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The RNA-binding Protein IMP-3 Is a Translational Activator of Insulin-like Growth Factor II Leader-3 mRNA during Proliferation of Human K562 Leukemia Cells

2005; Elsevier BV; Volume: 280; Issue: 18 Linguagem: Inglês

10.1074/jbc.m500270200

1083-351X

Baisong Liao, Yan Hu, David J. Herrick, Gary Brewer,

Growth Hormone and Insulin-like Growth Factors

IMP-3, a member of the insulin-like growth factor-II (IGF-II) mRNA-binding protein (IMP) family, is expressed mainly during embryonic development and in some tumors. Thus, IMP-3 is considered to be an oncofetal protein. The functional significance of IMP-3 is not clear. To identify the functions of IMP-3 in target gene expression and cell proliferation, RNA interference was employed to knock down IMP-3 expression. Using human K562 leukemia cells as a model, we show that IMP-3 protein associates with IGF-II leader-3 and leader-4 mRNAs and H19 RNA but not c-myc and β-actin mRNAs in vivo by messenger ribonucleoprotein immunoprecipitation analyses. IMP-3 knock down significantly decreased levels of intracellular and secreted IGF-II without affecting IGF-II leader-3, leader-4, c-myc, or β-actin mRNA levels and H19 RNA levels compared with the negative control siRNA treatment. Moreover, IMP-3 knock down specifically suppressed translation of chimeric IGF-II leader-3/luciferase mRNA without altering reporter mRNA levels. Together, these results suggest that IMP-3 knock down reduced IGF-II expression by inhibiting translation of IGF-II mRNA. IMP-3 knock down also markedly inhibited cell proliferation. The addition of recombinant human IGF-II peptide to these cells restored cell proliferation rates to normal. IMP-3 and IMP-1, two members of the IMP family with significant structural similarity, appear to have some distinct RNA targets and functions in K562 cells. Thus, we have identified IMP-3 as a translational activator of IGF-II leader-3 mRNA. IMP-3 plays a critical role in regulation of cell proliferation via an IGF-II-dependent pathway in K562 leukemia cells. IMP-3, a member of the insulin-like growth factor-II (IGF-II) mRNA-binding protein (IMP) family, is expressed mainly during embryonic development and in some tumors. Thus, IMP-3 is considered to be an oncofetal protein. The functional significance of IMP-3 is not clear. To identify the functions of IMP-3 in target gene expression and cell proliferation, RNA interference was employed to knock down IMP-3 expression. Using human K562 leukemia cells as a model, we show that IMP-3 protein associates with IGF-II leader-3 and leader-4 mRNAs and H19 RNA but not c-myc and β-actin mRNAs in vivo by messenger ribonucleoprotein immunoprecipitation analyses. IMP-3 knock down significantly decreased levels of intracellular and secreted IGF-II without affecting IGF-II leader-3, leader-4, c-myc, or β-actin mRNA levels and H19 RNA levels compared with the negative control siRNA treatment. Moreover, IMP-3 knock down specifically suppressed translation of chimeric IGF-II leader-3/luciferase mRNA without altering reporter mRNA levels. Together, these results suggest that IMP-3 knock down reduced IGF-II expression by inhibiting translation of IGF-II mRNA. IMP-3 knock down also markedly inhibited cell proliferation. The addition of recombinant human IGF-II peptide to these cells restored cell proliferation rates to normal. IMP-3 and IMP-1, two members of the IMP family with significant structural similarity, appear to have some distinct RNA targets and functions in K562 cells. Thus, we have identified IMP-3 as a translational activator of IGF-II leader-3 mRNA. IMP-3 plays a critical role in regulation of cell proliferation via an IGF-II-dependent pathway in K562 leukemia