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miR-431 Promotes Metastasis of Pancreatic Neuroendocrine Tumors by Targeting DAB2 Interacting Protein, a Ras GTPase Activating Protein Tumor Suppressor

2020; Elsevier BV; Volume: 190; Issue: 3 Linguagem: Inglês

10.1016/j.ajpath.2019.11.007

1525-2191

Tiantian Zhang, So-Young Choi, Tuo Zhang, Zhengming Chen, Yudan Chi, Shixia Huang, Jenny Xiang, Yi‐Chieh Nancy Du,

Chromatin Remodeling and Cancer

The incidence of pancreatic neuroendocrine tumor (PNET) is increasing, and it presents with various clinical manifestations and an unfavorable survival rate. A better understanding of the drivers of PNET tumorigenesis is urgently needed. Distinct miRNA signatures have been identified for different stages of tumorigenesis in both human and mouse PNETs. The functions of these miRNAs are poorly understood. miR-431 is the most up-regulated miRNA in the metastatic signature. However, it is unknown whether miR-431 contributes to metastasis of PNETs. Herein, we show that miR-431 overexpression activates Ras/extracellular signal-regulated kinase (Erk) signaling and promotes epithelial-mesenchymal transition, migration/invasion in vitro, and metastasis in both xenograft and spontaneous mouse models of PNET. Treatment of PNET cells with Erk inhibitor or locked nucleic acids sequestering miR-431 inhibits invasion. Four target prediction modules and dual-luciferase reporter assays were used to identify potential mRNA targets of miR-431. A Ras GTPase activating protein tumor suppressor (RasGAP), DAB2 interacting protein (DAB2IP), was discovered as an miR-431 target. Overexpression of DAB2IP's rat homolog, but not its mutant defective in Ras GTPase activating protein activity, reverses miR-431's effect on promoting invasion, Erk phosphorylation, and epithelial-mesenchymal transition of PNETs. Taken together, miR-431 silences DAB2IP to active Ras/Erk and promote metastasis of PNETs. miR-431 may be targeted to manage metastatic PNETs. The incidence of pancreatic neuroendocrine tumor (PNET) is increasing, and it presents with various clinical manifestations and an unfavorable survival rate. A better understanding of the drivers of PNET tumorigenesis is urgently needed. Distinct miRNA signatures have been identified for different stages of tumorigenesis in both human and mouse PNETs. The functions of these miRNAs are poorly understood. miR-431 is the most up-regulated miRNA in the metastatic signature. However, it is unknown whether miR-431 contributes to metastasis of PNETs. Herein, we show that miR-431 overexpression activates Ras/extracellular signal-regulated kinase (Erk) signaling and promotes epithelial-mesenchymal transition, migration/invasion in vitro, and metastasis in both xenograft and spontaneous mouse models of PNET. Treatment of PNET cells with Erk inhibitor or locked nucleic acids sequestering miR-431 inhibits invasion. Four target prediction modules and dual-luciferase reporter assays were used to identify potential mRNA targets of miR-431. A Ras GTPase activating protein tumor suppressor (RasGAP), DAB2 interacting protein (DAB2IP), was discovered as an miR-431 target. Overexpression of DAB2IP's rat homolog, but not its mutant defective in Ras GTPase activating protein activity, reverses miR-431's effect on promoting invasion, Erk phosphorylation, and epithelial-mesenchymal transition of PNETs. Taken together, miR-431 silences DAB2IP to active Ras/Erk and promote metastasis of PNETs. miR-431 may be targeted to manage metastatic PNETs. Pancreatic neuroendocrine tumors (PNETs) represent one-third of gastroenteropancreatic neuroendocrine tumors and are the second malignancy of the pancreas. The 5-year survival rate is approximately 55% when the tumors are localized