhosis and promotes HCC formation via interaction with mixed-lineage leukemia (MLL) histone methyltransferase complex and overexpression of homeobox A genes.17Xu B. Li S.H. Zheng R. Gao S.B. Ding L.H. Yin Z.Y. Lin X. Feng Z.J. Zhang S. Wang X.M. Jin G.H. 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Characteristic miR-24 expression in gastric cancers among atomic bomb survivors.Pathobiology. 2015; 82: 68-75Crossref PubMed Scopus (6) Google Scholar We propose that menin and miR-24 contribute to a regulatory negative-feedback loop that maintains cholangiocyte proliferation and that dysregulation leads to malignant growth. We have demonstrated the novel finding that menin is down-regulated in human CCA cell lines and advanced-stage human CCA samples and modulation of menin expression alters cholangiocarcinoma proliferation. We have also shown that miR-24 expression is up-regulated in human CCA cell lines and human CCA samples and modulation of miR-24 expression alters cholangiocarcinoma proliferation through changes in menin expression. Reagents were purchased from Sigma (St. Louis, MO), unless otherwise indicated. Human Men1 siRNA and control vectors were purchased from Santa Cruz Biotechnology (Dallas, TX) and transfected with Human MEN1 siRNA (sc-35922) (Santa Cruz Biotechnology). Human pCMV6-Entry MEN1 cDNA and control vector were purchased from Origene (Rockville, MD) and transfected with Lipofectamine 2000 Transfection reagent (ThermoFisher Scientific, Grand Island, NY). miRIDIAN microRNA Human hsa-miR-24-1 5p mimic (C-300495-07-0005), hairpin inhibitor (IH-300495-08-0005), and negative controls were purchased from GE Dharmacon (Lafayette, CO) and were transfected with Lipofectamine RNAiMAX Transfection reagent (ThermoFisher Scientific). The RNeasy Mini Kit for RNA purification and all of the following primers were purchased from Qiagen (Valencia, CA): Menin (MEN1, NM_000244), glyceraldehyde-3-phosphate dehydrogenase (NM_002046), MKI67 (NM_001145966), VEGF-A (NM_001025366), VEGF-C (NM_005429), VEGF receptor (VEGFR)-2 (NM_VEGFR-2), VEGFR-3 (NM_002020), angiopoietin 1 (ANG-1, NM_001146), angiopoietin 2 (ANG-2, NM_001118887), angiopoietin receptor 1 (TIE-1, NM_001253357), and angiopoietin receptor 2 (TIE-2, NM_000459). Antibodies used were as follows: hsa-miR-24-1 (002440 TaqMan; ThermoFisher Scientific), U6 snRNA (004394 TaqMan), Menin (A300-105A; Bethyl Laboratories, Montgomery, TX), Ck-19 (Ab15463; Abcam, Cambridge, MA), Ki-67 (Ab15580; Abcam), p-extracellular signal regulated kinase (ab50011; Abcam), and CD31 (89C2; Cell Signaling Technology, Danvers, MA). We studied six human CCA cell lines: Mz-ChA-1, TFK-1, SG231, CCLP-1, HuCC-T1, and HuH-28. The human intrahepatic CCA cell lines CCLP-1,24Shimizu Y. Demetris A.J. 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Bitter-Suermann D. Prellwitz W. Meyer zum Büschenfelde K.H. Biliary adenocarcinoma: characterisation of three new human tumor cell lines.J Hepatol. 1985; 1: 579-596Abstract Full Text PDF PubMed Scopus (168) Google Scholar was a gift from Dr. Greg Fitz (UT Southwestern Medical Center, Dallas, TX). The human intrahepatic biliary cell line, HuH-28,28Kusaka Y. Tokiwa T. Sato J. Establishment and characterization of a cell line from a human cholangiocellular carcinoma.Res Exp Med (Berl). 1988; 188: 367-375Crossref PubMed Scopus (43) Google Scholar and the human extrahepatic biliary TFK-1 cells29Saijyo S. Kudo T. Suzuki M. Katayose Y. Shinoda M. Muto T. Fukuhara K. Suzuki T. Matsuno S. Establishment of a new extrahepatic bile duct carcinoma cell line, TFK-1.Tohoku J Exp Med. 1995; 177: 61-71Crossref PubMed Scopus (124) Google Scholar were obtained from the Cancer Cell Repository (Tohoku University, Sendai, Japan); the cell lines were maintained as described.30Kanno N. Glaser S. Chowdhury U. Phinizy J.L. Baiocchi L. Francis H. LeSage G. Alpini G. Gastrin inhibits cholangiocarcinoma growth through increased apoptosis by activation of Ca2+-dependent protein kinase C-alpha.J Hepatol. 