Regulation of Epidermal Growth Factor Receptor Signaling in Human Cancer Cells by MicroRNA-7
2008; Elsevier BV; Volume: 284; Issue: 9 Linguagem: Inglês
10.1074/jbc.m804280200
ISSN1083-351X
AutoresRebecca J. Webster, Keith M. Giles, Karina J. Price, Priscilla M. Zhang, John S. Mattick, Peter J. Leedman,
Tópico(s)Circular RNAs in diseases
ResumoThe epidermal growth factor receptor (EGFR) is frequently overexpressed in cancer and is an important therapeutic target. Aberrant expression and function of microRNAs have been associated with tumorigenesis. Bioinformatic predictions suggest that the human EGFR mRNA 3′-untranslated region contains three microRNA-7 (miR-7) target sites, which are not conserved across mammals. We found that miR-7 down-regulates EGFR mRNA and protein expression in cancer cell lines (lung, breast, and glioblastoma) via two of the three sites, inducing cell cycle arrest and cell death. Because miR-7 was shown to decrease EGFR mRNA expression, we used microarray analysis to identify additional mRNA targets of miR-7. These included Raf1 and multiple other genes involved in EGFR signaling and tumorigenesis. Furthermore, miR-7 attenuated activation of protein kinase B (Akt) and extracellular signal-regulated kinase 1/2, two critical effectors of EGFR signaling, in different cancer cell lines. These data establish an important role for miR-7 in controlling mRNA expression and indicate that miR-7 has the ability to coordinately regulate EGFR signaling in multiple human cancer cell types. The epidermal growth factor receptor (EGFR) is frequently overexpressed in cancer and is an important therapeutic target. Aberrant expression and function of microRNAs have been associated with tumorigenesis. Bioinformatic predictions suggest that the human EGFR mRNA 3′-untranslated region contains three microRNA-7 (miR-7) target sites, which are not conserved across mammals. We found that miR-7 down-regulates EGFR mRNA and protein expression in cancer cell lines (lung, breast, and glioblastoma) via two of the three sites, inducing cell cycle arrest and cell death. Because miR-7 was shown to decrease EGFR mRNA expression, we used microarray analysis to identify additional mRNA targets of miR-7. These included Raf1 and multiple other genes involved in EGFR signaling and tumorigenesis. Furthermore, miR-7 attenuated activation of protein kinase B (Akt) and extracellular signal-regulated kinase 1/2, two critical effectors of EGFR signaling, in different cancer cell lines. These data establish an important role for miR-7 in controlling mRNA expression and indicate that miR-7 has the ability to coordinately regulate EGFR signaling in multiple human cancer cell types. The epidermal growth factor receptor (EGFR), 4The abbreviations used are: EGFR, epidermal growth factor receptor; KEGG, Kyoto Encyclopedia of Genes and Genomes; miRNA, microRNA; NSCLC, non-small cell lung cancer; RT, reverse transcription; qRT-PCR, quantitative RT-PCR; UTR, untranslated region; nt, nucleotide(s); WT, wild type; MT, mutant; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activate protein kinase; MEK, MAPK/ERK kinase; IRS2, insulin receptor substrate 2; PI3K, phosphatidylinositol 3-kinase; ARF4, ADP-ribosylation factor 4; PAK1, p21/Cdc42/Rac1-activated kinase 1; PBS, phosphate-buffered saline.4The abbreviations used are: EGFR, epidermal growth factor receptor; KEGG, Kyoto Encyclopedia of Genes and Genomes; miRNA, microRNA; NSCLC, non-small cell lung cancer; RT, reverse transcription; qRT-PCR, quantitative RT-PCR; UTR, untranslated region; nt, nucleotide(s); WT, wild type; MT, mutant; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activate protein kinase; MEK, MAPK/ERK kinase; IRS2, insulin receptor substrate 2; PI3K, phosphatidylinositol 3-kinase; ARF4, ADP-ribosylation factor 4; PAK1, p21/Cdc42/Rac1-activated kinase 1; PBS, phosphate-buffered saline. a member of the erbB receptor family, is widely expressed in human tissues and regulates important cellular processes, including proliferation, differentiation, and development (1Yano S. Kondo K. Yamaguchi M. Richmond G. Hutchison M. Wakeling A. Averbuch S. Wadsworth P. Anticancer Res. 2003; 23: 3639-3650PubMed Google Scholar). EGFR overexpression occurs in a range of solid tumors and is associated with disease progression, resistance to chemotherapy and radiation therapy, and poor prognosis (2Arteaga C.L. J. Clin. Oncol. 