and resected, but only approximately 15% when the tumors are not resectable.1Edge S. Byrd D.R. Compton C.C. Fritz A.G. Greene F.L. Trotti A. Exocrine and endocrine pancreas. AJCC Cancer Staging Manual.ed 7. Springer-Verlag, New York, NY2010: 241-249Google Scholar However, PNETs often display an indolent phenotype, resulting in late diagnosis at advanced stages when surgical resection with curative intent is no longer an alternative.2Zhang J.F.R. Iyer R. Seshadri M. Zajac-Kaye M. Hochwald S.N. Current understanding of the molecular biology of pancreatic neuroendocrine tumors.J Natl Cancer Inst. 2013; 105: 1005-1017Crossref PubMed Scopus (78) Google Scholar,3Buicko J.L. Finnerty B.M. Zhang T. Kim B.J. Fahey 3rd, T.J. Du Y.C. Insights into the biology and treatment strategies of pancreatic neuroendocrine tumors.Ann Pancreat Cancer. 2019; 2: 12Crossref PubMed Scopus (12) Google Scholar Sunitinib (a multitargeted protein tyrosine kinase inhibitor), everolimus (a mammalian target of rapamycin inhibitor), and lutetium Lu 177 dotatate (a radioactive peptide targeted to somatostatin receptor) are approved for the treatment of unresectable and progressive or metastatic PNETs.2Zhang J.F.R. Iyer R. Seshadri M. Zajac-Kaye M. Hochwald S.N. Current understanding of the molecular biology of pancreatic neuroendocrine tumors.J Natl Cancer Inst. 2013; 105: 1005-1017Crossref PubMed Scopus (78) Google Scholar,4Blumenthal G.M.C.P. Zhang J.J. Tang S. Sridhara R. Murgo A. Justice R. Pazdur R. FDA approval summary: sunitinib for the treatment of progressive well-differentiated locally advanced or metastatic pancreatic neuroendocrine tumors.Oncologist. 2012; 17: 1108-1113Crossref PubMed Scopus (93) Google Scholar,5Cives M. Strosberg J. Radionuclide therapy for neuroendocrine tumors.Curr Oncol Rep. 2017; 19: 9Crossref PubMed Scopus (95) Google Scholar All three drugs have their limitations. Both sunitinib and everolimus only extend median survival of PNET patients by approximately 6 months, and all patients developed resistance for both drugs.4Blumenthal G.M.C.P. Zhang J.J. Tang S. Sridhara R. Murgo A. Justice R. Pazdur R. FDA approval summary: sunitinib for the treatment of progressive well-differentiated locally advanced or metastatic pancreatic neuroendocrine tumors.Oncologist. 2012; 17: 1108-1113Crossref PubMed Scopus (93) Google Scholar,6Yao J.C.S.M. Ito T. Bohas C.L. Wolin E.M. Van Cutsem E. Hobday T.J. Okusaka T. Capdevila J. de Vries E.G. Tomassetti P. Pavel M.E. Hoosen S. Haas T. Lincy J. Lebwohl D. Öberg K. Everolimus for advanced pancreatic neuroendocrine tumors.N Engl J Med. 2011; 364: 514-523Crossref PubMed Scopus (2256) Google Scholar Lutetium Lu 177 dotatate can only be used for tumors expressing somatostatin receptors. As the incidences of PNETs are increasing,7Dasari A. Shen C. Halperin D. Zhao B. Zhou S. Xu Y. Shih T. Yao J.C. Trends in the incidence, prevalence, and survival outcomes in patients with neuroendocrine tumors in the United States.JAMA Oncol. 2017; 3: 1335-1342Crossref PubMed Scopus (1649) Google Scholar a better understanding of the mechanisms that underline PNET metastasis is vitally needed to improve therapeutic options. miRNAs are small RNAs that regulate the expression of complementary messenger RNAs and play multiple roles in cellular functions, including cell growth, differentiation, and development.8Bartel D.P. MicroRNAs: genomics, biogenesis, mechanism, and function.Cell. 2004; 116: 281-297Abstract Full Text Full Text PDF PubMed Scopus (29553) Google Scholar Metastasis promoter miRNAs9Ma L. Teruya-Feldstein J. Weinberg R.A. Tumour invasion and metastasis initiated by microRNA-10b in breast cancer.Nature. 