RESUMEN
Hepatocellular carcinoma (HCC) is a frequent form of cancer with a poor prognosis and with limited possibilities for medical intervention. Recent evidence has accumulated that long noncoding RNAs (lncRNAs) are important regulators of disease processes including cancer. Chromatin remodeling in cancer cells may result in an unusual expression of lncRNAs and indeed it has been shown that more than 7000 unannotated lncRNAs are expressed in HCCs. We identified a novel long intergenic noncoding RNA, Linc00176, that plays a role in proliferation and survival of HCC. Linc00176 regulates expression of more than 200 genes by the sponge function for tumor suppressor miRNAs, miR-9 and miR-185. Linc00176 is expressed at a high level only in HCC, and is activated by Myc, Max and AP-4 transcription regulators. Myc also upregulates miR-9 and miR-185. In Linc00176-depleted HCC, these miRNAs were released from Linc00176 and downregulated their target mRNAs. Thus, depletion of Linc00176 disrupted the cell cycle and induced necroptosis in HCC via released tumor suppressor miRNAs. These data indicate that atypically expressed lncRNAs may be useful targets for cancer therapy.
Asunto(s)
Carcinoma Hepatocelular/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/genética , MicroARNs/genética , ARN Largo no Codificante/metabolismo , Apoptosis/genética , Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular/genética , Conjuntos de Datos como Asunto , Regulación hacia Abajo , Genes Supresores de Tumor , Humanos , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Largo no Codificante/genética , ARN Mensajero/genética , ARN Interferente Pequeño/metabolismo , Regulación hacia ArribaRESUMEN
Hepatocellular carcinoma (HCC) is a frequent form of cancer with a poor prognosis and with limited possibilities of medical intervention. It has been shown that over 100 putative driver genes are associated with multiple recurrently altered pathways in HCC, suggesting that multiple pathways will need to be inhibited for any therapeutic method. mRNA processing is regulated by a complex RNA-protein network that is essential for the maintenance of homeostasis. THOC5, a member of mRNA export complex, has a role in less than 1% of mRNA processing, and is required for cell growth and differentiation, but not for cell survival in normal fibroblasts, hepatocytes and macrophages. In this report, we show that 50% depletion of THOC5 in human HCC cell lines Huh7 and HepG2 induced apoptosis. Transcriptome analysis using THOC5-depleted cells revealed that 396 genes, such as transmembrane BAX inhibitor motif containing 4 (TMBIM4), transmembrane emp24-like trafficking protein 10 (Tmed10) and D-tyrosyl-tRNA deacylase 2 (Dtd2) genes were downregulated in both cell lines. The depletion of one of these THOC5 target genes in Huh7 or HepG2 did not significantly induce cell death, suggesting that these may be fine tuners for HCC cell survival. However, the depletion of a combination of these genes synergistically increased the number of TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling)-positive HCC. It must be noted that the depletion of these genes did not induce cell death in the hepatocyte cell line, THLE-2 cells. THOC5 expression was enhanced in 78% of cytological differentiation grading G2 and G3 tumor in primary HCC. Furthermore, the expression of a putative glycoprotein, Tmed10, is correlated to THOC5 expression level in primary HCCs, suggesting that this protein may be a novel biomarker for HCC. These data imply that the suppression of the multiple THOC5 target genes may represent a novel strategy for HCC therapy.
Asunto(s)
Apoptosis/genética , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Proteínas Nucleares/genética , Interferencia de ARN , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular , Línea Celular Tumoral , Ciclina D1/genética , Ciclina D1/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Células Hep G2 , Humanos , Immunoblotting , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/metabolismo , Transporte de ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Hematopoiesis and commitment to a restricted lineage are guided by a timely expressed set of cytokine receptors and their downstream transcription factors. A member of the mRNA export complex, THOC5 (suppressors of the transcriptional defects of hpr1 delta by overexpression complex 5) is a substrate for several tyrosine kinases such as macrophage colony-stimulating factor (M-CSF) receptor and various leukemogenic tyrosine kinases, such as Bcr-Abl, or NPM-ALK. THOC5 tyrosine phosphorylation is elevated in stem cells from patients with chronic myeloid leukemia, suggesting that THOC5 may be involved in leukemia development. THOC5 is also an essential element in the maintenance of hematopoiesis in adult mice. In this report, we show that THOC5 is located in the nuclear speckles, and that it is translocated from the nucleus to cytoplasm during M-CSF-induced bone marrow-derived macrophage differentiation. Furthermore, we have identified THOC5 target genes by trancriptome analysis, using tamoxifen-inducible THOC5 knockout macrophages. Although only 99 genes were downregulated in THOC5-depleted macrophages, half of the genes are involved in differentiation and/or migration. These include well-known regulators of myeloid differentiation inhibitor of DNA binding (Id)1, Id3, Smad family member 6 (Smad6) and Homeobox (Hox)A1. In addition, a subset of M-CSF-inducible genes, such as Ets family mRNAs are THOC5 target mRNAs. Upon depletion of THOC5, unspliced v-ets erythroblastosis virus E26 oncogene homolog (Ets1) mRNA was accumulated in the nucleus. Furthermore, THOC5 was recruited to chromatin where Ets1 was transcribed and bound to unspliced and spliced Ets1 transcripts, indicating that THOC5 has a role in processing/export of M-CSF-inducible genes. In conclusion, regulation of immediate-early gene response by THOC5, a member of mRNA export complex contributes to the M-CSF-induced macrophage differentiation.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Genes Inmediatos-Precoces , Factor Estimulante de Colonias de Macrófagos/farmacología , Macrófagos/citología , Proteínas Nucleares/metabolismo , Transporte de ARN/genética , Transporte Activo de Núcleo Celular/efectos de los fármacos , Empalme Alternativo/efectos de los fármacos , Empalme Alternativo/genética , Animales , Células de la Médula Ósea/citología , Diferenciación Celular/genética , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Hematopoyesis/efectos de los fármacos , Hematopoyesis/genética , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Modelos Biológicos , Regiones Promotoras Genéticas/genética , Transporte de Proteínas/efectos de los fármacos , Proteína Proto-Oncogénica c-ets-1/genética , Procesamiento Postranscripcional del ARN/efectos de los fármacos , Transporte de ARN/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcripción Genética/efectos de los fármacos , Transcriptoma/genéticaRESUMEN
THOC5 is a member of the THO complex that is involved in processing and transport of mRNA. We have shown previously that hematopoietic stem cells have an absolute requirement for THOC5 for survival and that THOC5 is phosphorylated on tyrosine 225 as a consequence of leukemogenic protein tyrosine kinase (PTK) action. We have investigated pathways for THOC5 phosphorylation to develop an understanding of THO complex modulation by tyrosine kinase (TK) oncogenes in leukemias. We demonstrate that THOC5 phosphorylation is mediated by Src PTK and CD45 protein tyrosine phosphatase action and that this event is sensitive to oxidative status. We show that THOC5 phosphorylation is elevated in stem cells from patients with chronic myeloid leukemia (CML) and that this phosphorylation is sensitive to the frontline drugs used in CML treatment. Further we show that THOC5 Y225 phosphorylation governs mRNA binding. In addition, CXCL12 is shown to induce THOC5 Y225 phosphorylation, and site-directed mutagenesis demonstrates that this modulates motile response. In conclusion, we delineate a signaling pathway stimulated by leukemogenic PTKs, chemokines and oxidative stress that can affect THO complex mediation of gene expression describing mechanisms for post-transcriptional regulation of protein levels.