2001; 19: 32S-40SPubMed Google Scholar). Consequently, the EGFR and its downstream signaling effectors are major targets for new therapeutics such as monoclonal antibodies and tyrosine kinase inhibitors (3Ritter C.A. Arteaga C.L. Semin. Oncol. 2003; 30: 3-11Crossref PubMed Scopus (164) Google Scholar). However, the clinical responses of tumors to existing anti-EGFR agents are often limited, and thus a major research focus is the development of novel approaches to block EGFR expression and signaling (4Bianco R. Troiani T. Tortora G. Ciardiello F. Endocr. Relat. Cancer. 2005; 12: S159-S171Crossref PubMed Scopus (85) Google Scholar). MicroRNAs (miRNAs) are short, endogenous, non-coding RNA molecules that bind with imperfect complementarity to the 3′-untranslated regions (3′-UTRs) of target mRNAs, causing translational repression of the target gene or degradation of the target mRNA (5Bartel D.P. Cell. 2004; 116: 281-297Abstract Full Text Full Text PDF PubMed Scopus (29225) Google Scholar, 6Mattick J.S. Makunin I.V. Hum. Mol. Genet. 2005; 14: R121-R132Crossref PubMed Scopus (407) Google Scholar, 7Humphreys D.T. Westman B.J. Martin D.I. Preiss T. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 16961-16966Crossref PubMed Scopus (486) Google Scholar). miRNAs are involved in a range of processes that includes development, differentiation (8Chen J.F. Mandel E.M. Thomson J.M. Wu Q. Callis T.E. Hammond S.M. Conlon F.L. Wang D.Z. Nat. Genet. 2006; 38: 228-233Crossref PubMed Scopus (2204) Google Scholar), proliferation, and apoptosis (9Cheng A.M. Byrom M.W. Shelton J. Ford L.P. 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For instance, reduced expression of the let-7 family of miRNAs is associated with increased Ras oncogene expression and reduced survival in patients with non-small cell lung cancer (NSCLC) (13Johnson S.M. Grosshans H. Shingara J. Byrom M. Jarvis R. Cheng A. Labourier E. Reinert K.L. Brown D. Slack F.J. Cell. 2005; 120: 635-647Abstract Full Text Full Text PDF PubMed Scopus (3096) Google Scholar, 14Takamizawa J. Konishi H. Yanagisawa K. Tomida S. Osada H. Endoh H. Harano T. Yatabe Y. Nagino M. Nimura Y. Mitsudomi T. Takahashi T. Cancer Res. 2004; 64: 3753-3756Crossref PubMed Scopus (2140) Google Scholar). In contrast, increased expression of miR-21 in gliomas (15Chan J.A. Krichevsky A.M. Kosik K.S. Cancer Res. 2005; 65: 6029-6033Crossref PubMed Scopus (2213) Google Scholar), and breast, colon, lung, pancreas, prostate, and stomach cancers (16Volinia S. Calin G.A. Liu C.G. Ambs S. Cimmino A. Petrocca F. Visone R. Iorio M. Roldo C. Ferracin M. Prueitt R.L. Yanaihara N. Lanza G. Scarpa A. Vecchione A. Negrini M. Harris C.C. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 2257-2261Crossref PubMed Scopus (4916) Google Scholar) is associated with apoptosis resistance, reduced chemosensitivity, and increased tumor growth (17Si M.L. Zhu S. Wu H. Lu Z. Wu F. Mo Y.Y. Oncogene. 2006; 26: 2799-2803Crossref PubMed Scopus (1362) Google Scholar). Computational approaches to miRNA target prediction have used criteria such as sequence complementarity between target mRNAs and a “seed” region within the miRNA, and conservation of predicted miRNA-binding sites across 3′-UTRs from multiple species (reviewed in Refs. 18Rajewsky N. Nat. Genet. 2006; 38: S8-S13Crossref PubMed Scopus (935) Google Scholar, 19Maziére P. Enright A.J. Drug Discov. Today. 