2007; 449: 682-688Crossref PubMed Scopus (2226) Google Scholar and suppressor miRNAs10Tavazoie S.F. Alarcon C. Oskarsson T. Padua D. Wang Q. Bos P.D. Gerald W.L. Massague J. Endogenous human microRNAs that suppress breast cancer metastasis.Nature. 2008; 451: 147-152Crossref PubMed Scopus (1627) Google Scholar were first discovered in breast cancer. Subsequent studies revealed more miRNAs with functions in tumorigenesis and metastasis of other cancer types. A cross-species study of miRNAs has identified stage-specific miRNA expression signatures in human PNETs and the RIP1-Tag2 (RIP-Tag) transgenic mouse model of PNETs.11Olson P. Lu J. Zhang H. Shai A. Chun M.G. Wang Y. Libutti S.K. Nakakura E.K. Golub T.R. Hanahan D. MicroRNA dynamics in the stages of tumorigenesis correlate with hallmark capabilities of cancer.Genes Dev. 2009; 23: 2152-2165Crossref PubMed Scopus (241) Google Scholar Another comprehensive cross-species analysis of mRNA and miRNA transcriptomes of PNETs from the mouse model and human patients supports for the RIP-Tag mouse model as representative of human PNETs.12Sadanandam A. Wullschleger S. Lyssiotis C.A. Grotzinger C. Barbi S. Bersani S. Korner J. Wafy I. Mafficini A. Lawlor R.T. Simbolo M. Asara J.M. Blaker H. Cantley L.C. Wiedenmann B. Scarpa A. Hanahan D. A cross-species analysis in pancreatic neuroendocrine tumors reveals molecular subtypes with distinctive clinical, metastatic, developmental, and metabolic characteristics.Cancer Discov. 2015; 5: 1296-1313Crossref PubMed Scopus (115) Google Scholar Among those miRNAs expressed in different stages of PNET tumorigenesis, miR-431 is one of the up-regulated miRNAs in the metastasis-specific miRNA signature.11Olson P. Lu J. Zhang H. Shai A. Chun M.G. Wang Y. Libutti S.K. Nakakura E.K. Golub T.R. Hanahan D. MicroRNA dynamics in the stages of tumorigenesis correlate with hallmark capabilities of cancer.Genes Dev. 2009; 23: 2152-2165Crossref PubMed Scopus (241) Google Scholar However, the role of miR-431 during PNET tumorigenesis is unknown. In this study, we aim to determine whether miR-431 contributes to PNET metastasis. RCASBP-Y-DEST has been described.13Loftus S.K. Larson D.M. Watkins-Chow D. Church D.M. Pavan W.J. Generation of RCAS vectors useful for functional genomic analyses.DNA Res. 2001; 8: 221-226Crossref PubMed Scopus (48) Google Scholar After KpnI site was added before pCMV and the first BamHI site was destroyed in pcDNA6.2 GW-EmGFP-miR-lacZ vector,14Huse J.T. Brennan C. Hambardzumyan D. Wee B. Pena J. Rouhanifard S.H. Sohn-Lee C. le Sage C. Agami R. Tuschl T. Holland E.C. The PTEN-regulating microRNA miR-26a is amplified in high-grade glioma and facilitates gliomagenesis in vivo.Genes Dev. 2009; 23: 1327-1337Crossref PubMed Scopus (438) Google Scholar the pCMV-GFP-miR-lacZ part was subcloned from the modified pcDNA6.2 GW-EmGFP-miR-lacZ vector into pENTR3C using KpnI and XhoI sites. PCR-generated miR-431 was cloned into pENTR3C-GFP-lacZ using BamHI and BglII sites. pENTR3C-pCMV-GFP-miR-LacZ and pENTR3C-pCMV-GFP-miR-431were recombined with RCASBP-Y-DEST using LR Clonase (Invitrogen, Carlsbad, CA) to generate RCASBP-GFP-miR-LacZ and RCASBP-GFP-miR-431. Inserted regions in the new plasmids were confirmed by DNA sequencing. Chicken fibroblast DF115Himly M. Foster D.N. Bottoli I. Iacovoni J.S. Vogt P.K. The DF-1 chicken fibroblast cell line: transformation induced by diverse oncogenes and cell death resulting from infection by avian leukosis viruses.Virology. 1998; 248: 295-304Crossref PubMed Scopus (338) Google Scholar,16Schaefer-Klein J. Givol I. Barsov E.V. Whitcomb J.M. VanBrocklin M. Foster D.N. Federspiel M.J. Hughes S.H. The EV-O-derived cell line DF-1 supports the efficient replication of avian leukosis-sarcoma viruses and vectors.Virology. 