Extracellular Vesicle microRNAs from Acute Myeloid Leukemia Are Involved in the Regulation of Adherence Junction in Bone Marrow Microenvironment.
Authors: Yoshida Masahiro , Kobune Masayoshi , Miura Shogo , Ibata Soushi , Iyama Satoshi , Sato Tsutomu , Murase Kazuyuki , Takada Kohichi , Ono Kaoru , Hashimoto Akari , Tatekoshi Ayumi , Kamihara Yusuke , Sugama Yusuke , Kikuchi Shohei , Ikeda Hiroshi , Horiguchi Hiroto , Kawano Yutaka , Miyanishi Koji , Kuroda Hiroyuki , Maeda Masahiro , Kato Junji 
Published: October 2019

Abstract
Background. It has been shown that bone marrow (BM) microenvironment and physiological hematopoiesis were disturbed during development of acute myelogenous leukemia (AML). However, little is known the molecular mechanism involved in this process. We have recently demonstrated that high number of extracellular vesicles (EVs) including exosomes exist in BM interstitial fluid. Myeloid neoplasm derived EVs carry miRNAs and transmitted into mesenchymal stem cells (MSCs) (Highlights of ASH 2015, GSE64029). A subset of extracellular vesicular miRNAs including miR-7977 was increased in BM cavity and regulate the expression level of mRNA stabilizer poly(rC) binding protein 1 (PCBP1) and modulated the function of hematopoietic supporting capacity of MSCs. On the other hand, a relatively large subset of extra vesicular miRNAs was actually decreased in BM cavity of AML as compared with that of healthy volunteer. However, the clinical significance of these decreased miRNAs is unresolved. In the present study, miRNA pathway analysis was conducted to elucidate the role of decreased miRNAs in BM environment of AML.
Methods. To harvest EVs from 2 x 105primary AML CD34+ cells, normal BM CD34+ cells and leukemic cell lines including TF-1 and Kasumi-1, cells were cultured in serum-free StemPro®-34 medium with a cytokine cocktail on plates coated with fibronectin fragments. EV miRNA from the supernatant of CD34+ hematopoietic and leukemic cells were prepared. Microarray analysis of the miRNA profiles was done with the human miRNA Oligo chip (Human_miRNA_V20) and the 3D-Gene® miRNA labeling kit. For analysis of transcriptome in CD34+/CD38- normal and AML cells, data sets (GSE24395) was downloaded as a matrix by GEOquery package (Bioconductor, R commander version 3.2.2). The data sets (GSE24395) and our data sets (GSE64029) were normalized by rma method using limma package before analysis. Clustering analysis were performed with amap package to find a subset of miRNA decreased. For miRNA pathway analysis, DIANA-mirPath version 3 was utilized.
Results. Thirty EV miRNAs derived from AML were decreased as compared with those from normal CD34+ cells. Especially, miR-92a-3p, miR-125a-3p, miR-4448, miR-4484 and miR-4270 were remarkably reduced. The miRNA pathway analysis indicated that a KEGG pathway (hsa04520), adherens junction strongly correlated with these decreased miRNAs (P=0.00000025). The miRNAs including miR-4448, miR-4484 and miR-4270 could be associated with downstream molecules of adherens junction including beta-catenin, IGF-1R, WAVE, Vinculin, TGF-beta and Smad4. Importantly, we found that CD34+CD38- AML stem cells highly expressed JAM family molecules (JAM2 and JAM3) associated with adherens junction (GSE24395). These results suggested that these miRNAs regulate the pathway of adherens junction in BM microenvironment. In addition, adherens junction could be enhanced concomitant with reduction of miRNAs released from AML.
Conclusion. EV miRNAs derived from AML are involved in the regulation of the pathway of adherens junction in BM microenvironment. Adherens junction is known to contribute to quiescence, apoptosis and drug resistance via HIPPO signaling pathway, EV miRNAs may have an important role on the development and drug resistance of AML.

Disclosures
No relevant conflicts of interest to declare.