2007; 12: 452-458Crossref PubMed Scopus (236) Google Scholar). Recently, additional features that determine target site functionality have been identified (20Grimson A. Farh K.K. Johnston W.K. Garrett-Engele P. Lim L.P. Bartel D.P. Mol. Cell. 2007; 27: 91-105Abstract Full Text Full Text PDF PubMed Scopus (3011) Google Scholar). Nevertheless, the imperfect complementarity of miRNA and target sequences means that identification and functional validation of authentic miRNA targets remains a major challenge. It has been suggested that miRNAs may have the ability to regulate hundreds or even thousands of target mRNAs (21Lewis B.P. Burge C.B. Bartel D.P. Cell. 2005; 120: 15-20Abstract Full Text Full Text PDF PubMed Scopus (9777) Google Scholar) and that much of this regulation could occur at the level of mRNA decay (22Krützfeldt J. Rajewsky N. Braich R. Rajeev K.G. Tuschl T. Manoharan M. Stoffel M. Nature. 2005; 438: 685-689Crossref PubMed Scopus (3338) Google Scholar). Because specific miRNAs have the potential to regulate the expression of several members of a signaling pathway or cellular process (23Stark A. Brennecke J. Russell R.B. Cohen S.M. PLoS Biol. 2003; 1: E60Crossref PubMed Scopus (623) Google Scholar), we hypothesized a role for miRNAs in aberrant EGFR expression and signaling in human cancers. In this study, we identify EGFR and Raf1 as direct targets of miR-7 in cancer cells, with miR-7 blocking EGFR and Raf1 expression by inducing mRNA decay. Furthermore, we demonstrate the potential for miR-7 to act as a tumor suppressor by coordinately regulating the EGFR signaling pathway at multiple levels to repress protein kinase B (Akt) and extracellular signal-regulated kinase 1/2 (ERK1/2) activity in human cancer cell lines. Cell Culture, miRNA Precursors, and Normal Tissue RNA—A549, MDA-MB-468, U87, DU145, and U373 cell lines were obtained from the American Type Culture Collection (ATCC) and cultured at 37 °C in 5% CO2 with Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Synthetic miRNA precursor molecules corresponding to human miR-7 (Pre-miR miRNA Precursor Product ID: PM10047) and a negative control miRNA (miR-NC; Pre-miR miRNA Precursor Negative Control #1, Product ID: AM17110) were obtained from Ambion. Total RNA (First-Choice) from normal brain, normal lung, and normal breast tissue was purchased from Ambion. Luciferase Plasmid Construction—pGL3-miR-7-target was generated by ligating annealed DNA oligonucleotides corresponding to the perfect hsa-miR-7 target site (forward: 5′-CAA CAA AAT CAC TAG TCT TCC A-3′ and 5′-TGG AAG ACT AGT GAT TTT GTT G-3′) to unique SpeI and ApaI sites that were inserted 3′ of the luciferase open reading frame of pGL3-control (Promega) firefly luciferase reporter vector (designated pGL3-control-MCS (24Giles K.M. Daly J.M. Beveridge D.J. Thomson A.M. Voon D.C. Furneaux H.M. Jazayeri J.A. Leedman P.J. J. Biol. Chem. 2003; 278: 2937-2946Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar)). Full-length EGFR 3′-UTR reporter plasmid was synthesized by GenScript Corp. using the entire EGFR 3′-UTR (nt 3879-5616 of GenBank™ accession number NM_005228) and the pMIR-REPORT luciferase plasmid backbone (Ambion). Wild-type (WT) EGFR target reporter plasmids pGL3-EGFR-A, -B, and -C were generated by cloning annealed oligonucleotides corresponding to nt 4214-4260, nt 4302-4348, and nt 4585-4631, respectively, of EGFR (GenBank™ accession number NM_005228) mRNA 3′-UTR into SpeI and ApaI sites in pGL3-control-MCS. Plasmid pGL3-EGFR-D contained a PCR-generated EGFR 3′-UTR sequence that spanned the predicted miR-7 target sites B and C. Mutant (MT) reporters were also generated that included three nucleotide substitutions to impair binding of the miR-7 seed sequence to its target. Plasmids pGL3-RAF1-WT and pGL3-RAF1-MT were constructed by cloning annealed DNA oligonucleotides corresponding to nt 2965-3030 of the Raf1 mRNA 3′-UTR (GenBank™ accession number NM_002880), into the SpeI and ApaI sites in pGL3-control-MCS. The sequence of all plasmids was confirmed by sequencing. Transfections and Luciferase Assays—Cells were seeded 24 h prior to transfection in 6-well plates or 10-cm dishes and transfected using Lipofectamine 2000 (Invitrogen) with miRNA precursor molecules at final concentrations ranging from 0.1 to 30 nm. Cells were harvested at 12-24 h (for RNA