1998; 248: 305-311Crossref PubMed Scopus (166) Google Scholar and N134 cell lines17Du Y.C. Lewis B.C. Hanahan D. Varmus H. Assessing tumor progression factors by somatic gene transfer into a mouse model: Bcl-xL promotes islet tumor cell invasion.PLoS Biol. 2007; 5: e276Crossref PubMed Scopus (63) Google Scholar have been described. N134/GFP-miR-LacZ and N134/GFP-miR-431 were generated following previously described procedure.18Zhang G. Chi Y. Du Y.C. Identification and characterization of metastatic factors by gene transfer into the novel RIP-Tag; RIP-tva Murine model.J Vis Exp. 2017; 128: 55890Google Scholar QGP1 cell line was kindly provided by Chris Harris (Rutgers University Cancer Institute of New Jersey, New Brunswick, NJ).19Tang L.H. Contractor T. Clausen R. Klimstra D.S. Du Y.C. Allen P.J. Brennan M.F. Levine A.J. Harris C.R. Attenuation of the retinoblastoma pathway in pancreatic neuroendocrine tumors due to increased cdk4/cdk6.Clin Cancer Res. 2012; 18: 4612-4620Crossref PubMed Scopus (82) Google Scholar,20Evers B.M. Townsend Jr., C.M. Upp J.R. Allen E. Hurlbut S.C. Kim S.W. Rajaraman S. Singh P. Reubi J.C. Thompson J.C. Establishment and characterization of a human carcinoid in nude mice and effect of various agents on tumor growth.Gastroenterology. 1991; 101: 303-311Abstract Full Text PDF PubMed Scopus (180) Google Scholar QGP1 cells were infected with viruses carrying thymidine kinase/green fluorescent protein (GFP)/luciferase fusion reporter,21Ponomarev V. Doubrovin M. Serganova I. Vider J. Shavrin A. Beresten T. Ivanova A. Ageyeva L. Tourkova V. Balatoni J. Bornmann W. Blasberg R. Gelovani Tjuvajev J. A novel triple-modality reporter gene for whole-body fluorescent, bioluminescent, and nuclear noninvasive imaging.Eur J Nucl Med Mol Imaging. 2004; 31: 740-751Crossref PubMed Scopus (232) Google Scholar which was kindly provided by Drs. Inna Serganova and Ronald Blasberg (Memorial Sloan Kettering Cancer Center, New York, NY). GFP+ N134/GFP-miR-LacZ, N134/GFP-miR-431, and QGP1/TGL cells were sorted using FACS-DiVa Cell Sorter (BD Biosciences, San Jose, CA) in the Weill Cornell Medicine (WCM) Flow Cytometry Core. Rat DAB2IP expression construct, GTPase activating protein (GAP) mutant of rat DAB2IPexpression construct, and the control vector (pCI-neo) were gifts from Dr. Jer-Tsong Hsieh (University of Texas Southwestern Medical Center, Dallas, TX)22Wang Z. Tseng C.P. Pong R.C. Chen H. McConnell J.D. Navone N. Hsieh J.T. The mechanism of growth-inhibitory effect of DOC-2/DAB2 in prostate cancer: characterization of a novel GTPase-activating protein associated with N-terminal domain of DOC-2/DAB2.J Biol Chem. 2002; 277: 12622-12631Crossref PubMed Scopus (127) Google Scholar and they were transiently transfected into N134/GFP-miR-431 cells by Lipofectamine 3000 (Invitrogen) following the instructions of the manufacturer. Forty-eight hours after transfection, cells were either used for the invasion assay described below or lysed in radioimmunoprecipitation assay buffer (50 mmol/L Tris, pH 7.4, 150 mmol/L NaCl, 1% NP-40, and 0.5% sodium deoxycholate) supplemented with a protease inhibitor mixture and PhosSTOP (Roche, Pleasanton, CA) for Western blot analysis. DF1 and N134 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 0.2 mmol/L l-glutamine, and 1% penicillin/streptomycin. QGP1 cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 0.2 mmol/L l-glutamine, and 1% penicillin/streptomycin. DNA synthesis was measured by Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Invitrogen), according to the manufacturer's protocol. Two miRCURY LNA Power miRNA inhibitors (Qiagen/Exiqon, Germantown, MD) were number 4103450102 for has-miR-431-5p (sequence: 5'-GCATGACGGCCTGCAAGAC-3') and negative control A, number 199006102 (sequence: 5'-TAACACGTCTATACGCCCA-3'). For miRNA