Aberrant Epigenetic Down-regulating MicroRNA-663 Enhances H-ras Oncogene Expression In K562 Cell Line

MicroRNAs (miRNAs) are small non-coding RNA approximately 22 nucleotides in length, which target 3’untranslated region (3’UTR) of mRNA specifically to prevent translation of mRNA or degrade mRNA.Aberrant methylation in the promoter region and miRNAs expression were asscociated with leukemogenesis in many researches.From gene regulation step,DNA methylation regulated gene expression before transcription and miRNAs regulated gene expression in transcriptive and translational level.The miRNAs and DNA methylation were both important ways of epigenetic regulation for gene expression. Although the miRNAs and DNA methylation were independent of each other, they were not isolated from each other.They were related to each other and regulated gene expression in suitable developmental time and cell or tissue.In the study, expression of up-regulated miRNAs was screened using microarray assay after DNA demethylation in K562 cell line.The up-regulated miRNAs were selected by bioinformatic analysis, then were detected by realtime PCR for confirming the differential expressions.The miR-663 was found up-regulated after DNA demethylation in K562 cell line. Expression level of miR-663 was detected in leukaemia cell lines, white blood cells from bone marrow of healthy donor and from peripheral blood of pretreatment chronic myelogenous leukemia (CML) patient. The methylated stutus of the CpG island in the upstream region of miR-663 precusor (pre-miR-663) was analyzed in K562 cells and white blood cells from bone marrow of healthy donor by bisulfite-sequencing.The promoter activity of upstream sequences of pre-miR-663 was determined by dual-luciferase reporter assay system.The potential target genes of miR-663 were predicted through bioinformatic analysis.The candidate gene of H-ras and SRC were selected for further study. The luciferase recombinant plasmid incerted the 3’UTR of potential target gene, recombinant plasmid of miR-663 expression and luciferase plasmid of pRL-TK were cotransfected into HEK29T cells.Compare with the control,overexpression miR-663 decreased fluorescence of the target gene.The 3’UTR of miR-663 target gene was mutated,then was inserted into luciferase recombinant plasmid. Compare with the control, ascertained if the reduction in luciferase activity induced by miR-663 overexpression was reversed after mutation of the miR-663 target 3’UTR。The expression of target protein was determined by western blot in K562 cells of overexpression miR-663 and the control. Affections on cell proliferation, cell cycle and apoptosis for the K562 cells were analyzed, in which miR-663 was overexpressed, fuction of miR-663 was suppressed, target gene was knocked down by small interfering RNA (siRNA) respectively.The miR-663 and H-ras mRNA expression level were detected in white cells of bone marrow from healthy donors and CML patients.The results in the study showed that upstream CpG island of pre-miR-663 had promoter activity.The CpG island in the upstream region of pre-miR-663 was aberrant methylation in K562 cell line compared with white blood cells from bone marrow of healthy donor. DNA demethylation could upregulate the expression of miR-663 in K562 cell line, suggesting that miR-663 should be potentially regulated by upstream CpG island.The miRNA-663 could regulate the target gene of H-ras.Overexpression of miR-663 could suppress proliferation of the K562 cell line at least in part due to enhance the cell apoptosis.The miR663 expression level was lower in white cells of bone marrow from CML patients than in white cells of bone marrow from healthy donors.