cells. The human insulin-like growth factor II (IGF-II) 1The abbreviations used are: IGF-II, insulin-like growth factor II; KOC, human KH domain-containing protein overexpressed in cancer; Vg1-RBP, Vg1 mRNA-binding protein; CRD-BP, c-myc mRNA-coding region determinant-binding protein; IMP, IGF-II mRNA-binding protein; ZBP, zipcode-binding protein of chicken β-actin mRNA; siRNA, short interfering RNA; RNAi, RNA interference; ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum albumin; PBS, phosphate-buffered saline; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; UTR, untranslated region; RIP, messenger ribonucleoprotein immunoprecipitation; qRT-PCR, real-time, quantitative, reverse transcription-PCR; KH, K-homology; MTS, [3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium]. mRNA-binding protein (IMP) family consists of IMP-1, IMP-2, and IMP-3. The three closely related IMP family members contain six RNA binding motifs, including two RNA recognition motifs and four heterogeneous nuclear ribonucleoprotein K-homology (KH) domains (1Nielsen J. Christiansen J. Lykke-Andersen J. Johnsen A.H. Wewer U.M. Nielsen F.C. Mol. Cell. Biol. 1999; 19: 1262-1270Crossref PubMed Scopus (565) Google Scholar). IMP-3 has amino acid identities of 65.7 and 59.7% with IMP-1 and IMP-2, respectively. The sequence similarities of the RNA binding domains among the IMP proteins, especially within the KH domains, are much higher (2Mori H. Sakakibara S. Imai T. Nakamura Y. Iijima T. Suzuki A. Yuasa Y. Takeda M. Okano H. J. Neurosci. Res. 2001; 64: 132-143Crossref PubMed Scopus (47) Google Scholar). All three IMPs can bind to human IGF-II mRNA with high affinity in vitro (1Nielsen J. Christiansen J. Lykke-Andersen J. Johnsen A.H. Wewer U.M. Nielsen F.C. Mol. Cell. Biol. 1999; 19: 1262-1270Crossref PubMed Scopus (565) Google Scholar, 3Nielsen J. Kristensen M.A. Willemoes M. Nielsen F.C. Christiansen J. Nucleic Acids Res. 2004; 32: 4368-4376Crossref PubMed Scopus (89) Google Scholar). IMP-1 is identical to the mouse c-myc coding region determinant-binding protein (CRD-BP) (4Bernstein P.L. Herrick D.J. Prokipcak R.D. Ross J. Genes Dev. 1992; 6: 642-654Crossref PubMed Scopus (227) Google Scholar, 5Doyle G.A. Betz N.A. Leeds P.F. Fleisig A.J. Prokipcak R.D. Ross J. Nucleic Acids Res. 1998; 26: 5036-5044Crossref PubMed Scopus (144) Google Scholar, 6Ioannidis P. Trangas T. Dimitriadis E. Samiotaki M. Kyriazoglou I. Tsiapalis C.M. Kittas C. Agnantis N. Nielsen F.C. Nielsen J. Christiansen J. Pandis N. Int. J. Cancer. 2001; 94: 480-484Crossref PubMed Scopus (58) Google Scholar) and the chicken β-actin mRNA-binding protein-1 (ZBP-1) (7Ross A.F. Oleynikov Y. Kislauskis E.H. Taneja K.L. Singer R.H. Mol. Cell. Biol. 1997; 17: 2158-2165Crossref PubMed Google Scholar). IMP-3 is identical to the KH domain-containing protein overexpressed in cancer (KOC) (8Mueller-Pillasch F. Lacher U. Wallrapp C. Micha A. Zimmerhackl F. Hameister H. Varga G. Friess H. Buchler M. Beger H.G. Vila M.R. Adler G. Gress T.M. Oncogene. 1997; 14: 2729-2733Crossref PubMed Scopus (235) Google Scholar) and the Xenopus laevis Vg1 mRNA-binding protein (Vg1-RBP/Vera) (9Elisha Z. Havin L. Ringel I. Yisraeli J.K. EMBO J. 1995; 14: 5109-5114Crossref PubMed Scopus (85) Google Scholar, 10Havin L. Git A. Elisha Z. Oberman F. Yaniv K. Schwartz S.P. Standart N. Yisraeli J.K. Genes Dev. 1998; 12: 1593-1598Crossref PubMed Scopus (183) Google Scholar). Whereas no orthologs for IMP-2 have been found, p62, a human hepatocellular carcinoma autoantigen, seems to be a splice variant (11Zhang J.Y. Chan E.K. Peng X.X. Tan E.M. J. Exp. Med. 1999; 189: 1101-1110Crossref PubMed Scopus (180) Google Scholar). So far, at least five RNA targets for IMP-1 have been reported, including