microRNA-34b/c on chromosome 11q23 is aberrantly methylated in chronic lymphocytic leukemia

Abstract

A commonly deleted region in chronic lymphocytic leukemia (CLL) is the 11q22-23 region, which encompasses the ATM gene. Evidence suggests that tumor suppressor genes other than ATM are likely to be involved in CLL with del(11q). A microRNA (miR) cluster including the miR-34b and miR-34c genes is located, among other genes, within the commonly deleted region (CDR) at 11q. Interestingly, these miRs are part of the TP53 network and have been shown to be epigenetically regulated. In this study, we investigated the expression and methylation status of these miRs in a well-characterized cohort of CLL, including cases with/without 11q-deletion. We show that the miR-34b/c promoter was aberrantly hypermethylated in a large proportion of CLL cases (48%, 25/52 cases). miR-34b/c expression correlated inversely to DNA methylation (P = 0.003), and presence of high H3K37me3 further suppressed expression regardless of methylation status. Furthermore, increased miR-34b/c methylation inversely correlated with the presence of 11q-deletion, indicating that methylation and del(11q) independently silence these miRs. Finally, 5-azacytidine and trichostatin A exposure synergistically increased the expression of miR-34b/c in CLL cells, and transfection of miR-34b or miR-34c into HG3 CLL cells significantly increased apoptosis. Altogether, our novel data suggest that miR-34b/c is a candidate tumor suppressor that is epigenetically silenced in CLL.

Keywords: DNA methylation; chronic lymphatic leukemia; epigenetics; micro-RNA.

Figure 1. DNA methylation as measured by MS-MCA (x-axis) compared with bisulfite pyrosequencing (y-axis) in normal controls (n = 4) and CLL samples (n = 28).

Figure 2. Expression of miR-34a (n = 8), miR-34b (n = 16), and miR-34c (n = 15) by RQ-PCR in CLL samples. ΔΔCT for each miR compared with the reference gene RNU6 was calculated. P values indicate difference in expression between miRs.

Figure 3. Expression of miR-34b and miR-34b in unmethylated (n = 9) vs. methylated (n = 6) CLL samples by RQ-PCR. Expression levels are presented as in Figure 2 and p-values show the difference in expression between indicated groups.

Figure 4. Effect on miR-34b and miR-34b expression by Decitabine (DAC) and trichostatin A (TSA) alone and in combination. HG3 cells were cultured either with DAC at 0,005 μM/L for 72 h,, with TSA at 500nM/L for the last 24 h or in combination with DAC for 48 h followed by TSA for 24 h. Reference gene for RQ-PCR analysis was RNU6.

Figure 5. (A) Higher normalized levels of H3K27me3 at the miR-34b/c promoter in normal CD19+ lymphocytes (n = 3) compared with CLL samples (n = 6)(P = 0.048). Error bars show ± 2 SD (B) miR-34b/c expression in relation to DNA methylation (x-axis) and H3K27me3 levels (dichotomized at median) (z-axis). Sample numbers are indicated in the figure and individual measurements for each sample are given in Table S2. (C) Nucleosome occupancy measured by DHSS, normalized to GADPH. There is no difference between unmethylated and methylated CLL samples, P = 0.4 and 0.3 for promoter and TSS respectively. Error bars show ± 2 SD.

Figure 6. Results of miR-34b and miR-34c overexpression on apoptosis measured as Annexin V positivity by FACS in the HG3 cells incubated for 24 h. Box plots indicate the percentages of apoptosis of six individual experiments (P = 0.036 for miR-34b and P = 0.026 for miR-34c compared with control).