Abstract
Micro RNAs (miRs) are small non-coding RNAs of 19-25 bases in length having the ability to modulate the expression of other genes. MiRs are frequently involved in carcinogenesis and its expression analysis to predict the phenotype of malignancies. Transcriptional silencing of tumor suppressor genes in cancer cells is often associated with hypermethylation of the promoter CpG islands. Three different DNMTs play major roles in establishing and maintaining DNA methylation patterns. It has been demonstrated that miR-29 family down-regulate DNMTs expression and several tumor suppressor miRs are hypermethylated and consequently down-regulated in cancer cells. Many studies have evaluated miR expression, epigenetics and the prognosis of acute myeloid leukemia (AML), but interrelationships between expression and methylation of miRs and DNMTs expression in AML patients’ samples has not been fully elucidated.
BM samples obtained from 54 of AML patients are subjected to the study after informed consent. Control BM samples are obtained from12 patients suffering from lymphoma. This study was approved by IRB of Gunma University Hospital. DNA, RNA and miR were extracted from BM mononuclear cells. The expression levels of miR 29a, 29b, 29c, 34a, 34b, 34c and mRNAs of DNMT1, 3A, 3B, primary miR (pri-miR) 29a/b-1, pri-miR 34a, 34b/c, c-myc, p21 were quantified by real time PCR with either Taqman-probe or SYBR green. Methylation specific PCR (MSP) was carried out with bisulfite converted DNA to see methylation status of for miR 34a, 34b/c.
The expression levels of miR29a, 29b, 29c, pri-miR29a/b-1, 34a, 34b, 34b star and 34c were significantly reduced in cells of AML (median 0.55, 0.19, 0.34, 50.98, 213.40, 1.61, 260.09 and 288.49 respectively) than of control (3.08, 4.78, 3.57, 2082.49, 472.68, 28.99 and 1781.33,) (p<0.001). Contrary, the expression levels of pri-miR34a and miR 34a star, were not significantly different between the AML and control samples. The promoter of miR34a and miR34b/c have CpG island which are often hyper-methylated thus we examined methylation of these miRs using MSP. We found that the promoters of miR34a and miR34b/c are more frequently methylated in AML than in control (38% vs 11%, p=0.005, 69% vs 17%, p<0.001). These results implied that miR expressions are epigenetically regulated through DNA methylation in AML. MiR34a, miR34b/c and pri-miR34a levels were not significantly different in between methylated and un-methylated group of AML, even though the miRs tended to be lower in methylated group. When the correlation between DNMTs and miRs expression levels were analyzed in AML, DNMTs expression tended to be negatively correlated with miR 34 family expression, but significant negative correlation were observed in DNMT1 and miR34b (r=-0.365, p=0.014) DNMT3B and miR34b, c (r=-0.556, p=0.017, r=-0.494, p=0.037, respectively). MiR34 family transcription is up-regulated by TP53. We analyzed the correlation between miR34 family and well-known TP53 target p21, but there was no significant correlation. C-myc is also known to suppress miR29 family which is negative regulator of DNMTs. C-myc level is significantly higher in AML than control (0.019 vs. 0.007, p=0.001) and miR29 family is lower in AML, but the correlation between c-myc and miR29 expressions in AML did not reach statistical significance. C-myc is positively correlated with DNMT1 and 3A expressions (r=0.753, r=0.610, p<0.001). There were significant positive correlations among miRs expressions in AML samples; miR29a-29b r=0.717, 29a-29c r=0.739, 29a-34a r=0.568, p<0.001, 29a-34b r=0.309, p=0.029, miR29b-29c r=0.893, 29b-34a r=0.596, 29b-34b r=0.621, 29b-34c r=0.518, 29c-34a r=0.646, 29c-34b r=0.655, 29c-34c r=0.500, p<0.001. When the cell lines were treated with 5 deoxy-azacytidine, miR34a, b, c expression levels increased after the treatment, suggesting that these miRs expression was regulated at least in part by promoter hypermethylation.
We found significant reduction of miRs expression in AML and the reduction in part associated with hypermethylation. Direct correlations in between miR expression, methylation and DNMT could not be shown in this study, however, we somehow showed interrelationships between c-myc and DNMTs, miR29 and miR34 family. C-myc, miR29, DNMT, methylation, miR34 axis may play an important role in AML development and progression.
No relevant conflicts of interest to declare.
Author notes
Asterisk with author names denotes non-ASH members.