analyses, total RNA was extracted from various cell lines using microRNeasy mini kit (Qiagen). miRNAs were reverse transcribed by TaqMan miRNA reverse transcription kit (number 4366596; Applied Biosystems, Foster City, CA) using 10 ng of total RNA, and the expression levels of mature miRNAs were quantified using the TaqMan miRNA expression assay (number 4427975; Applied Biosystems). Mouse snoRNA202 and human SNORD48 (previously known as RNU48) were used as endogenous controls for normalization. For mRNA expression analyses, mRNA was isolated from cells grown on 6- or 10-cm plates using Trizol with DNase I treatment (Invitrogen) or RNeasy mini kit (Qiagen) containing genomic DNA Eliminator spin columns. Total RNA (5 μg) was reverse transcribed using cDNA First-Strand Synthesis Kit (Invitrogen), and 20 ng of the resulting cDNA was then mixed with SYBR Green PCR Master Mix (Applied Biosystems) and the appropriate primers. Each reaction was performed in quadruplicate, and mRNA expression was quantified by performing real-time PCR amplification using an ABI Prism 7900HT Real-Time PCR System (Applied Biosystems). The primer sequences are as follows: CDH1 (mouse, forward: 5′-CAGGTCTCCTCATGGCTTTGC-3′; reverse: 5′-CTTCCGAAAAGAAGGCTGTCC-3′), CDH2 (mouse, forward: 5′-CATCAACCGGCTTAATGGTG-3′; reverse: 5′-ACTTTCACACGCAGGATGGA-3′), ZEB2 (mouse, forward: 5′-ATGGCAACACATGGGTTTAGTGGC-3′; reverse: 5′-ATTGGACTCTGAGCAGATGGGTGT-3′), SNAI2 (mouse, forward: 5′-CACATTCGAACCCACACATTGCCT-3′; reverse: 5′-TGTGCCCTCAGGTTTGATCTGTCT-3′), ACTB (mouse, forward: 5′-ATAGGAGTCCTTCTGACCCATTCC-3′; reverse: 5′-ATGACGATATCGCTGCGCTGGT-3′), and B2M (mouse, forward: 5′-ATGCTGAAGAACGGGAAAAA-3′; reverse: 5′-CAGTCTCAGTGGGGGTGAAT-3′) with the comparative CT method (ΔΔCT). Both ACTB and B2M were used as endogenous controls for normalization. Generation of RIP-Tag; RIP-tva mice has been described.17Du Y.C. Lewis B.C. Hanahan D. Varmus H. Assessing tumor progression factors by somatic gene transfer into a mouse model: Bcl-xL promotes islet tumor cell invasion.PLoS Biol. 2007; 5: e276Crossref PubMed Scopus (63) Google Scholar Nonobese diabetic/scid-IL2Rgc knockout mice were generated by the Jackson Laboratory (Bar Harbor, ME). All mice were housed in accordance with institutional guidelines. All procedures involving mice were approved by the institutional animal care and use committee. For the experimental metastasis assay, 1 × 106 N134 cells in 150 μL phosphate-buffered saline were injected into the tail veins of male nonobese diabetic/scid-IL2Rgc knockout mice at the age of 7 to 8 weeks. For in vivo infection, viral supernatant was passed through 0.45-μm filters and was concentrated by high-speed ultracentrifugation at 95,400 × g for 1.5 hours before intracardiac injection into RIP-Tag; RIP-tva mice at 7 weeks of age. Mice were sacrificed when they were 16 weeks of age or sick. Intracardiac injection of concentrated RCAS viruses to RIP-Tag; RIP-tva mice and viral titer determination by end-point dilution of DF1 producer cells were performed as described.17Du Y.C. Lewis B.C. Hanahan D. Varmus H. Assessing tumor progression factors by somatic gene transfer into a mouse model: Bcl-xL promotes islet tumor cell invasion.PLoS Biol. 2007; 5: e276Crossref PubMed Scopus (63) Google Scholar,18Zhang G. Chi Y. Du Y.C. Identification and characterization of metastatic factors by gene transfer into the novel RIP-Tag; RIP-tva Murine model.J Vis Exp. 2017; 128: 55890Google Scholar Tissues were removed and fixed in 10% buffered formalin overnight at room temperature. Formalin-fixed, paraffin-embedded sections (5 μm thick) were deparaffinized and rehydrated by passage through a graded xylene/ethanol series before staining. Hematoxylin and eosin staining was