ABSTRACT
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.
Subject(s)
Cell Differentiation/drug effects , Cell Differentiation/genetics , Leukemia, Myeloid/genetics , MicroRNAs/genetics , Phorbol Esters/pharmacology , Phosphorylcholine/analogs & derivatives , Polymethacrylic Acids/pharmacology , Cell Line, Tumor , Genetic Heterogeneity/drug effects , Humans , K562 Cells , Phosphorylcholine/pharmacologySubject(s)
Antineoplastic Agents/therapeutic use , Biomarkers, Tumor , Imatinib Mesylate/therapeutic use , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , MicroRNAs/genetics , Protein Kinase Inhibitors/therapeutic use , Adolescent , Adult , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/adverse effects , Child , Drug Resistance, Neoplasm/genetics , Female , Gene Expression Profiling , Gene Expression Regulation, Leukemic , Humans , Imatinib Mesylate/administration & dosage , Imatinib Mesylate/adverse effects , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/diagnosis , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/mortality , Male , Middle Aged , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/adverse effects , Treatment Outcome , Young AdultABSTRACT
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.