IGF-II, c-myc, β-actin, tau, and H19. IMP-1 can affect stability, localization, and translation of its target RNAs (1Nielsen J. Christiansen J. Lykke-Andersen J. Johnsen A.H. Wewer U.M. Nielsen F.C. Mol. Cell. Biol. 1999; 19: 1262-1270Crossref PubMed Scopus (565) Google Scholar, 3Nielsen J. Kristensen M.A. Willemoes M. Nielsen F.C. Christiansen J. Nucleic Acids Res. 2004; 32: 4368-4376Crossref PubMed Scopus (89) Google Scholar, 4Bernstein P.L. Herrick D.J. Prokipcak R.D. Ross J. Genes Dev. 1992; 6: 642-654Crossref PubMed Scopus (227) Google Scholar, 7Ross A.F. Oleynikov Y. Kislauskis E.H. Taneja K.L. Singer R.H. Mol. Cell. Biol. 1997; 17: 2158-2165Crossref PubMed Google Scholar, 12Runge S. Nielsen F.C. Nielsen J. Lykke-Andersen J. Wewer U.M. Christiansen J. J. Biol. Chem. 2000; 275: 29562-29569Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 13Atlas R. Behar L. Elliott E. Ginzburg I. J. Neurochem. 2004; 89: 613-626Crossref PubMed Scopus (126) Google Scholar). IMP-1 binds to the 5′-UTR of IGF-II leader-3 mRNA and inhibits its translation. IMP-1 also binds to the 3′-terminus of the untranslated H19 RNA and regulates its subcellular location. CRD-BP/IMP-1 binds to the coding region instability determinant of c-myc mRNA and stabilizes c-myc mRNA in a cell-free mRNA decay system. Chicken ZBP-1/IMP-1 binds to the "zipcode" segment in the 3′-UTR of β-actin mRNA and localizes the mRNA to the lamellipodia of fibroblasts (1Nielsen J. Christiansen J. Lykke-Andersen J. Johnsen A.H. Wewer U.M. Nielsen F.C. Mol. Cell. Biol. 1999; 19: 1262-1270Crossref PubMed Scopus (565) Google Scholar, 3Nielsen J. Kristensen M.A. Willemoes M. Nielsen F.C. Christiansen J. Nucleic Acids Res. 2004; 32: 4368-4376Crossref PubMed Scopus (89) Google Scholar, 4Bernstein P.L. Herrick D.J. Prokipcak R.D. Ross J. Genes Dev. 1992; 6: 642-654Crossref PubMed Scopus (227) Google Scholar, 7Ross A.F. Oleynikov Y. Kislauskis E.H. Taneja K.L. Singer R.H. Mol. Cell. Biol. 1997; 17: 2158-2165Crossref PubMed Google Scholar, 12Runge S. Nielsen F.C. Nielsen J. Lykke-Andersen J. Wewer U.M. Christiansen J. J. Biol. Chem. 2000; 275: 29562-29569Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Tau mRNA is an axonally localized mRNA in neurons (14Aronov S. Aranda G. Behar L. Ginzburg I. J. Neurosci. 2001; 21: 6577-6587Crossref PubMed Google Scholar, 15Aronov S. Aranda G. Behar L. Ginzburg I. J. Cell Sci. 2002; 115: 3817-3827Crossref PubMed Scopus (106) Google Scholar, 16Binder L.I. Frankfurter A. Rebhun L.I. J. Cell Biol. 1985; 101: 1371-1378Crossref PubMed Scopus (1240) Google Scholar). IMP-1 can bind to the 3′-UTR of tau mRNA, but the functional significance for the binding is unknown (13Atlas R. Behar L. Elliott E. Ginzburg I. J. Neurochem. 2004; 89: 613-626Crossref PubMed Scopus (126) Google Scholar). IMP-1 is thought to be an oncofetal protein since it is mainly expressed during embryogenesis and in some tumors; it is reduced or absent in adult tissues (6Ioannidis P. Trangas T. Dimitriadis E. Samiotaki M. Kyriazoglou I. Tsiapalis C.M. Kittas C. Agnantis N. Nielsen F.C. Nielsen J. Christiansen J. Pandis N. Int. J. Cancer. 2001; 94: 480-484Crossref PubMed Scopus (58) Google Scholar, 17Leeds P. Kren B.T. Boylan J.M. Betz N.A. Steer C.J. Gruppuso P.A. Ross J. Prokipcak R.D. Herrick D.J. Bernstein P.L. Oncogene. 1997; 14: 1279-1286Crossref PubMed Scopus (93) Google Scholar, 18Ross J. Lemm I. Berberet B. Doyle G.A. Bourdeau-Heller J.M. Coulthard S. Meisner L.F. Lee C.H. Leeds P. Kren B.T. Boylan J.M. Betz N.A. Steer C.J. Gruppuso P.A. Prokipcak R.D. Herrick D.J. Bernstein P.L. Oncogene. 