performed. Immunochemistry was performed using VECTASTAIN Elite ABC kit, following the manufacturer's instructions. The primary antibody used was rabbit antisynaptophysin (1:500; VP-S284; Vector Labs, Burlingame, CA). Cells at 80% confluency from 4- to 10-cm plates were combined and lysed in 1 mL lysis buffer (25 mmol/L Tris-HCl, pH 7.2, 150 mmol/L NaCl, 5 mmol/L MgCl2, 1% NP-40, and 5% glycerol). Of total lysate of each cell line, 6% was saved before the pull-down assay. Protein of each cell line (500 μg) was processed with the Active Ras Pull-Down and Detection Kit (number 16117; Thermo Scientific, Waltham, MA). Activated Ras proteins were precipitated with a GST-fusion protein of the Ras-binding domain of Raf1 along with glutathione agarose resin, following the manufacturer's instruction. Precipitated proteins were eluted from beads using 2 × loading buffer (12 mmol/L Tris, pH 6.8, 5% glycerol, 0.4% SDS, 140 mmol/L 2-mercaptoethanol, and 0.02% bromophenol blue), separated by SDS-PAGE, and analyzed by Western blot analysis with anti-Ras antibody provided in the kit. The intensities of bands are quantified by Fiji software version 2.0.0-rc-41/1.40d (NIH, Bethesda, MD). The relative ratios were normalized to bands from the first lane. Cells were lysed in NP-40 buffer (100 mmol/L NaCl, 100 mmol/L Tris, pH 8.2, and 0.5% NP-40) supplemented with a protease inhibitor mixture and PhosSTOP (Roche). Proteins were quantified by Bradford assay (Bio-Rad, Hercules, CA). Equal amounts of proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. To visualize equal protein loading, blots were stained with Ponceau S. Blots were incubated in 5% nonfat milk in Tris-buffered saline and Tween 20, probed with primary antibodies to phosphorylated extracellular signal-regulated kinase 1/2 (p-Erk1/2; 1:1000; 4376; Cell Signaling Technology, Danvers, MA), total Erk1/2 (1:1000; 9102; Cell Signaling Technology), cadherin 1 (1:1000; 610181; BD Biosciences), α-tubulin (1:1000; T5168; Sigma, St. Louis, MO), Slug (1:1000; ab27568; Abcam, Cambridge, MA), and DAB2 interacting protein (DAB2IP; 1:800; gift from Dr. Jer-Tsong Hsieh), and then incubated with horseradish peroxidase–conjugated secondary antibodies. Protein bands were visualized by enhanced chemical luminescence (Pierce, Rockford, IL). For migration assays, 0.5 × 106 or 1 × 106 of cells were seeded in the upper chambers of 8-μm porous polycarbonate membranes with DMEM containing 2% FBS, 0.2 mmol/L l-glutamine, and 1% penicillin/streptomycin. The lower chambers were filled with DMEM containing 10% FBS, 0.2 mmol/L l-glutamine, and 1% penicillin/streptomycin. After 72 hours, cells migrating to the bottom chambers were fixed, stained with 0.1% crystal violet for 30 minutes, and counted in eight fields under ×10 magnification. For invasion assays, 2.5 to 5 × 104 cells were seeded in the Matrigel-coated transwell chambers (BD Biosciences) with DMEM containing 1% FBS, 0.2 mmol/L l-glutamine, and 1% penicillin/streptomycin. The lower chambers were filled with DMEM containing 10% FBS, 0.2 mmol/L l-glutamine, and 1% penicillin/streptomycin. After 48 hours, cells on the opposite side of the chambers were fixed with 4% paraformaldehyde for 20 minutes, stained with 0.1% crystal violet for 30 minutes, and counted in eight fields under ×20 magnification. For SCH772984 (Selleck Chemicals LLC, Houston, TX) treatment, N134 cells were pretreated with dimethyl sulfoxide (vehicle control) or 1 μmol/L SCH772984 for 7 days. A total of 2.5 × 104 cells were seeded in Matrigel-coated transwell chambers with either dimethyl sulfoxide or 1 μmol/L SCH772984 in both the upper and lower chambers. For invasion assays of transiently transfected cells, 106 cells were seeded in the Matrigel-coated transwell chambers (BD