Epigenetic inactivation of mir-34b/c in addition to mir-34a and DAPK1 in chronic lymphocytic leukemia

Abstract

BackgroundTP53 mutation/deletion is uncommon in chronic lymphocytic leukemia (CLL). We postulated that components of TP53-centered tumor suppressor network, miR-34b/c, in addition to DAPK1 and miR-34a might be inactivated by DNA hypermethylation. Moreover, we tested if miR-34b/c methylation might correlate with miR-203 or miR-124-1 methylation in CLL.MethodsmiR-34b/c, miR-34a and DAPK1 methylation was studied in 11 normal controls, 7 CLL cell lines, and 78 diagnostic CLL samples by methylation-specific polymerase chain reaction. MEC-1 cells were treated with 5-Aza-2’-deoxycytidine for reversal of methylation-associated miRNA silencing. Tumor suppressor properties of miR-34b were demonstrated by over-expression of precursor miR-34b in MEC-1 cells.ResultsmiR-34b/c promoter was unmethylated in normal controls, but completely methylated in 4 CLL cell lines. miR-34b/c expression was inversely correlated with miR-34b/c methylation. Different MSP statuses of miR-34b/c, including complete methylation and complete unmethylation, were verified by quantitative bisulfite pyrosequencing. 5-Aza-2’-deoxycytidine treatment resulted in promoter demethylation and miR-34b re-expression in MEC1 cells. Moreover, over-expression of miR-34b resulted in inhibition of cellular proliferation and increased cell death. In primary CLL samples, miR-34a, miR-34b/c and DAPK1 methylation was detected in 2.6%, 17.9% and 34.6% of patients at diagnosis respectively. Furthermore, 39.7%, 3.8% and 2.6% patients had methylation of one, two or all three genes respectively. Overall, 46.2% patients had methylation of at least one of these three genes. Besides, miR-34b/c methylation was associated with methylation of miR-34a (P = 0.03) and miR-203 (P = 0.012) in CLL.ConclusionsTaken together, miR-34b/c is a tumor suppressor miRNA frequently methylated, and hence silenced in CLL. Together with DAPK1 methylation, miR-34b/c methylation is implicated in the disruption of the TP53-centered tumor suppressor network. Moreover, the association of miRNA methylation warrants further study.

Hypermethylation of potential tumor suppressor miR-34b/c is correlated with late clinical stage in patients with soft tissue sarcomas

“…Strong evidence demonstrates a positive correlation between the appearance of target gene methylation and the reduction in the level of target protein expression (Bauer et al, 2010;Rodriguez-Paredes and Esteller, 2011). Previous studies have shown that hypermethylation of miR-34b/c contributed to the down-regulation protein expression in lung adenocarcinoma, colon cancer and chronic lymphocytic leukemia (Nadal et al, 2013;Roy et al, 2012;Wang et al, 2014). In the present study, aberrant miR-34b/c methylation occurs in STSs, it has been hypothesized that miR-34b/c hypermethylation was related to the down-regulation of miR-34b/c gene expression.…”

“…Similarly, miR-34b/c expression was inversely correlated with miR-34b/c methylation in chronic lymphocytic leukemia. MiR-34b re-expression by demethylation treatment resulted in the repression of cellular proliferation and increased cell death in MEC1 cells (Wang et al, 2014). Moreover, a recent study identified that the abnormal methylation of miR-34b/c was associated with a high probability of recurrence as well as poor overall survival and disease-free survival.…”

MicroRNAs in chronic lymphocytic leukemia: miRacle or miRage for prognosis and targeted therapies?

“…These miRNAs are targets of the tumor suppressor p53 (TP53) 16 , which is often deregulated or mutated in CLL. miR-34b/c expression is significantly lower in CLL patients with del11q 17 and besides being deleted in CLL, miR-34b/c is also often epigenetically inactivated through hypermethylation 18,19 . Furthermore, an inverse correlation has been found between the presence of del11q and miR-34b/c hypermethylation, suggesting that there are different modes of silencing miR-34b/c that are independent of each other 18 .…”

MicroRNAs in Disease

“…The p53 transcription factor is a direct regulator of miR-34a and miR-34b/c, while these miRNAs target and regulate p53 expression [ 81 ]. In chronic lymphocytic leukemia, miR-34a, miR-34b/c and DAPK1 were found to be epigenetically inactivated by hypermethylation of their promoter to disrupt the tumor suppressive p53 pathway [ 82 ]. Hypermethylation of miR-34b/c is also considered as a potential diagnostic factor in Stage I non-small cell lung carcinoma [ 83 ].…”