2001; 20: 6544-6550Crossref PubMed Scopus (81) Google Scholar, 19Doyle G.A. Bourdeau-Heller J.M. Coulthard S. Meisner L.F. Ross J. Lee C.H. Leeds P. Kren B.T. Boylan J.M. Betz N.A. Steer C.J. Gruppuso P.A. Prokipcak R.D. Herrick D.J. Bernstein P.L. Cancer Res. 2000; 60: 2756-2759PubMed Google Scholar). Recent investigations show diverse functions for IMP-1 in various cell types. For example, overexpression of IMP-1 in mammary epithelial cells of transgenic mice induces mammary tumors and increases IGF-II mRNA levels by 100-fold without affecting cellular IGF-II protein levels (20Tessier C.R. Doyle G.A. Clark B.A. Pitot H.C. Ross J. Cancer Res. 2004; 64: 209-214Crossref PubMed Scopus (92) Google Scholar). IMP-1 knock-out mice exhibit dwarfism and translation inhibition of both leader-3 and leader-4 isoforms of IGF-II mRNA (21Hansen T.V. Hammer N.A. Nielsen J. Madsen M. Dalbaeck C. Wewer U.M. Christiansen J. Nielsen F.C. Mol. Cell. Biol. 2004; 24: 4448-4464Crossref PubMed Scopus (178) Google Scholar). Our previous studies showed that IMP-1 knockdown by RNA interference promotes cell proliferation via up-regulation of IGF-II mRNA and protein levels, which may involve a nuclear mechanism (22Liao B. Patel M. Hu Y. Charles S. Herrick D.J. Brewer G. J. Biol. Chem. 2004; 279: 48716-48724Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Thus, IMP-1 appears to be a phylogenetically conserved and multi-functional RNA-binding protein. The human KH domain-containing protein KOC was originally identified in human pancreatic cancer (8Mueller-Pillasch F. Lacher U. Wallrapp C. Micha A. Zimmerhackl F. Hameister H. Varga G. Friess H. Buchler M. Beger H.G. Vila M.R. Adler G. Gress T.M. Oncogene. 1997; 14: 2729-2733Crossref PubMed Scopus (235) Google Scholar). Xenopus Vg1 mRNA-binding protein (Vg1-RBP) was identified by using human KOC as a probe, and it has an amino acid sequence identity of 84% with KOC. Thus, Vg1-RBP is thought to be a Xenopus homologue of human KOC. X. laevis Vegetal 1 (Vg1) mRNA encodes a transforming growth factor β-like protein that stimulates the formation of mesoderm at the vegetal cortex. Vg1-RBP binds to the 3′-UTR of Vg1 mRNA and localizes the mRNA to the vegetal pole of X. laevis oocytes at stages III and IV (for review, see Refs. 23Deshler J.O. Highett M.I. Schnapp B.J. Science. 1997; 276: 1128-1131Crossref PubMed Scopus (234) Google Scholar and 24Mueller-Pillasch F. Pohl B. Wilda M. Lacher U. Beil M. Wallrapp C. Hameister H. Knochel W. Adler G. Gress T.M. Mech. Dev. 1999; 88: 95-99Crossref PubMed Scopus (171) Google Scholar). Vg1-RBP is also implicated in regulation of the migration of neural crest cells during early neural development (25Yaniv K. Fainsod A. Kalcheim C. Yisraeli J.K. Development. 2003; 130: 5649-5661Crossref PubMed Scopus (79) Google Scholar). Human IMP-3 was isolated in RD rhabdomyosarcoma cells and found to be identical to KOC. IMP-3/KOC can bind to the 5′-UTR of IGF-II leader-3 mRNA and the 3′-UTRs of all the IGF-II mRNA isoforms (1Nielsen J. Christiansen J. Lykke-Andersen J. Johnsen A.H. Wewer U.M. Nielsen F.C. Mol. Cell. Biol. 1999; 19: 1262-1270Crossref PubMed Scopus (565) Google Scholar, 3Nielsen J. Kristensen M.A. Willemoes M. Nielsen F.C. Christiansen J. Nucleic Acids Res. 2004; 32: 4368-4376Crossref PubMed Scopus (89) Google Scholar). However, the functional significance is not clear. Transgenic overexpression of IMP-3/KOC results in remodeling of the exocrine pancreas (26Wagner M. Kunsch S. Duerschmied D. Beil M. Adler G. Mueller F. Gress T.M. Gastroenterology. 