RESUMEN
Chronic myelocytic leukemia cell line K562 undergoes differentiation by phorbol esters to megakaryocytes and we have used this system to understand miRNA processing leading to isomiR generation. PMA treatment significantly altered the production of miRNA in K562 cells. Expression of 24.4% of miRNAs were found to be stimulated whereas expression of 10% miRNAs were inhibited by PMA treatment. Our results suggest that miRNA precursors are processed into isomiRs in a deterministic manner. The relative levels of different isomiRs of a miRNA remained mainly unchanged even after PMA treatment irrespective of overall changes in expression (either up-regulation or down-regulation). However, not all miRNAs behave in the same way, about 7% showed a variation of isomiR profiles after PMA treatment. Most of the later class of miRNAs were found to be oncogenic miRNAs. Further, it was also found that number of isomiRs was independent of abundance of a miRNA. Functional importance of different isomiRs was demonstrated using three different isomiRs of miR-22. Our results showed that different isomiRs could inhibit expression of targets genes with different efficiencies. Our study suggests that the heterogeneity of a miRNA population generated during processing is in general regulated and that variation in the generation of an isomiR can be a functionally important regulatory feature.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Leucemia Mieloide/genética , MicroARNs/genética , Ésteres del Forbol/farmacología , Fosforilcolina/análogos & derivados , Ácidos Polimetacrílicos/farmacología , Línea Celular Tumoral , Heterogeneidad Genética/efectos de los fármacos , Humanos , Células K562 , Fosforilcolina/farmacologíaAsunto(s)
Antineoplásicos/uso terapéutico , Biomarcadores de Tumor , Mesilato de Imatinib/uso terapéutico , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , MicroARNs/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , Adolescente , Adulto , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Niño , Resistencia a Antineoplásicos/genética , Femenino , Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Humanos , Mesilato de Imatinib/administración & dosificación , Mesilato de Imatinib/efectos adversos , Leucemia Mielógena Crónica BCR-ABL Positiva/diagnóstico , Leucemia Mielógena Crónica BCR-ABL Positiva/mortalidad , Masculino , Persona de Mediana Edad , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/efectos adversos , Resultado del Tratamiento , Adulto JovenRESUMEN
Expression of many miRNAs is altered in different cancers and these changes are thought to play a key role in formation and progression of cancer. In chronic myelogenous leukemia (CML) a number of miRNAs are known to be down regulated as compared to normal cells. In this report we have investigated the mechanism of this down regulation by using PMA induced differentiation of CML cell line K562 to megakaryocytes as an experimental system. On treatment with PMA, expression of many down regulated miRNAs including miR-22 is induced. PMA also induces expression of several transcription factors, including FosB, EGR1 and EGR2. Our results using a number of approaches, such as promoter reporter assay, FosB knock down and Chip assay, suggest that the expression of miR-22 is regulated transcriptionally by FosB.
Asunto(s)
Diferenciación Celular/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , MicroARNs/biosíntesis , Proteínas Proto-Oncogénicas c-fos/genética , Diferenciación Celular/efectos de los fármacos , Proteína 1 de la Respuesta de Crecimiento Precoz/biosíntesis , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Proteína 2 de la Respuesta de Crecimiento Precoz/biosíntesis , Proteína 2 de la Respuesta de Crecimiento Precoz/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Megacariocitos/efectos de los fármacos , MicroARNs/genética , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacología , Ácidos Polimetacrílicos/farmacología , Proteínas Proto-Oncogénicas c-fos/biosíntesisRESUMEN
MicroRNAs control cellular processes by regulating expression of their target genes. Here we report that neuro-epithelial transforming gene 1 (NET1) is a target of tumor suppressor microRNA 22 (miR-22). miR-22 is downregulated in peripheral blood mononuclear cells derived from chronic myeloid leukemia (CML) patients and in CML cell line K562. NET1 was identified as one of the targets of miR-22 using both in vitro and in vivo experiments. Either mutations or naturally occurring single-nucleotide polymorphisms in NET1 3'-UTR that map at the miR-22 binding site were found to affect binding of miR-22 to NET1 mRNA. Over expression of NET1 in K562 cells resulted in increased proliferation. However decreased proliferation and alteration in cell cycle were observed on either overexpression of miR-22 or knockdown of NET1 expression respectively. We also found that overexpression of miR-22 or NET1 knockdown inhibits actin fiber formation, probably by downregulation of NET1 as NET1 knockdown also resulted in depletion of actin fiber formation. We suggest that the oncogenic properties of CML cells are probably due to deregulated expression of NET1 as a result of altered expression of miR-22.