2001; 34: 284-291Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar The human immortalized, nonmalignant, cholangiocyte cell line, H69, was obtained from Dr. Gregory J. Gores (Mayo Clinic, Rochester, MN).31Francis H. DeMorrow S. Venter J. Onori P. White M. Gaudio E. Francis T. Greene Jr., J.F. Tran S. Meininger C.J. Alpini G. Inhibition of histidine decarboxylase ablates the autocrine tumorigenic effects of histamine in human cholangiocarcinoma.Gut. 2012; 61: 753-764Crossref PubMed Scopus (56) Google Scholar Real-time PCR analysis32Han Y. Demorrow S. Invernizzi P. Jing Q. Glaser S. Renzi A. Meng F. Venter J. Bernuzzi F. White M. Francis H. Lleo A. Marzioni M. Onori P. Alvaro D. Torzilli G. Gaudio E. Alpini G. Melatonin exerts by an autocrine loop antiproliferative effects in cholangiocarcinoma: its synthesis is reduced favoring cholangiocarcinoma growth.Am J Physiol Gastrointest Liver Physiol. 2011; 301: G623-G633Crossref PubMed Scopus (44) Google Scholar of H69 and all CCA cell lines was evaluated using 1 μg of RNA. Glyceraldehyde-3-phosphate dehydrogenase was used as a control primer along with the SYBR Green real-time PCR kit (SABiosciences, Frederick, MD). A ΔΔCT analysis was performed using H69 as the control sample.33DeMorrow S. Glaser S. Francis H. Venter J. Vaculin B. Vaculin S. Alpini G. Opposing actions of endocannabinoids on cholangiocarcinoma growth: recruitment of Fas and Fas ligand to lipid rafts.J Biol Chem. 2007; 282: 13098-13113Crossref PubMed Scopus (91) Google Scholar Data are expressed as relative mRNA levels ± SEM (n = 3). We evaluated the expression of MEN1 mRNA in cDNAs obtained from three control patients and three CCA patients (Table 1); these samples were obtained from Dr. Pietro Invernizzi (Humanitas Research Hospital, Rozzano, Italy) under a protocol number approved by the Ethics Committee of the Humanitas Research Hospital; the protocol was also reviewed by the Veterans' Administration Institutional Review Board (Temple, TX) and Research & Development Committee. The use of human tissue was also approved by the Texas A&M Health Science Center College of Medicine Institutional Review Board (College Station, TX). Real-time PCR was performed in triplicate.Table 1Human CCA Biopsy SamplesHuman CCA biopsy sample∗Sex was not available.GradeAge, yearsDiagnosis2173Intrahepatic cholangiocarcinoma.Degree: G1.Histopathological staging: pT1.8266Intrahepatic cholangiocarcinoma.Grade: G2.Growth pattern: solid.Histopathological staging: pT2a.6367Cancer of the bile ducts: type, histological tubule-papillary; degree, histological G3.Histopathological staging TNM v7: pT1 N1.CCA, cholangiocarcinoma; TNM v7, Tumor Node Metastasis, 7th edition.∗ Sex was not available. Open table in a new tab CCA, cholangiocarcinoma; TNM v7, Tumor Node Metastasis, 7th edition. For immunoblotting analysis,34Han Y. Onori P. Meng F. DeMorrow S. Venter J. Francis H. Franchitto A. Ray D. Kennedy L. Greene J. Renzi A. Mancinelli R. Gaudio E. Glaser S. Alpini G. Prolonged exposure of cholestatic rats to complete dark inhibits biliary hyperplasia and liver fibrosis.Am J Physiol Gastrointest Liver Physiol. 2014; 307: G894-G904Crossref PubMed Scopus (31) Google Scholar protein was obtained and quantified from whole cell lysates from nonmalignant and CCA cell lines, and 40 μg was loaded into each well. Blots were blocked overnight and then stained with menin antibody at a 1:2000 dilution and β-actin antibody at a 1:5000 dilution. Data are expressed as fold change (means ± SEM, n = 3) of the relative expression after normalization with β-actin (housekeeping). Measurement of menin expression was also performed using flow cytometry, as described.34Han Y. Onori P. Meng F. DeMorrow S. Venter J. Francis H. Franchitto A. Ray D. Kennedy L. Greene J. Renzi A. Mancinelli R. Gaudio E. Glaser S. Alpini G. Prolonged exposure of cholestatic rats to complete dark inhibits biliary hyperplasia and liver fibrosis.Am J Physiol Gastrointest Liver Physiol. 