Abstract
MicroRNAs (miRNAs) are short noncoding RNAs regulating a variety of biological processes by post-transcriptionally silencing via targeting mRNA. Recently there are many reports demonstrating that epigenetic alterations correlate to the characteristics of tumor cells, and that miRNAs were also reported to be regulated by methylation of CpG islands within the promoter region. MiR-203 is epigenetically silenced in human BCR-ABL1-positive leukemic cell lines and primary chronic myelogenous leukemia (CML) cells by the methylation of promoter region. In this study, we analyzed the effect of imatinib, a tyrosine-kinase inhibitor specific to BCR-ABL1 protein, on the expression of miRNA in BCR-ABL1-positive cells.
Two CML cell lines (K562 and KU812) and one AML cell line (HL60) were treated with imatinib for 72 hours. Microarray analysis of miRNAs was conducted by 3D-Gene (TORAY) in K562 cells with/without imatinib. Methylation specific PCR and bisulfite direct sequencing was performed to evaluate methylation status of promoter region of miR-203. Validation of expressions of miRNAs, including miR-203, and mRNAs was analyzed by RT-qPCR. The expression of BCR-ABL protein was confirmed by Western blotting. The function of miR-203 for cell survival was evaluated by the transfection of anti-miRNA.
The microarray analysis showed that 48 miRNAs of CpG-rich 212 miRNAs were upregulated over 2-fold after imatinib treatment, in K562 cells. The demethylated state of the promoter region of miR-203, one of 48 miRNAs, was confirmed by bisulfite direct sequencing. The expression of BCR-ABL mRNA, which is one of the target of miR-203, was inhibited with imatinib to 52% and 26% of the level in control cultures in K562 cells and KU812 cells, respectively. The expression of BCR-ABL protein was also inhibited. The addition of anti-miR-203 increased the expression level of BCR-ABL protein to 68.1% in the K562 cell culture with imatinib treatment. The expression of DNA methyltransferase (DNMT) mRNA was analyzed, and the expressions of DNMT1 and DNMT3B were significantly decreased after imatinib treatments in CML cell lines, whereas the expression of DNMT3A was not changed.
We report, for the first time, that imatinib up-regulated miR-203 by inducing demethylation of the promoter region of miR-203 in CML cells. MiR-203 is the important miRNA to inhibit ABL1 and BCR-ABL1 mRNA, and imatinib-induced demethylation of miR-203 is the possible mechanism to suppress growth of BCR-ABL1-positive leukemic cells. It was suggested that the demethylation was partially caused by down regulation of DNMT1 and DNMT3B after imatinib treatments in CML cell lines. In conclusion, imatinib not only inhibits the activity of tyrosine kinase but also induces DNA demethylation of miR-203 in CML cells.
No relevant conflicts of interest to declare.
Author notes
Asterisk with author names denotes non-ASH members.