extraction) or 3 days (for protein extraction). For reporter gene assays, cells were seeded in 24-well plates and co-transfected using Lipofectamine 2000 (Invitrogen) with 100 ng of firefly luciferase reporter DNA and 5 ng of pRL-CMV Renilla luciferase reporter DNA as a transfection control. Lysates were assayed for firefly and Renilla luciferase activities 24 h after transfection using the Dual Luciferase Report Assay System (Promega) and a Fluostar OPTIMA microplate reader (BMG Labtech). Expression values were normalized to Renilla luciferase. RT-PCR—Total RNA was extracted from cell lines with TRIzol reagent (Invitrogen) and RNeasy columns (Qiagen) and treated with DNase I (Promega) to eliminate contaminating genomic DNA. For qRT-PCR analysis of EGFR and Raf1 mRNA expression, 1 μg of total RNA was reverse transcribed to cDNA with random hexamers and Thermoscript (Invitrogen). Real-time PCR for Raf1 and GAPDH was performed using a Corbett 3000 RotorGene instrument (Corbett Research) with Quanti-Tect SYBR Green PCR mixture (Qiagen) with primers from Primer Bank (25Wang X. Seed B. Nucleic Acids Res. 2003; 31: e154Crossref PubMed Scopus (676) Google Scholar) (EGFR-F, 5′-GCG TTC GGC ACG GTG TAT AA-3′; EGFR-R, 5′-GGC TTT CGG AGA TGT TGC TTC-3′; RAF1-F, 5′-GCA CTG TAG CAC CAA AGT ACC-3′; RAF1-R, 5′-CTG GGA CTC CAC TAT CAC CAA TA-3′; GAPDH-F, 5′-ATG GGG AAG GTG AAG GTC G-3′; GAPDH-R, 5′-GGG GTC ATT GAT GGC AAC AAT A-3′). Expression of EGFR and Raf1 mRNA relative to GAPDH mRNA was determined using the 2-ΔΔCT method (26Livak K.J. Schmittgen T.D. Methods. 2001; 25: 402-408Crossref PubMed Scopus (121148) Google Scholar). For analysis of miR-7 expression by qRT-PCR, reverse transcription and PCR were carried out using TaqMan miRNA assay kits (Applied Biosystems) for hsa-miR-7 (Part #4373014) and U44 snRNA (Part #4373384) with a Corbett 3000 RotorGene thermocycler (Corbett Research) according to the manufacturer's instructions. Statistical analyses of qRT-PCR data were performed using GenEx software (MultiD). Western Blotting—Cytoplasmic protein extracts were prepared as described (24Giles K.M. Daly J.M. Beveridge D.J. Thomson A.M. Voon D.C. Furneaux H.M. Jazayeri J.A. Leedman P.J. J. Biol. Chem. 2003; 278: 2937-2946Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar), resolved on NuPAGE 4-12% Bis-Tris gels (Invitrogen), and transferred to polyvinylidene difluoride membranes (Roche Applied Science). Membranes were probed with anti-EGFR mouse monoclonal antibody (1:1,000, Neomarkers, catalog #MS-400-P1), anti-Raf1 mouse monoclonal antibody (1:1,000, Santa Cruz Biotechnology, sc-7267), anti-IRS2 rabbit monoclonal antibody (1:500, Cell Signaling Technology, #4502), anti-Akt rabbit monoclonal antibody (1:1,000, Cell Signaling Technology, #9272), anti-phospho-Akt (Ser-473) rabbit monoclonal antibody (1:500, Cell Signaling Technology, #4060), anti-p44/42 (ERK1/2) MAPK mouse monoclonal antibody (1:750, Cell Signaling Technology, #4696), anti-phospho-p44/42 (P-ERK1/2) MAPK (Thr-202/Tyr-204) rabbit monoclonal antibody (1:750, Cell Signaling Technology, #9101), or anti-β-actin mouse monoclonal antibody (1:15,000, Abcam ab6276-100), prior to detection with ECL Plus detection reagent (GE Healthcare) and ECL-Hyperfilm (GE Healthcare). Cell Cycle Analysis and Cell Counting—Following trypsinization, cells were permeabilized, stained with propidium iodide, and analyzed on an EPICS XL-MCL (Coulter Corp. flow cytometer). Cell cycle analysis was performed using MultiPlus AV MultiParameter data analysis software (Phoenix Flow Systems). Cells were seeded in 6-cm dishes and assessed 3 days after miR-7 or miR-NC transfection by light microscopy. Five representative fields of view were photographed for each condition. Cells in each field of view were counted manually. Microarray Expression Profiling and Analysis—Total RNA was isolated from A549 cells transfected for 24 h with miR-7 or miR-NC (30 nm) using TRIzol reagent (Invitrogen) and RNeasy columns (Qiagen) and assessed for quality and integrity using a 2100 Bioanalyzer (Agilent Technologies). Gene expression profiling by microarray hybridization was performed with two experimental replicates by the Lotterywest State Microarray Facility using Human Genome U133 Plus 2.0 array chips (Affymetrix). The raw data were processed using GeneSifter software (VizX Labs). An “all groups must pass” restriction was imposed, with a threshold quality score of “P” (present) required for inclusion in subsequent analysis. Data were normalized to the all means fluorescence and were log2-transformed. Pairwise analysis of the probe intensities of miR-7- and miR-NC-treated sample data sets was performed using Student's t tests (two-tailed, unpaired), and used to identify transcripts that were significantly up-regulated or down-regulated with miR-7 transfection (p < 0.05) by at least a factor of 2. These represented potential miR-7 target transcripts. Investigation of the enrichment of gene sets for predicted miRNA targets was conducted using the L2L microarray analysis tool (27Newman, J. C., and Weiner, A. M. (2005) Genome Biology http://genomebiology.com/2005/6/9/R81Google Scholar). miRanda (28Enright, A. J., John, B., Gaul, U., Tuschl, T., Sander, C., and Marks, D. S. (2003) Genome Biology http://genomebiology.com/2003/5/1/R1Google Scholar), PicTar (29Krek A. Grün D. Poy M.N. Wolf R. Rosenberg L. Epstein E.J. MacMenamin P. da Piedade I. Gunsalus K.C. Stoffel M Rajewsky N. Nat. Genet. 2005; 37: 495-500Crossref PubMed Scopus (3870) Google Scholar), and TargetScan (21Lewis B.P. Burge C.B. Bartel D.P. Cell. 2005; 120: 15-20Abstract Full Text Full Text PDF PubMed Scopus (9777) Google Scholar) were used for miR-7 target predictions. Analysis of the enrichment of gene sets for functional Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways was performed with GeneSifter software (VizX Labs). Microarray expression data have been deposited in the Gene Expression Omnibus under Accession Number GSE14507. EGFR 3′-UTR Contains Multiple, Specific Target Sequences for miR-7—As EGFR expression is regulated in part via cis-acting 3′-UTR mRNA stability sequences (30Balmer L.A. Beveridge D.J. Jazayeri J.A. Thomson A.M. Walker C.E. Leedman P.J. Mol. Cell. Biol. 2001; 21: 2070-2084Crossref PubMed Scopus (69) Google Scholar), we sought to identify miRNAs that could regulate EGFR gene expression in human cells. TargetScan (21Lewis B.P. Burge C.B. Bartel D.P. Cell. 2005; 120: 15-20Abstract Full Text Full Text PDF PubMed Scopus (9777) Google Scholar) identified three putative miR-7 target sites (A, B, and C, Fig. 1A). The 3′-end of each site contains the hexamer motif UCUUCC, which is complementary to the seed region (nt 2-7) of human miR-7 (hsa-miR-7) (Fig. 1B). When contextual features of miRNA target sites associated with functionality were taken into account, such as their proximity to AU-rich sequences and positioning away from the center of 3′-UTRs (20Grimson A. Farh K.K. Johnston W.K. Garrett-Engele P. Lim L.P. Bartel D.P. Mol. Cell. 2007; 27: 91-105Abstract Full Text Full Text PDF PubMed Scopus (3011) Google Scholar), EGFR sites B and C ranked in the top 6% and 4%, respectively, of all predicted miR-7 target sites in TargetScan. Target site A ranked only in the top 56% of TargetScan predicted sites for miR-7. This suggested that sites B and C were more likely to represent functional targets of miR-7 than site A. The miRanda algorithm (28Enright, A. J., John, B., Gaul, U., Tuschl, T., Sander, C., and Marks, D. S. (2003) Genome Biology http://genomebiology.com/2003/5/1/R1Google Scholar) also predicted EGFR to be a miR-7 target, whereas PicTar (29Krek A. Grün D. Poy M.N. Wolf R. Rosenberg L. Epstein E.J. MacMenamin P. da Piedade I. Gunsalus K.C. Stoffel M Rajewsky N. Nat. Genet. 2005; 37: 495-500Crossref PubMed Scopus (3870) Google Scholar) did not. Although miR-7 is normally expressed in the brain, lens, pituitary, and hypothalamus (31Sempere, L. 