2014; 307: G894-G904Crossref PubMed Scopus (31) Google Scholar Briefly, H69 and Mz-ChA-1 cells (the CCA lines implanted into athymic mice) were harvested from culture plates using TrypLE solution (ThermoFisher Scientific), and washed with appropriate dilution of fixation buffer (eBioscience, San Diego, CA). Cells were resuspended at a minimum of 5 × 105 cells/mL in 1× permeabilization buffer (eBioscience, Inc., San Diego, CA) and incubated for 15 minutes at room temperature with menin antibody at a dilution of 1:100. Then, Alexa Fluor 488 conjugated secondary antibody was added to suspension at a dilution of 1:50 and cells were incubated for 15 minutes at room temperature in the dark. Cells incubated without antibody or with only Alexa Fluor 488 conjugated secondary antibody were used as negative controls. Cells were analyzed using FACSCalibur (Becton Dickinson, San Jose, CA), with CellQuest Pro software version 5.2 (BD Bioscience, San Jose, CA). At least 10,000 events in the light scatter (side scatter/forward scatter) were acquired. The expression of menin was identified and gated on FL1-A/Count plots. The relative quantity of the selected proteins (mean selected protein fluorescence intensity) is expressed as mean FL1-A (samples)/mean FL1-A (secondary antibodies only) (n = 3). Mz-ChA-1 cells were targeted for the transient knockdown of menin expression using a human MEN1 siRNA (Santa Cruz Biotechnology) (sc-35922) along with siRNA Transfection Reagent (Santa Cruz Biotechnology) (sc-29528), according to the vendor's instructions. Mz-ChA-1 cells were targeted for menin overexpression using the pCMV6-Entry expression vector containing MEN1 cDNA was purchased from Origene32Han Y. Demorrow S. Invernizzi P. Jing Q. Glaser S. Renzi A. Meng F. Venter J. Bernuzzi F. White M. Francis H. Lleo A. Marzioni M. Onori P. Alvaro D. Torzilli G. Gaudio E. Alpini G. Melatonin exerts by an autocrine loop antiproliferative effects in cholangiocarcinoma: its synthesis is reduced favoring cholangiocarcinoma growth.Am J Physiol Gastrointest Liver Physiol. 2011; 301: G623-G633Crossref PubMed Scopus (44) Google Scholar and was transfected with the Lipofectamine 2000 Transfection reagent (ThermoFisher Scientific), according to protocol. Transfected cell lines were allowed to grow for 24 to 48 hours before harvesting. The menin-containing plasmid was amplified using ElectroMAX DH5α Competent Cells (Qiagen) and Invitrogen imMedia agar and liquid media and isolated using Qiagen mini kit, according to vendors' instructions. Plasmid yields were quantified using ND-1000 NanoDrop (ThermoFisher Scientific). The pCMV6-Entry vector without the Men1 insert was used as a control. Transfection efficacy was assessed by measuring menin expression by real-time quantitative PCR and flow cytometry.33DeMorrow S. Glaser S. Francis H. Venter J. Vaculin B. Vaculin S. Alpini G. Opposing actions of endocannabinoids on cholangiocarcinoma growth: recruitment of Fas and Fas ligand to lipid rafts.J Biol Chem. 2007; 282: 13098-13113Crossref PubMed Scopus (91) Google Scholar, 35Francis H. Glaser S. DeMorrow S. Gaudio E. Ueno Y. Venter J. Dostal D. Onori P. Franchitto A. Marzioni M. Vaculin S. Vaculin B. Katki K. Stutes M. Savage J. Alpini G. Small mouse cholangiocytes proliferate in response to H1 histamine receptor stimulation by activation of the IP3/CaMK I/CREB pathway.Am J Physiol Cell Physiol. 2008; 295: C499-C513Crossref PubMed Scopus (112) Google Scholar Cell proliferation was measured by Ki-67 real-time PCR expression, migration via wound healing,36Fronza M. Heinzmann B. Hamburger M. Laufer S. Merfort I. Determination of the wound healing effect of Calendula extracts using the scratch assay with 3T3 fibroblasts.J Ethnopharmacol. 2009; 126: 463-467Crossref PubMed Scopus (211) Google Scholar invasion via Boyden chamber, and angiogenesis via VEGF-A, VEGF-C, VEGFR-2, VEGFR-3, ANG-1, ANG-2, TIE-1, and TIE-2 real-time PCR expression. Mz-ChA-1 cells were plated onto 6-well plates, as described above, and transfected 24 hours later. Twenty-four hours after transfection, cells were tryspinized and reseeded onto a new 6-well plate with twice the number of cells. Twenty-four hours later, a wound was initiated in the cell monolayer using a small pipette tip and a 19-inch gauge needle.36Fronza M. Heinzmann B. Hamburger M. Laufer S. Merfort I. Determination of the wound healing effect of Calendula extracts using the scratch assay with 3T3 fibroblasts.J Ethnopharmacol. 