Abstract
BackgroundThe miR-9 family microRNAs have been identified as a tumor suppressor miRNA in cancers. We postulated that miR-9-1, miR-9-2 and miR-9-3 might be inactivated by DNA hypermethylation in chronic lymphocytic leukemia (CLL).MethodsMethylation of miR-9-1, miR-9-2 and miR-9-3 was studied in eight normal controls including normal bone marrow, buffy coat, and CD19-sorted peripheral blood B-cells from healthy individuals, seven CLL cell lines, and seventy-eight diagnostic CLL samples by methylation-specific polymerase chain reaction.ResultsThe promoters of miR-9-3 and miR-9-1 were both unmethylated in normal controls, but methylated in five (71.4%) and one of seven CLL cell lines respectively. However, miR-9-2 promoter was methylated in normal controls including CD19 + ve B-cells, hence suggestive of a tissue-specific but not tumor-specific methylation, and thus not further studied. Different MSP statuses of miR-9-3, including complete methylation, partial methylation, and complete unmethylation, were verified by quantitative bisulfite methylation analysis. 5-Aza-2′-deoxycytidine treatment resulted in miR-9-3 promoter demethylation and re-expression of pri-miR-9-3 in I83-E95 and WAC3CD5+ cells, which were homozygously methylated for miR-9-3. Moreover, overexpression of miR-9 led to suppressed cell proliferation and enhanced apoptosis together with downregulation of NFκB1 in I83-E95 cells, supporting a tumor suppressor role of miR-9-3 in CLL. In primary CLL samples, miR-9-3 was detected in 17% and miR-9-1 methylation in none of the patients at diagnosis. Moreover, miR-9-3 methylation was associated with advanced Rai stage (≥ stage 2) (P = 0.04).ConclusionsOf the miR-9 family, miR-9-3 is a tumor suppressor miRNA relatively frequently methylated, and hence silenced in CLL; whereas miR-9-1 methylation is rare in CLL. The role of miR-9-3 methylation in the constitutive activation of NFκB signaling pathway in CLL warrants further study.
Highlights
DNA methylation refers to the chemical modification of the cytosine ring in a CpG dinucleotide by the addition of a methyl group (-CH3) to the number 5 carbon of the cytosine pyrimidine ring in the CpG dinucleotide, resulting in the formation of 5-methylcytosine [1]
Taken together, our results revealed that miR-9-3 was a tumor suppressor miRNA frequently methylated in chronic lymphocytic leukemia (CLL). miR-9-3 was a tumor suppressor miRNA hypermethylated in CLL, which was associated with down-regulation of NFκB1 protein, and might contribute to constitutive activation of NFκB signaling pathway in CLL
M-Methylation-specific polymerase chain reaction (MSP) of miR-9-2 showed that the positive control (PC) and 3 normal CD19 + ve B-cell controls (N1-N3) were completely methylated (B)
Summary
DNA methylation refers to the chemical modification of the cytosine ring in a CpG dinucleotide by the addition of a methyl group (-CH3) to the number 5 carbon of the cytosine pyrimidine ring in the CpG dinucleotide, resulting in the formation of 5-methylcytosine [1]. Global DNA hypomethylation, but aberrant, gene-specific DNA pathogenesis or prognosis of chronic lymphocytic leukemia (CLL) [7,8,9,10,11]. Mature microRNA (miRNAs) are endogenous, singlestranded, non-protein-coding small RNAs measuring 19 to 25 nucleotides (nts), which suppress the expression of proteins that they target [12,13]. Tumor suppressor miRNAs have shown to be silenced by aberrant DNA hypermethylation in cancers [1,16,17]. Previous studies identified methylation of some tumor suppressor miRNAs, including miR203, miR-124-1, miR-181a/b, miR-107 and miR-424, to be involved in CLL leukemogenesis [18]. The miR-9 family microRNAs have been identified as a tumor suppressor miRNA in cancers. We postulated that miR-9-1, miR-9-2 and miR-9-3 might be inactivated by DNA hypermethylation in chronic lymphocytic leukemia (CLL)
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