Extravesicular Mir-7977 Released from Leukemic Cells Inhibites the PCBP1-Mediated mRNA Stabilization and Hippo-YAP Signaling Pathway in Bone Marrow Mesenchymal Stromal Cells

[Background] We and others have revealed that various abnormalities of the bone marrow (BM) environment such as aberrant cytokine expression and impaired microRNA (miRNA) biogenesis are observed in patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). However, underlying mechanisms to induce BM stromal dysfunction have not yet been clarified. Recently, we have found that abundant extracellular vesicles including exosomes, were detected in the BM interstitial fluid. Furthermore, we have shown that exosomal miR-7977 was highly released from AML cells (GSE64029) and was taken up by BM mesenchymal stem cells (MSCs). Based on these findings, we hypothesized that exosomal miR-7977 could be involved in the alteration of BM stromal function of MDS/AML.

[Materials and Methods] To gain an insight into the function of exosomal miR-7977 in BM, we firstly performed transduction of a miR-7977 mimic into BM MSCs and compared transcriptomes between control-transduced and miR-7977-transduced MSCs (GSE108186). Subsequently, we selected the differentially expressed genes (DEGs) and conduced gene set enrichment analysis (GSEA).

[Results] Regarding DEGs, we focused on poly(rC) binding protein 1 (PCBP1), since PCBP1 was predicted to be a target gene of miR-7977 by an online database for miRNA target prediction using miRDB. The reductions in PCBP1 mRNA and protein were confirmed in MSCs after miR-7977 transfer by real time PCR. Because PCBP1 is known to be involved in splicing, and the expression levels of several genes, we analyzed the expression of several hematopoietic factors and found that expression level of stem cell factor (SCF), angiopoietin-1 (ANGPT1) and Jagged-1 (JAG1) were remarkably decreased. Regarding the pathway analysis, GSEA showed that the gene sets of Yes-associated protein 1 (YAP1)_up were significantly enriched (p<0.001, q<0.25), suggesting that miR-7977 modulates the Hippo-YAP signaling pathway. Visualization of pathway and network showed that miR-7977 significantly reduced the expression of Hippo core kinase, STK4. The transfer of miR-7977 mimic inactivated the Hippo-YAP signaling pathway as proven by GFP-tagged YAP nuclear trans localization and TEAD reporter assay. The miR-7977-transduced MSCs showed elevated saturation density and enhanced entry into the cell cycle.

[Conclusion] Collectively, these results indicated that miR-7977 is an important factor that inhibits normal hematopoiesis via PCBP1 reduction and up-regulate the growth of functionally-disturbed MSCs via inactivation of Hippo-YAP signaling pathway in MDS/AML.