Biosciences) with DMEM containing 1% FBS, 0.2 mmol/L l-glutamine, and 1% penicillin/streptomycin. The lower chambers were filled with DMEM containing 10% FBS, 0.2 mmol/L l-glutamine, and 1% penicillin/streptomycin. After 72 hours of incubation, cells on the opposite side of the chambers were fixed by 4% paraformaldehyde for 20 minutes, stained with 0.1% crystal violet for 30 minutes, and counted in all the fields under ×20 magnification. Experiment was conducted in triplicates. To analyze cell proliferation, 0.5 × 106 cells were seeded directly onto 24-well plates in triplicate. After 72 hours, cells were fixed by 4% paraformaldehyde for 20 minutes, stained with 0.1% crystal violet for 30 minutes, and lysed by 500 μL methanol with gentle shaking at the speed of 200 rpm for 20 minutes. Cell lysate from each well (200 μL) was transferred to a 96-well plate, and OD560 was read. For QGP1 cell invasion assay, a control inhibitor (LNA-CTRL) or miR-431 inhibitor (LNA-miR-431) was transfected to QGP1 cells. After 4 days, 7.5 × 104 cells were seeded in Matrigel-coated transwell chambers. Forty-eight hours later, cells were counted as described.23Choi S. Chen Z. Tang L.H. Fang Y. Shin S.J. Panarelli N.C. Chen Y.T. Li Y. Jiang X. Du Y.C. Bcl-xL promotes metastasis independent of its anti-apoptotic activity.Nat Commun. 2016; 7: 10384Crossref PubMed Scopus (54) Google Scholar Total RNA was extracted using the RNeasy Plus mini kit (74134; Qiagen) and QIAshredder kit (79654; Qiagen), according to the manufacturer's protocol. After isolation, the total RNA integrity was checked using an Agilent Technologies (Santa Clara, CA) 2100 Bioanalyzer. cDNA libraries were generated using the Illumina TruSeq RNA Sample Preparation kit and sequenced with pair-end 51 bp on HiSeq4000 sequencer (Illumina, San Diego, CA). Tophat version 2.0.1124Kim D. Pertea G. Trapnell C. Pimentel H. Kelley R. Salzberg S.L. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions.Genome Biol. 2013; 14: R36Crossref PubMed Scopus (8791) Google Scholar was used to align sequencing reads to the mm9 mouse reference genome, and Cufflinks version 2.1.125Trapnell C. Williams B.A. Pertea G. Mortazavi A. Kwan G. van Baren M.J. Salzberg S.L. Wold B.J. Pachter L. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation.Nat Biotechnol. 2010; 28: 511-515Crossref PubMed Scopus (10631) Google Scholar,26Trapnell C. Hendrickson D.G. Sauvageau M. Goff L. Rinn J.L. Pachter L. Differential analysis of gene regulation at transcript resolution with RNA-seq.Nat Biotechnol. 2013; 31: 46-53Crossref PubMed Scopus (2429) Google Scholar was used to measure transcript abundances in fragments per kilobase of exon model per million mapped reads and to identify differentially expressed genes. The RNA-sequencing (RNA-Seq) data sets of this study are available in Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo; accession number GSE139119). Gene set enrichment analysis was used to determine whether an a priori defined set of genes shows statistically significant, concordant differences between N134 cells expressing GFP-miR-431 and N134 cells expressing GFP-miR-LacZ (control). Reverse phase protein array (RPPA) was performed as described previously with minor modifications.27Chang C.H. Zhang M. Rajapakshe K. Coarfa C. Edwards D. Huang S. Rosen J.M. Mammary stem cells and tumor-initiating cells are more resistant to apoptosis and exhibit increased DNA repair activity in response to DNA damage.Stem Cell Reports. 