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Cell. 2007; 129: 1401-1414Abstract Full Text Full Text PDF PubMed Scopus (2995) Google Scholar), its expression is significantly decreased in pituitary adenomas and in a panel of central nervous system cancer cell lines relative to normal tissue (34Bottoni A. Zatelli M.C. Ferracin M. Tagliati F. Piccin D. Vignali C. Calin G.A. Negrini M. Croce C.M. Degli Ulberti E.C. J. Cell. Physiol. 2007; 210: 370-377Crossref PubMed Scopus (185) Google Scholar, 35Gaur A. Jewell D.A. Liang Y. Ridzon D. Moore J.H. Chen C. Ambros V.R. Israel M.A. Cancer Res. 2007; 67: 2456-2468Crossref PubMed Scopus (597) Google Scholar). This raises the possibility that it may function as a tumor suppressor in these systems by inhibiting oncogene expression. The three putative EGFR 3′-UTR miR-7 target sites are poorly conserved between human, mouse, and rat (Fig. 1B). Binding sites that are not conserved between species are often excluded in an attempt to reduce the number of false-positives in target prediction sets. However, the evolution of miRNAs and their target mRNAs suggests that this exclusion could also increase the rate of false-negative predictions (19Maziére P. Enright A.J. Drug Discov. Today. 2007; 12: 452-458Crossref PubMed Scopus (236) Google Scholar). For instance, in mice, miR-7b regulates Fos oncogene translation via a 3′-UTR target site that is not present in human Fos mRNA (36Lee H.J. Palkovits M. Young W.S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 15669-15674Crossref PubMed Scopus (77) Google Scholar). To investigate the interaction between miR-7 and its predicted EGFR mRNA 3′-UTR target sites, we generated reporter vectors containing sequences with complementarity to miR-7 downstream of a luciferase open reading frame (Fig. 1C). These included a sequence with perfect complementarity to the miR-7 sequence (miR-7 target), the full-length wild-type EGFR 3′-UTR (EGFR 3′-UTR), four wild-type EGFR 3′-UTR sequences (A-D), and the same four EGFR sequences with three point mutations predicted to disrupt miR-7 binding in each of the seed match regions (Fig. 1D). In human EGFR-overexpressing NSCLC cells (A549) transfected with synthetic miR-7 precursor (miR-7), expression of the perfect target reporter was reduced, an effect that was not observed following transfection with a negative control miRNA precursor (miR-NC) (Fig. 1E). Similarly, miR-7, but not miR-NC, reduced expression of a reporter that contained the full-length EGFR 3′-UTR (Fig. 1E). We next investigated the relative contribution of each putative miR-7 target site in the EGFR 3′-UTR to the regulation of gene expression by miR-7. In A549 cells, miR-7 reduced the expression of reporters bearing putative target sites B and C, but not of the corresponding mutant reporters (Fig. 1F). In contrast, putative target site A did not mediate a change in reporter expression by miR-7 (Fig. 1F). This suggested that site A alone was not a target for miR-7 binding. The presence of sites B and C in the same reporter construct (plasmid construct EGFR D, Fig. 1C), conferred additive, although not synergistic, repression with miR-7, which was not observed with the EGFR D mutant reporter (Fig. 1F). Together, these data indicate that the EGFR 3′-UTR is a specific target of miR-7 and that two of the three predicted miR-7-binding sites in the EGFR mRNA 3′-UTR are likely to be specific targets of miR-7. Furthermore, these data suggest that miR-7 may repress EGFR expression via target sites B and C in an additive fashion. miR-7 Inhibits EGFR Expression in Human Cancer Cell Lines and Has Reduced Expression in U87 Glioblastoma Cells—We next sought to determine the effect of miR-7 on EGFR mRNA and protein expression in EGFR-positive A549, U87 (glioblastoma), MDA-MB-468 (breast cancer), and DU145 (prostate cancer) cell lines. Compared with transfection with miR-NC precursor, transfection with miR-7 precursor reduced EGFR mRNA expression significantly in A549, U87, MDA-MB-468, and DU145 cells at 24 h post-transfection by qRT-PCR (Fig. 2A), consistent with miR-7 promoting EGFR mRNA decay. On the other hand, Lee and co-workers (36Lee H.J. Palkovits M. Young