Association Between Micro-RNA and Epigenetic Modifiers DNA Methyltransferases (DNMTs), Histone Deacetylases (HDACs) in Multiple Myeloma (MM) and Monoclonal Gammmopathy with Undetermined Significance (MGUS)
Authors: Handa Hiroshi , Hattori Hikaru , Takahashi Noriyuki , Sasaki Yoshiko , Saitoh Takayuki , Osaki Yohei , Tahara Kenichi , Koiso Hiromi , Mitsui Takeki , Shimizu Hiroaki , Takizawa Makiko , Yokohama Akihiko , Tsukamoto Norifumi , Alkebsi Lobna , Nojima Yoshihisa , Murakami Hirokazu 
Published: October 2019

Abstract
Abstract 3942

Background:
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 has been shown. Transcriptional silencing of tumor suppressor genes in cancer cells is often associated with hypermethylation of their promoter CpG islands and histone deacetylation. Three different DNMTs play major roles in establishing and maintaining DNA methylation patterns, DNMT1, 3a, 3b. Histone acetylation is carried out by histone acetyltransferases and acetyl groups can be removed by histone deacetylases (HDACs). Cancer cells often show high expression of epigenetic modifiers DNMTs and HDACs. It has been demonstrated that miR-15a, b miR-16, miR-29 family down-regulate DNMTs and Sp-1 regulating HDACs expression. Many studies evaluate miR expression and its association with chromosomal abnormalities and the prognosis of multiple myeloma (MM), but the relationship between miR and its target gene expression in the patients has not been reported.

Methods:
MM cell lines and BM samples obtained from 46 of MM patients, 23 of MGUS patients are subjected to the study after informed consent. Normal plasma cells are obtained from the bone marrow samples of 13 patients with malignant lymphoma. Sequential BM samples during the treatment were obtained from 6 patients. RNA and miR were extracted from plasma cells separated by CD138 antibody and magnetic beads. MiR15a, 15b, 16, 29a, 29b, 29c and mRNA expression of DNMT1, 3A, 3B, HDAC 1–11 were quantified by real time PCR with either Taqman-probe or SYBR green. To verify the role of these miRs, miR mimics and miR inhibitor were transduced to MM cell lines with lipofectamine, mRNA expressions of their target genes were analyzed with real time PCR.

Results:
The expression level of miR15a and 16 was reduced in plasma cells of MM (6.4±3.1, 19.5±7.5) than of MGUS (34.8±15.4, 72.1±36.5) and of normal subjects (74.4±1.5, 25.5±12.9)(p=0.02, p=0.02). MiR 15b, 29a, 29b, 29c expressions were not significantly different among MM, MGUS and normal subjects. Conversely, DNMT1, 3A were elevated in MM (7.2±4.5, 8.5±3.3) than in MGUS (0.8±0.2, 1.7±0.6) and normal subjects (0.4±0.3, 2.1±1.8) (p=0.03). DNMT3B expression was not different among subjects. HDAC3, 7, 9 expression were elevated in MM (0.9±0.4, 6.7±2.8, 138±59) than in MGUS (0.46±0.24, 1.4±0.7, 46.8±43.3) (p=0.03, p=0.001, p=0.001) and normal subjects (0.18±0.17, 1.1±0.97, 20.8±9.9) (p=0.01, p=0.017, p=0.012). HDACs expressions were not significantly different in between plasma cells of MGUS and of normal subjects. In the MM cell lines, miR 29a and 29b expression were inversely correlated with DNMT1 mRNA expression (r=0.96, p=0.0003: r=0.86, p=0.014). In the patient samples, DNMT1 was inversely correlated with miR15a, miR15b, miR16, miR29a, miR29b (r=-0.435, p=0.003; r=-0.341, p=0.02, r=-0.332, p=0.03, r=-0.419, p=0.005, r=-0.407, p=0.006), DNMT3A was inversely correlated with miR15a, miR29a, miR29b, miR29c (r=-0.365, p=0.02; r=-0.315, p=0.04; r=-0.371, p=0.01; r=-0.315, p=0.04), DNMT3B was inversely correlated with miR15a, miR15b, miR29a, miR29b (r=-0.418, p=0.005; r=-0.385, p=0.01; r=-0.353, p=0.02; r=-0.358, p=0.02). HDAC1, 3, 7 were inversely correlated only with miR15a (r=-0.25, p=0.039, r=-0.27, p=0.025, r=-0.25, p=0.038). Four of 6 patients' plasma cells showed decreasing miR 15a, 15b, 16 and 29 families and increasing DNMT1 expression during the treatment. Those patients acquired drug resistance. One patient's sample whose miR expression increased during the treatment showed reduced DNMT1 expression. The effects of transduction of miR mimics and inhibitors to MM cell lines were various among cell lines, miR16, 29a, b showed ability to reduce DNMTs expression.

Conclusion:
We found significant reduction of miRs expression and elevation DNMT1, 3A and HDAC3, 7, 9 in MM and possibly associated with MM progression. Negative correlation with the expression level of the DNMTs, HDACs and miRs in patients' samples were seen. The sequential samples during the treatment and transduction experiment in vitro suggest that miRs regulate epigenetic modifiers expression It has been reported that miRs expressions themselves are epigenetically controlled, thus these factors may affect each other and be related to MM progression.

Disclosures:
No relevant conflicts of interest to declare.