2015; 5: 378-391Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 28Creighton C.J. Huang S. Reverse phase protein arrays in signaling pathways: a data integration perspective.Drug Des Devel Ther. 2015; 9: 3519-3527PubMed Google Scholar, 29Holdman X.B. Welte T. Rajapakshe K. Pond A. Coarfa C. Mo Q. Huang S. Hilsenbeck S.G. Edwards D.P. Zhang X. Rosen J.M. Upregulation of EGFR signaling is correlated with tumor stroma remodeling and tumor recurrence in FGFR1-driven breast cancer.Breast Cancer Res. 2015; 17: 141Crossref PubMed Scopus (51) Google Scholar, 30Welte T. Kim I.S. Tian L. Gao X. Wang H. Li J. Holdman X.B. Herschkowitz J.I. Pond A. Xie G. Kurley S. Nguyen T. Liao L. Dobrolecki L.E. Pang L. Mo Q. Edwards D.P. Huang S. Xin L. Xu J. Li Y. Lewis M.T. Wang T. Westbrook T.F. Rosen J.M. Zhang X.H. Oncogenic mTOR signalling recruits myeloid-derived suppressor cells to promote tumour initiation.Nat Cell Biol. 2016; 18: 632-644Crossref PubMed Scopus (134) Google Scholar Protein lysates were prepared from cultured cells with modified Tissue Protein Extraction Reagent (Life Technologies, Carlsbad, CA) and a cocktail of protease and phosphatase inhibitors (Roche). The lysates were diluted into 0.5 mg/mL in SDS sample buffer and denatured on the same day. The Aushon 2470 Arrayer (Aushon BioSystems, Billerica, MA) with a 40-pin (185-μm) configuration was used to spot samples and control lysates onto nitrocellulose-coated slides (Grace Bio-Labs, Bend, OR) using an array format of 960 lysates/slide (2880 spots/slide). The slides were processed as described and probed with a set of 224 antibodies against total proteins and phosphoproteins using an automated slide stainer Autolink 48 (Dako, Santa Clara, CA). Each slide was incubated with one specific primary antibody, and a negative control slide was incubated with antibody diluent without any primary antibody. Primary antibody binding was detected using a biotinylated secondary antibody followed by streptavidin-conjugated IRDye680 fluorophore (LI-COR Biosciences, Lincoln, NE). Total protein content of each spotted lysate was assessed by fluorescent staining with Sypro Ruby Protein Blot Stain, according to the manufacturer's instructions (Molecular Probes, Eugene, OR). Fluorescence-labeled slides were scanned on a GenePix 4400 AL scanner, along with accompanying negative control slides, at an appropriate photomultiplier tubes voltage setting to obtain optimal signal for this specific set of samples. The images were analyzed with GenePix Pro 7.2 (Molecular Devices, San Jose, CA). Total fluorescence signal intensities of each spot were obtained after subtraction of the local background signal for each slide and were then normalized for variation in total protein, background, and non-specific labeling using a group-based normalization method, as described.27Chang C.H. Zhang M. Rajapakshe K. Coarfa C. Edwards D. Huang S. Rosen J.M. Mammary stem cells and tumor-initiating cells are more resistant to apoptosis and exhibit increased DNA repair activity in response to DNA damage.Stem Cell Reports. 2015; 5: 378-391Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar For each spot on the array, the background-subtracted foreground signal intensity was subtracted by the corresponding signal intensity of the negative control slide (omission of primary antibody) and then normalized to the corresponding signal intensity of total protein for that spot. Each image, along with its normalized data, was evaluated for quality through manual inspection and control samples. Antibody slides that failed the quality inspection were either repeated at the end of the staining runs or removed before data reporting. Total 211 antibodies remained in the list. The 3′ untranslated region (UTR) of mouse DAB2IP was amplified by PCR and cloned into a pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega, Madison, WI). HEK 293T cells were cotransfected with miRNA mimic (Invitrogen) and pmirGLO Dual-Luciferase 3′ UTR vector using the Lipofectamine 2000 Transfecti