RESUMO
Prostate cancer antigen 3 (PCA3) is the most specific prostate cancer biomarker but its function remains unknown. Here we identify PRUNE2, a target protein-coding gene variant, which harbors the PCA3 locus, thereby classifying PCA3 as an antisense intronic long noncoding (lnc)RNA. We show that PCA3 controls PRUNE2 levels via a unique regulatory mechanism involving formation of a PRUNE2/PCA3 double-stranded RNA that undergoes adenosine deaminase acting on RNA (ADAR)-dependent adenosine-to-inosine RNA editing. PRUNE2 expression or silencing in prostate cancer cells decreased and increased cell proliferation, respectively. Moreover, PRUNE2 and PCA3 elicited opposite effects on tumor growth in immunodeficient tumor-bearing mice. Coregulation and RNA editing of PRUNE2 and PCA3 were confirmed in human prostate cancer specimens, supporting the medical relevance of our findings. These results establish PCA3 as a dominant-negative oncogene and PRUNE2 as an unrecognized tumor suppressor gene in human prostate cancer, and their regulatory axis represents a unique molecular target for diagnostic and therapeutic intervention.
Assuntos
Antígenos de Neoplasias/genética , Íntrons/genética , Proteínas de Neoplasias/genética , Neoplasias da Próstata/genética , RNA Longo não Codificante/genética , Proteínas Supressoras de Tumor/genética , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Animais , Antígenos de Neoplasias/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Células MCF-7 , Masculino , Camundongos SCID , Dados de Sequência Molecular , Proteínas de Neoplasias/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Ligação Proteica , Interferência de RNA , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Longo não Codificante/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Supressoras de Tumor/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Phage display is a resourceful tool to, in an unbiased manner, discover and characterize functional protein-protein interactions, create vaccines, and engineer peptides, antibodies, and other proteins as targeted diagnostic and/or therapeutic agents. Recently, our group has developed a new class of internalizing phage (iPhage) for ligand-directed targeting of organelles and to identify molecular pathways within live cells. This unique technology is suitable for applications ranging from fundamental cell biology to drug development. This unit describes the methods for generating and screening the iPhage display system, and explains how to select and validate candidate internalizing homing peptide.
Assuntos
Técnicas de Visualização da Superfície Celular/métodos , Técnicas Citológicas/métodos , Organelas/química , Organelas/metabolismo , Linhagem Celular Tumoral , Clonagem Molecular , Escherichia coli , HumanosRESUMO
Techniques that are largely used for protein interaction studies and the discovery of intracellular receptors, such as affinity-capture complex purification and the yeast two-hybrid system, may produce inaccurate data sets owing to protein insolubility, transient or weak protein interactions or irrelevant intracellular context. A versatile tool for overcoming these limitations, as well as for potentially creating vaccines and engineering peptides and antibodies as targeted diagnostic and therapeutic agents, is the phage-display technique. We have recently developed a new technology for screening internalizing phage (iPhage) vectors and libraries using a ligand/receptor-independent mechanism to penetrate eukaryotic cells. iPhage particles provide a unique discovery platform for combinatorial intracellular targeting of organelle ligands along with their corresponding receptors and for fingerprinting functional protein domains in living cells. Here we explain the design, cloning, construction and production of iPhage-based vectors and libraries, along with basic ligand-receptor identification and validation methodologies for organelle receptors. An iPhage library screening can be performed in â¼8 weeks.