2003; 124: 1901-1914Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Although IMP family members share high structural similarity, recombinant mouse IMP-3/KOC only binds to the 5′-UTR of IGF-II leader-3 mRNA and not to some IMP-1 target RNAs such as c-myc and β-actin by in vitro UV cross-linking assay. This suggests functional differences among the IMP members (2Mori H. Sakakibara S. Imai T. Nakamura Y. Iijima T. Suzuki A. Yuasa Y. Takeda M. Okano H. J. Neurosci. Res. 2001; 64: 132-143Crossref PubMed Scopus (47) Google Scholar). IMP-3 seems to be involved in tumorigenesis and embryonic development. High levels of IMP-3 mRNA were detected in pancreatic cancer cell lines and tissues as well as other human tumors such as gastric cancer, soft tissue sarcoma, colon carcinoma, RD rhabdomyosarcoma cells, and K562 human leukemia cells (1Nielsen J. Christiansen J. Lykke-Andersen J. Johnsen A.H. Wewer U.M. Nielsen F.C. Mol. Cell. Biol. 1999; 19: 1262-1270Crossref PubMed Scopus (565) Google Scholar, 8Mueller-Pillasch F. Lacher U. Wallrapp C. Micha A. Zimmerhackl F. Hameister H. Varga G. Friess H. Buchler M. Beger H.G. Vila M.R. Adler G. Gress T.M. Oncogene. 1997; 14: 2729-2733Crossref PubMed Scopus (235) Google Scholar, 22Liao B. Patel M. Hu Y. Charles S. Herrick D.J. Brewer G. J. Biol. Chem. 2004; 279: 48716-48724Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 27Zhang J.Y. Chan E.K. Peng X.X. Lu M. Wang X. Mueller F. Tan E.M. Clin. Immunol. 2001; 100: 149-156Crossref PubMed Scopus (63) Google Scholar, 28Yaniv K. Yisraeli J.K. Gene (Amst.). 2002; 287: 49-54Crossref PubMed Scopus (127) Google Scholar, 29Mueller F. Bommer M. Lacher U. Ruhland C. Stagge V. Adler G. Gress T.M. Seufferlein T. Br. J. Cancer. 2003; 88: 699-701Crossref PubMed Scopus (37) Google Scholar). The IMP-3 transcript has similar expression patterns with IMP-1 during mouse embryonic development. For example, they are expressed at early stages, peak around embryonic day 12.5, decrease until birth, and then are low or absent in adult tissues (1Nielsen J. Christiansen J. Lykke-Andersen J. Johnsen A.H. Wewer U.M. Nielsen F.C. Mol. Cell. Biol. 1999; 19: 1262-1270Crossref PubMed Scopus (565) Google Scholar, 21Hansen T.V. Hammer N.A. Nielsen J. Madsen M. Dalbaeck C. Wewer U.M. Christiansen J. Nielsen F.C. Mol. Cell. Biol. 2004; 24: 4448-4464Crossref PubMed Scopus (178) Google Scholar). This fetal expression profile overlaps that of IGF-II leader-3 mRNA, which implies a regulatory effect of IMP-3 on IGF-II gene expression in combination with the binding of IMP-3 to IGF-II leader-3 mRNA (1Nielsen J. Christiansen J. Lykke-Andersen J. Johnsen A.H. Wewer U.M. Nielsen F.C. Mol. Cell. Biol. 1999; 19: 1262-1270Crossref PubMed Scopus (565) Google Scholar, 21Hansen T.V. Hammer N.A. Nielsen J. Madsen M. Dalbaeck C. Wewer U.M. Christiansen J. Nielsen F.C. Mol. Cell. Biol. 2004; 24: 4448-4464Crossref PubMed Scopus (178) Google Scholar). Moreover, human and mouse IMP-3 and Xenopus Vg1-RBP have similar fetal expression patterns. For instance, high levels of the transcripts were seen in the gut, pancreas, kidney, skin, snout, placenta, and brain during mouse development (24Mueller-Pillasch F. Pohl B. Wilda M. Lacher U. Beil M. Wallrapp C. Hameister H. Knochel W. Adler G. Gress T.M. Mech. Dev. 1999; 88: 95-99Crossref PubMed Scopus (171) Google Scholar). Together, these studies indicate that IMP-3 may play a pivotal role in tumorigenesis and development. However, the regulatory effects on expression of its target mRNAs and the functional significance remain to be elucidated. In this study we knocked down IMP-3 expression by RNA interference (RNAi) in human K562 leukemia cells, which were utilized for the identification of IMP-1 function, to examine IMP-3 function (22Liao B. Patel M. Hu Y. Charles S. Herrick D.J. Brewer G. J. Biol. Chem. 2004; 279: 48716-48724Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). We found that IMP-3 knockdown inhibited translation of IGF-II leader-3 mRNA without affecting its mRNA levels. Furthermore, IMP-3 knockdown reduced cell proliferation through an IGF-II-dependent mechanism. Preparation