Epigenetic silencing of a long non-coding RNA KIAA0495 in multiple myeloma

Abstract

In multiple myeloma, a long non-coding RNA, KIAA0495 (alias PDAM/TP73-AS1), had been found progressively downregulated from normal plasma cell to benign monoclonal gammopathy of undetermined significance to symptomatic myeloma. Herein, by methylation-specific PCR, the putative KIAA0495 promoter was found unmethylated in all healthy controls (N = 14) but methylated in 50 % of myeloma cell lines (N = 10). KIAA0495 methylation was shown inversely correlated with KIAA0495 expression. However, KIAA0495 methylation was detected in none of both primary myeloma samples at diagnosis (N = 61) and at relapse/progression (N = 16). Collectively, despite frequently methylated in cell lines, KIAA0495 methylation appeared unimportant in the pathogenesis or progression of myeloma.

Fig. 1

Methylation of KIAA0495 in myeloma. a Sequencing analysis of M-MSP products showed that the cytosine [C] residues of CpG dinucleotides were methylated and remained unchanged after bisulfite conversion, whereas all the other non CpG C residues were unmethylated and converted to thymidine [T], indicating complete bisulfite conversion and specificity of MSP. b M-/U-MSP showed KIAA0495 was completely methylated in positive control with methylated DNA, but completely unmethylated in 14 healthy donor controls, including peripheral buffy coat (N1-N10), marrow buffy coat (N11-N13), and CD138-sorted marrow plasma cells (N14). c M-/U-MSP showed KIAA0495 was partially methylated in KMS-12-PE, LP-1, NCI-H929, OCI-MY5, and OPM-2, whereas it was completely unmethylated in MOLP-8, RPMI-8226, U-266, WL-2, and JJN-3. d Quantitative bisulfite pyrosequencing interrogating the methylation intensity on a stretch of 7 neighboring CpG dinucleotides indicated consistent resutls as shown by the MSP statuses (MM, MU, and UU)

Fig. 2

Methylation and expression of KIAA0495 in myeloma cells. a Semi-quantitative RT-PCR analysis showed expression of KIAA0495 in MOLP-8, RPMI-8226, U-266, WL-2, and JJN-3, which were completely unmethylated for KIAA0495, but not in KMS-12-PE, LP-1, NCI-H929, OCI-MY5, and OPM-2, which were partially methylated for KIAA0495 (P = 0.0079). Sequencing analysis confirmed the authenticity of the RT-PCR of KIAA0495. b qRT-PCR analysis showed the expression of KIAA0495 was significantly higher, as indicated by a lower ΔC_t, in methylated than completely unmethylated myeloma cell lines (P = 0.000155). c Two partially methylated myeloma cell lines, LP-1 and OCI-MY5, were separately treated with 1 μmol/l of 5-AzadC for 3 days, after which, 5-AzadC was removed and cells were cultured for additional 6 days. On day 3, upon treatment with 5-AzadC, KIAA0495 was demethylated as indicated by the decreased percentage of methylation intensity by quantitative bisulfite pyrosequencing, with concommitent KIAA0495 re-expression as measured by qRT-PCR. On day 9, upon removal of 5-AzadC for 6 days, methylation of KIAA0495 was restored with re-suppression of KIAA0495

Fig. 3

Methylation of KIAA0495 in primary myeloma marrow samples at diagnosis (Dx) and at relapse/progression (R)