Effect of 5-Aza-2′-deoxycytidine (5-AzadC) treatment on lymphoma and myeloma cells.

<p>(A) M-/U-MSP analysis of <i>miR-124-1</i> promoter methylation status and stem-loop qRT-PCR analysis of the mature <i>miR-124</i> expression. 5-AzadC treatment resulted in progressive demethylation of <i>miR-124-1</i> promoter, and re-expression of the mature <i>miR-124</i> in cell lines harbouring homozygous <i>miR-124-1</i> methylation. (B) ChIP analysis for trimethyl H3K4, trimethyl H3K9, acetyl H3K9, trimethyl H3K27 in <i>miR-124-1</i> promoter. 5-AzadC treatment led to augmentation of euchromatin code of trimethyl H3K4. (C) Western blot analysis of CDK6 in response to 5-AzadC treatment. Bottom row showed densitometric quantization of the Western blot, indicating relative CDK6 expression under actin normalization.</p

Promoter methylation of <i>miR-124-1</i> and expression of <i>miR-124</i> in primary samples.

<p>(A) Methylation of <i>miR-124-1</i> in primary samples. (B) M-/U-MSP analysis of <i>miR-124-1</i> promoter methylation status and (C) Stem-loop qRT-PCR analysis of the mature <i>miR-124</i> expression in 25 primary NHL samples with matched DNA and RNA. ΔC_t, C_t<i>miR-124</i> -C_t<i>RNU48</i>.</p

Methylation of <i>miR-124-1</i>.

<p>(A) Schematic diagram showing the distribution of CpG dinucleotides (solid vertical lines) over the precursor (solid black box) and mature <i>miR-124-1</i>. Sequence analysis of the M-MSP product from bisulfite-treated positive control DNA showed that the cytosine [C] residues of CpG dinucleotides were methylated and remained unchanged, whereas all the other C residues were unmethylated and were converted to thymidine [T], indicating complete bisulfite conversion and specificity of MSP. Grey bars indicated the amplification regions of the MSP, ChIP, and BGS primers. (B) U-MSP showed that the methylated positive control [P] was totally methylated, and all five normal controls (N1–N5) were unmethylated. In the M-MSP, the methylated control was positive (methylated) but all normal controls were negative (unmethylated). For the cell lines, SUP-T1, SUP-M2 (ALK+), SU-DHL-1 (ALK+), KARPAS-299 (ALK+), KMS-12-PE, LP-1, OPM-2, and WL-2 were completely methylated of <i>miR-124-1</i>. (C) Bisulfite genomic sequencing for the bisulfite-treated promoter region of <i>miR-124-1</i> of normal controls (N1–N5), lymphoma and myeloma cell lines of different methylation statuses (MM, UM, or UU), and the methylated positive control were depicted. Unmethylated (empty circle) and methylated (filled circle) CpG dinucleotides were shown by eight independent clones for each sample.</p