of siRNA—The human IMP-3 and IMP-1 SMARTpool siRNA duplexes were designed and chemically synthesized by Dharmacon Research (Lafayette, CO). The SMARTpool siRNA is a mixture of four different siRNA duplexes targeting distinct coding region sequences of IMP-3 (GenBank™ accession number NM_006547) or IMP-1 (GenBank™ accession number AF117106). The sequences of the SMARTpool siRNAs are proprietary. The negative control siRNA contained nucleotides randomly arranged (5′-aac ugg gua agc ggg cgc aaa-3′). BLAST searches against human genome sequences in GenBank™ were performed by Dharmacon to ensure specificity of the siRNAs. The siRNA duplexes were dissolved in 1× universal RNA oligo buffer (20 mm KCl, 6 mm HEPES-KOH (pH 7.5), 0.2 mm MgCl2). Cell Culture and siRNA Transfection—K562 cells (ATCC) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mm glutamine (Invitrogen) at 37 °C in 5% CO2. 5-10 × 106 K562 cells were transfected with either 200 nm IMP-3 siRNA or both IMP-3 and IMP-1 siRNAs (double knockdown) or control siRNA (negative control) or an equal volume of 1× universal RNA oligo buffer (mock control) by electroporation using a Gene Pulser (Bio-Rad) as described previously (22Liao B. Patel M. Hu Y. Charles S. Herrick D.J. Brewer G. J. Biol. Chem. 2004; 279: 48716-48724Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Transfected cells were maintained in regular culture medium without antibiotics for the times indicated in the figure legends. Western Blot Analysis—Cytoplasmic and nuclear fractions were prepared using the CelLytic NuCLEAR™ extraction kit (Sigma). Total protein concentration of the extracts was quantified by Bradford assay using the protein assay reagent (Bio-Rad) following the manufacturer's instructions. For Western blot analysis, cytoplasmic (40 μg) and nuclear (20 μg) lysates were size-fractionated by SDS-PAGE and transferred onto nitrocellulose membranes (Fisher). Antibodies used and their dilutions are as follows: c-Myc (Oncogene) 1:300; α-tubulin (Sigma) 1:10,000; lamin A/C (Upstate Biotechnology) 1:1,300; IMP-1,-2, -3 (as described previously (22Liao B. Patel M. Hu Y. Charles S. Herrick D.J. Brewer G. J. Biol. Chem. 2004; 279: 48716-48724Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar)) 1:5,000; goat anti-mouse IgG (H+L) horse-radish peroxidase conjugate (Promega) 1:2,500; goat anti-rabbit IgG horseradish peroxidase conjugate (Sigma) 1:3,000. Western blot analysis was carried out using the SuperSignal® West chemiluminescent substrate kit (Pierce) according to the manufacturer's protocol. The blots were stripped and reprobed with anti-α-tubulin or lamin A/C antibody. α-Tubulin and lamin A/C served as the loading controls for cytoplasmic and nuclear protein fractions, respectively. Western blot results were quantified by using a DC120 Kodak Zoom Digital Image system (Eastman Kodak Co.). Enzyme-Linked Immunosorbent Assay (ELISA)—IGF-II concentrations in cytoplasmic lysates and culture media of K562 cells were examined by ELISA using the non-extraction IGF-II ELISA kit (Diagnostic Systems Laboratories, Inc.) according to the manufacturer's instructions as described previously (22Liao B. Patel M. Hu Y. Charles S. Herrick D.J. Brewer G. J. Biol. Chem. 2004; 279: 48716-48724Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Media samples were pretreated with buffers provided in the kit to dissociate IGF-II and IGF-binding proteins before the assay. For detection of intracellular IGF-II protein, cytoplasmic lysates were prepared using the CellLytic NuCLEAR extraction kit (Sigma). The concentration of the total protein in the lysates was measured by the Bradford assay using the protein assay reagent (Bio-Rad) following the manufacturer's instructions. Dual Luciferase