2009; 126: 463-467Crossref PubMed Scopus (211) Google Scholar Phase contrast images were taken at 0, 3, 6, 24, and 48 hours with a Nikon Eclipse TS100 microscope, and NIS-elements D version 4.00.07 software was used to measure the width of the wound (Nikon, Melville, NY). Data are expressed as fold-change of relative widths compared with 0-hour measurements of their respective transfected cell type ± SEM (n = 6). Statistical significance was evaluated between control and transfected cell types at respective time points. Migration of Mz-ChA-1 cells was evaluated using QCM ECMatrix Cell Invasion Assay (Millipore, Billerica, MA), according to manufacturer's protocol. Fluorescence was read at 480/520 nm using a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA). Data are expressed as fold-change of transfected cell lines relative to control cell lines ± SEM (n = 7). Cell proliferation was measured by the CellTiter 96 Aqueous nonradioactive cell proliferation assay (Promega, Madison, WI).32Han Y. Demorrow S. Invernizzi P. Jing Q. Glaser S. Renzi A. Meng F. Venter J. Bernuzzi F. White M. Francis H. Lleo A. Marzioni M. Onori P. Alvaro D. Torzilli G. Gaudio E. Alpini G. Melatonin exerts by an autocrine loop antiproliferative effects in cholangiocarcinoma: its synthesis is reduced favoring cholangiocarcinoma growth.Am J Physiol Gastrointest Liver Physiol. 2011; 301: G623-G633Crossref PubMed Scopus (44) Google Scholar Absorbance was measured at 490 nm on a microplate spectrophotometer (VersaMax; Molecular Devices). Data were expressed as the degree of change of treated cells compared with vehicle-treated controls (n = 7). A proliferation assay was used to assess the effects of altered MEN1 and miR-24 expression on CCA cell growth. miRNA was isolated from samples using the Ambion mirVana miRNA isolation kit (ThermoFisher Scientific), per protocol.34Han Y. Onori P. Meng F. DeMorrow S. Venter J. Francis H. Franchitto A. Ray D. Kennedy L. Greene J. Renzi A. Mancinelli R. Gaudio E. Glaser S. Alpini G. Prolonged exposure of cholestatic rats to complete dark inhibits biliary hyperplasia and liver fibrosis.Am J Physiol Gastrointest Liver Physiol. 2014; 307: G894-G904Crossref PubMed Scopus (31) Google Scholar cDNA libraries were generated using TaqMan microRNA RT kit, per protocol (ThermoFisher Scientific) and TaqMan MicroRNA Assay hsa-miR-24 primers (ThermoFisher Scientific). TaqMan Universal PCR Master mix was used for real-time PCR, and U6 snRNAs were used for endogenous control (ThermoFisher Scientific). A ΔΔCT analysis was performed using normal H69 as controls. Data are expressed as fold-change of relative microRNA levels ± SEM (n = 3). miRNA-sequencing data were obtained from nine human CCA patients from the The Cancer Genome Atlas Research Network (http://cancergenome.nih.gov, last accessed April 7, 2016) via the Genomic Data Commons Data Portal The Cancer Genome Atlas–CHOL. Briefly, the manifest files as well as the miRNA expression quantification files were downloaded for all 36 CCA patient samples contained in the The Cancer Genome Atlas database. File manipulation was performed with Python, Python Software Foundation, Python Language Reference version 2.7. (http://www.python.org, last accessed April 7, 2016). Example coding for this process can be found at https://github.com/ehrl1ch/RNA-Seq (last accessed April 7, 2016). miR-24 expression is displayed as normalized miRNA transcript count for each tumor sample compared with its matched human control. A t-test was performed to assess statistical significance. Experiments were performed to test whether miR-24 directly interacts with menin to change its expression. Luciferase constructs were obtained from Dr. Judy S. Crabtree (Louisiana State University, Baton Rouge, LA). These constructs consisted of a 1600-bp fragment of human MEN1 3′-untranslated region cloned into a pmirGLO vector (pmirGLO-MEN1). The plasmid was amplified and isolated according to the protocol described above. A total of 5 × 105 Mz-ChA-1 cells per as
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