W.S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 15669-15674Crossref PubMed Scopus (77) Google Scholar) observed that miR-7 regulated translation of Fos mRNA in the mouse hypothalamus. These contrasting results suggest that miR-7 may regulate the stability and/or translation of target mRNAs. Furthermore, compared with transfection with either transfection reagent alone or miR-NC, transfection with miR-7 for 72 h reduced EGFR protein expression in A549, U87, MDA-MB-468, and DU145 cells, shown by immunoblotting (Fig. 2B). This effect was observed with concentrations of miR-7 precursor as low as 1 nm (data not shown). In addition to investigations of the regulation of EGFR expression by miR-7 in cancer cells, we examined the expression of miR-7 in two EGFR-positive glioblastoma cell lines (U87 and U373) using TaqMan miRNA qRT-PCR assays. We observed that the expression of miR-7 was significantly down-regulated in U87 and U373 cells compared with RNA extracted from total normal human brain tissue (Fig. 2C). This result is consistent with the possibility that reduced miR-7 levels could be associated with elevated expression of EGFR in primary cerebral tumors. We also compared expression of miR-7 in normal brain, normal lung, and normal breast, to its expression in A549, MDA-MB-468, and DU145 cell lines (Fig. 2C and supplemental Fig. 1), and found very low expression of miR-7 in the normal lung and breast compared with normal brain, suggesting that miR-7 expression is restricted to specific tissues that include those belonging to the central nervous system. Supporting this finding, we observed that miR-7 was expressed at low levels in A549, MDA-MB-468, and DU145 cells compared with normal brain. Functional Effects of miR-7 in EGFR-positive Human Cancer Cell Lines—Based on our data showing that miR-7 can regulate EGFR expression in cancer cells, we hypothesized that miR-7 might compromise the growth and viability of the same cells. To test this hypothesis, we transfected A549 cells with miR-7 or miR-NC precursors and measured cell cycle progression using propidium iodide staining and flow cytometry, and cell numbers using cell counts at 3 days post-transfection. Transfection with miR-7 induced cell cycle arrest at G1 (Fig. 3A) and caused a significant decrease in A549 cell numbers and cell viability (Fig. 3B). The reduction in viable cell number with miR-7 transfection did not appear to involve apoptosis, as evidenced by, firstly, the absence of a sub-G1 cell population by propidium iodide staining and flow cytometry (Fig. 3A) and, secondly, the lack of activation of the executioner caspases 3 and 7 (data not shown). Thus, our results suggest that miR-7 not only inhibits EGFR gene expression by targeting its mRNA for degradation but induces a program of gene expression to reduce cell viability and trigger non-apoptotic cell death. In view of the important role of EGFR in the protein kinase B (Akt) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathways, two pathways linked to cancer, we examined the ability of miR-7 transfection to repress Akt and ERK1/2 activity in different cancer cell lines. Following transfection of A549, U87, MDA-MB-468, and DU145 cell lines with miR-7, we observed reductions in basal and EGF-induced phosphorylation of Akt (Fig. 3, C and D, and supplemental Fig. 2A) in all four cell lines. The capacity of miR-7 to regulate ERK1/2 activity appeared to be cell-specific, because miR-7 decreased EGF-stimulated ERK1/2 phosphorylation in U87 cells but did not alter ERK1/2 phosphorylation in A549, MDA-MB-468, or DU145 cells (Fig. 3, E and F, and supplemental Fig. 2B). Together, these observations are consistent with miR-7's ability to inhibit cancer cell cycle progression and reduce cell viability, and suggest that, in addition to the regulation of EGFR expression, miR-7 can regulate the activity of important downstream signaling effectors of EGFR, including Akt and ERK1/2, in multiple EGFR-positive cancer cell lines. miR-7 Re
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