Reporter Assay—Luciferase activity was examined by a dual luciferase reporter assay using the dual luciferase reporter assay kit (Promega) on a TD-20/20 luminometer (Turner Designs, Sunnyvale, CA) as described previously (22Liao B. Patel M. Hu Y. Charles S. Herrick D.J. Brewer G. J. Biol. Chem. 2004; 279: 48716-48724Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The plasmids pcDNA-IGF-II leader-3/luciferase (pcDNA-IGF-II-L3-Luc) and pcDNA-IGF-II leader-4/luciferase (pcDNA-IGF-II-L4-Luc) were kindly provided by Dr. Jan Christiansen. These plasmids contain the firefly luciferase coding region and the complete leader 3 (1164 bp) or leader 4 (94 bp) exon, respectively, in the pcDNA3.1 basic vector (Invitrogen) (1Nielsen J. Christiansen J. Lykke-Andersen J. Johnsen A.H. Wewer U.M. Nielsen F.C. Mol. Cell. Biol. 1999; 19: 1262-1270Crossref PubMed Scopus (565) Google Scholar). 1 × 107 K562 cells were co-transfected with 1 μg of pRL-SV40 Renilla luciferase control vector (Promega), 10 μg of either pcDNA-IGF-II-L3-Luc or pcDNA-IGF-II-L4-Luc, and 200 nm IMP-3 siRNA or negative control siRNA or both IMP-1 and IMP-3 siRNAs by electroporation as described above. pRL-SV40 served as an internal control. pGL-Promoter vector containing the firefly luciferase coding region (Promega) was used as a control for the effects of 5′-UTR sequences on gene expression. Luciferase activity was measured at 48 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity in the same cell extract and expressed as a ratio of firefly/Renilla luciferase activity. Quantitative Real-time Reverse Transcription-PCR (qRT-PCR)—H19 RNA and IGF-II, IGF-II leader-3, IGF-II leader-4, c-myc, IMP-1, IMP-3, β-actin, firefly luciferase, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were examined by qRT-PCR. One-step qRT-PCR was performed using the QuantiTect Probe RT-PCR kit (Qiagen) on a MX-4000 Multiplex Quantitative PCR system (Stratagene, La Jolla, CA) according to the manufacturer's instructions. The use of dual-fluorescence-labeled probes greatly increased the specificity of the qRT-PCR. The PCR primers and probes were designed using web-based Primer-3 software (www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi) and were synthesized by Integrated DNA Technologies (Coralville, IA). The Tm values for the probes and primers are ∼70 and 60 °C, respectively. They are as follows: c-myc probe, 5′-6-FAM™-CGG GCA CTT TGC ACT GGA ACT TAC A-TAMRA™-3′, c-myc forward primer, 5′-ACG AAA CTT TGC CCA TAG CA-3′, c-myc reverse primer, 5′-GCA AGG AGA GCC TTT CAG AG-3′; IGF-II probe, 5′-6-FAM™-TGG ACA CCC TCC AGT TCG TCT GTG-TAMRA™-3′, IGF-II forward primer, 5′-AAG TCG ATG CTG GTG CTT CT-3′, IGF-II reverse primer, 5′-CGG AAA CAG CAC TCC TCA A-3′; IGF-II leader-3 probe, 5′-6-FAM™-TTC TCT CCC GCT GTG CGC CT-TAMRA™-3′, IGF-II leader-3 forward primer, 5′-ATT ACA CGC TTT CTG TTT CTC TCC-3′, IGF-II leader-3 reverse primer, 5′-AAA TGA GGT CAG CTG TTG TAT CAA G-3′; IGF-II leader-4 probe, 5′-6-FAM™-CCT CCT CCT CCT GCC CCA GC-TAMRA™-3′, IGF-II leader-4 forward primer, 5′-TCT CCT GTG AAA GAG ACT TCC AG-3′, IGF-II leader-4 reverse primer, 5′-CAA GAA GGT GAG AAG CAC CAG-3′; firefly luciferase probe, 5′-6-FAM™-TCC GTT CGG TTG GCA GAA GC-TAMRA™-3′, firefly luciferase forward primer, 5′-AGA GAT ACG CCC TGG TTC CT-3′, firefly luciferase reverse primer, 5′-CCA ACA CCG GCA TAA AGA AT-3′; IMP-3 probe, 5′-6-Cy5™-TGC TTG CCA GGT TGC CCA GA-BHQ™-3′, IMP-3 forward primer, 5′-AGT TGT TGT CCC TCG TGA CC-3′, IMP-3 reverse primer, 5′-GTC CAC TTT GCA GAG CCT TC-3′; IMP-1 probe, 5′-Cy5™-GTT GCA GGG CCG AGC AGG AA-BHQ™-3′, IMP-1 forward primer, 5′-AAC CCT GAG AGG ACC ATC ACT-3′, IMP-1 reverse primer, 5′-AGC TGG GAA AAG ACC TAC AGC-3′; H19 RNA probe, 5′-6-FAM™-CCT GAC TCA GGA ATC