Analysis of Regulatory miRNAs of Histone Demethylase JMJD3 In MDS CD34+ Hematoprogenitor Cells

Myelodysplastic syndromes (MDS) represent a very complex group of myeloid leukemias with diverse genetic and epigenetic characteristics. We have identified JMJD3, a histone demethylase, aberrantly upregulated in bone marrow CD34+ hematopoietic progenitor cells of MDS (Blood 2009). JMJD3 is a jmjc domain containing histone demethylase that removes methyl groups from Lys-27 of histone H3, and is also known to positively regulate the methylation on Lys-4 of histone H3. This results in activation of upstream regulators of NF-kB signaling, suggesting that JMJD3 plays a crucial role in the pathogenesis of MDS. Therefore, insight on the regulation of JMJD3 expression in MDS is of importance. Aberrant expression of miRNAs, including loss of expression, which leads to up-regulation of the expression of its target genes, has been identified in various types of malignancies including MDS. We hypothesized that alteration of regulatory miRNAs could be involved in the upregulation of JMJD3 in MDS CD34+ cells. Identification of JMJD3 regulatory miRNAs may help further dissect the molecular mechanisms underlying the pathogenesis of MDS.

To identify regulatory miRNAs targeting JMJD3, we performed an analysis using microRNA Genomics Resource (miRGen) and identified 40 candidate miRNAs that potentially interact with the 3′ untranslate region (3′-UTR) of JMJD3 RNA. We then characterized the interactions of these candidate miRNAs to JMJD3 by using a luciferase assay system by cloning the 3′-UTR region of JMJD3 gene into a luciferase reporter construct. By co-transfecting each of the 27 available miRNAs from the 40 candidates together with the luciferase reporter construct into human megakaryocytic blast cell line Meg-O1, we identified 7 mirRNAs (has-mir -29a, -29b, -29c, -99b, -101, -377, and -767-5p) that negatively regulate JMJD3 3′UTR associated luciferase activity. We then examined the expression for these 7 key potential regulatory miRNAs of JMJD3 in a cohort of 36 patient bone marrow CD34+ cells by microRNA Taqman probe based Q-PCR analysis. The median age of these patients was 67 years (32 to 85); 42% had low or INT-1 disease; 21% were diploid and 28% had an alteration of chromosome 5 or 7. Analysis showed that 16 of the 36 MDS CD34+ cell samples examined showed lost expression of mirRNA hsa-mir767-5p, and 7 had down-regulated expression of the same microRNA. For these 23 MDS samples with repressed mir-767-5p expression, 11 of them (~48%) had upregulated expression of JMJD3, as demonstrated by Q-PCR analysis Consistently, in miRNA transfected Meg-O1 cells, Q-PCR analysis indicates that over-expression of mir767-5p is accompanied with decrease of JMJD3 RNA by 68% based on Q-PCR analysis using 3 different probes for JMJD3. Of interest, 4 of the 5 patients with monosomy of chromosome 7 had downregulation of mir767-5p.

These results support the hypothesis that mir767-5p is a key regulatory miRNA targeting JMJD3 in hematopoietic progenitor cells. Since one important mechanism for miRNA on target gene expression is through inhibiting the translation from mRNA to protein, we are now developing an antibody against endogenous JMJD3, with which we will be able to further correlate the level of mir767-5p and the expression of JMJD3 protein in both cell lines and primary MDS samples. Furthermore, correlation between alteration of mir767-5p with clinical, cytogenetic and molecular features in MDS patients will be characterized in a larger cohort of patients. These will further determine the role of mir767-5p for the regulation of JMJD3 and its role during the pathogenesis of MDS. By pursuing the studies described above, a potential link between two key epigenetic regulatory components, histone methylation regulator (JMJD3) and microRNAs, involved in pathogenesis of MDS, will be established.

No relevant conflicts of interest to declare.