GGC TCT GGA-TAMRA™-3′, H19 RNA forward primer, 5′-ATG GTG CTA CCC AGC TCA AG-3′, H19 RNA reverse primer, 5′-TGT TCC GAT GGT GTC TTT GA-3′; β-actin probe, 5′-FAM™-TTG GAG CGA GCA TCC CCC A-TAMRA™-3′, β-actin forward primer, 5′-ACT GGA ACG GTG AAG GTG AC-3′, β-actin reverse primer, 5′-AGA GAA GTG GGG TGG CTT TT-3′; GAPDH probe, 5′-Cy3™-CCA TGG CAC CGT CAA GGC TG-BHQ™-3′, GAPDH forward primer, 5′-TTT AAC TCT GGT AAA GTG GAT ATT GTT G-3′, GAPDH reverse primer, 5′-ATT TCC ATT GAT GAC AAG CTT CC-3′. GAPDH was used as a quantitative, internal control in the same tube (2-multiplex PCR). Parallel reactions with no template or no reverse transcriptase were run as controls. Total RNA samples were prepared with TRIzol reagent (Invitrogen) and digested with RNase-free DNase I (Roche Applied Science). The qRT-PCR conditions were 50 °C for 30 min, 95 °C for 15 min, and 50 cycles of 94 °C for 20 s and 60 °C for 1 min. The qRT-PCR products were visualized by 2% agarose gel electrophoreses to ensure the correct size. Each sample was run in triplicate, and the experiment was performed three times. The data were analyzed using software included with the MX-4000 instrument. Messenger Ribonucleoprotein Immunoprecipitation (RIP) Assay—Identification of endogenous RNA-protein complexes was performed by RIP assay as described previously with modifications (30Lal A. Mazan-Mamczarz K. Kawai T. Yang X. Martindale J.L. Gorospe M. EMBO J. 2004; 23: 3092-3102Crossref PubMed Scopus (397) Google Scholar). Briefly, 5 × 107 K562 cells were harvested and lysed for 15 min at 4 °C in 200 μl of cold lysis buffer (100 mm KCl, 5 mm MgCl2, 10 mm HEPES (pH 7.0), 0.5% Nonidet P-40, 10 μm dithiothreitol) supplemented with RNase and protease inhibitors (1 ml of lysis buffer contains 5.25 μl of 40 units/ml RNase OUT (Invitrogen), 2 μl of 0.2% vanadyl ribonucleoside complexes (New England Biolabs), 40 μl of complete protease inhibitor mixture (Roche Applied Science)). Lysate was centrifuged for 10 min at 12,000 rpm, and supernatant was transferred to a fresh 1.5-ml tube. To pre-clear the cytoplasmic lysates, 20 μg of non-immune rabbit IgG (Sigma) was added to the supernatant and kept on ice for 45 min, then incubated with 50 μl of a 50% (v/v) suspension of protein A-Sepharose beads for 3 h at 4 °C with rotation. This was centrifuged at 12,000 rpm, and supernatant was recovered (pre-cleared lysates). Total protein concentration in the lysates was measured by Bradford assay as described above. For immunoprecipitation, 1.5 mg of cytoplasmic lysate proteins were incubated with 100 μl of a 50% suspension of protein A-Sepharose beads (Sigma) pre-coated with the same amount of either non-immune rabbit IgG (Sigma) or anti-human IMP-3 antibody (3-20 μg) in 800 μl of NT-2 buffer (150 mm NaCl, 1 mm MgCl2, 50 mm Tris-HCl (pH 7.4), 0.05% Nonidet P-40) containing RNase inhibitor and protease inhibitors for 3 h at 25 °C with rotation. Beads were washed 10 times using NT-2 buffer, digested with 20 units of RNase-free DNase I (Promega) in 100 of μl of NT-2 buffer for 20 min at 30 °C, washed with NT-2 buffer, and further digested with 0.5 mg/ml protease K (Ambion) in 100 μl of NT-2 buffer containing 0.1% SDS at 55 °C for 30 min. RNA was extracted with phenol/chloroform and precipitated with ethanol. Glycogen (Roche Applied Science) was added to facilitate precipitation of RNA. qRT-PCR was performed to examine RNAs associated with cytoplasmic IMP-3 as described above. Cell Proliferation Assay—Cell counts were determined by trypan blue (Sigma) exclusion assay according to the manufacturer's protocol. Cell proliferation was also examined by MTS assay using the CellTiter 96® AQueous One solution cell proliferation assay kit (Promega). Briefly, 20 μl of the One Solutio