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Multiple microRNAs (miRNAs) regulate epithelial-mesenchymal transition and endothelial-mesenchymal transition (EndMT). Here we report that microRNA-27b (miR-27b) positively regulates transforming growth factor-β (TGF-β)-induced EndMT of MS-1 mouse pancreatic microvascular endothelial cells. TGF-β induced miR-23b/24-1/27b expression, and inhibition of miR-27 suppressed TGF-β-mediated induction of mesenchymal genes. Genome-wide miRNA target analysis revealed that miR-27 targets Elk1, which acts as a competitive inhibitor of myocardin-related transcription factor-serum response factor signalling and as a myogenic repressor. miR-27b was also found to regulate several semaphorin receptors including Neuropilin 2, Plexin A2 and Plexin D1. These results suggest important roles of miR-27 in TGF-β-driven EndMT., (© The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Transforming growth factor-β (TGF-β) plays central roles in endothelial-mesenchymal transition (EndMT) involved in development and pathogenesis. Although EndMT and epithelial-mesenchymal transition are similar processes, roles of microRNAs in EndMT are largely unknown. Here, we report that constitutively active microRNA-31 (miR-31) is a positive regulator of TGF-β-induced EndMT. Although the expression is not induced by TGF-β, miR-31 is required for induction of mesenchymal genes including α-SMA, actin reorganization and MRTF-A activation during EndMT. We identified VAV3, a regulator of actin remodeling and MRTF-A activity, as a miR-31 target. Global transcriptome analysis further showed that miR-31 positively regulates EndMT-associated unique secretory phenotype (EndMT-SP) characterized by induction of multiple inflammatory chemokines and cytokines including CCL17, CX3CL1, CXCL16, IL-6 and Angptl2. As a mechanism for this phenomenon, TGF-β and miR-31 suppress Stk40, a negative regulator of NF-κB pathway. Interestingly, TGF-β induces alternative polyadenylation (APA)-coupled miR-31-dependent Stk40 suppression without concomitant miR-31 induction, and APA-mediated exclusion of internal poly(A) sequence in Stk40 3'UTR enhances target efficiency of Stk40. Finally, miR-31 functions as a molecular hub to integrate TGF-β and TNF-α signaling to enhance EndMT. These data confirm that constitutively active microRNAs, as well as inducible microRNAs, serve as phenotypic modifiers interconnected with transcriptome dynamics during EndMT., (© 2015 The Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.)

The precise control of microRNA (miRNA) biosynthesis is crucial for gene regulation. Lin28A and Lin28B are selective inhibitors of biogenesis of let-7 miRNAs involved in development and tumorigenesis. Lin28A selectively inhibits let-7 biogenesis through cytoplasmic uridylation of precursor let-7 by TUT4 terminal uridyl transferase and subsequent degradation by Dis3l2 exonuclease. However, a role of this uridylation pathway remains unclear in let-7 blockade by Lin28B, a paralog of Lin28A, while Lin28B is reported to engage a distinct mechanism in the nucleus to suppress let-7. Here we revisit a functional link between Lin28B and the uridylation pathway with a focus on let-7 metabolism in cancer cells. Both Lin28A and Lin28B interacted with Dis3l2 in the cytoplasm, and silencing of Dis3l2 upregulated uridylated pre-let-7 in both Lin28A- and Lin28B-expressing cancer cell lines. In addition, we found that amounts of let-7 precursors influenced intracellular localization of Lin28B. Furthermore, we found that MCPIP1 (Zc3h12a) ribonuclease was also involved in degradation of both non-uridylated and uridylated pre-let-7. Cancer transcriptome analysis showed association of expression levels of Lin28B and uridylation pathway components, TUT4 and Dis3l2, in various human cancer cells and hepatocellular carcinoma. Collectively, these results suggest that cytoplasmic uridylation pathway actively participates in blockade of let-7 biogenesis by Lin28B., (© 2015 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.)

Asymmetric selection of single-stranded guide RNAs from double-stranded RNA precursors is crucial in RNA silencing-mediated gene regulation. However, the precise mechanisms of small-RNA asymmetry are unclear, especially because asymmetric selection can still occur when the putative asymmetry sensors Drosophila R2D2 and mammalian Dicer are depleted. Here we report a direct contribution of mammalian Argonaute 2 (Ago2) to microRNA (miRNA) asymmetry. Ago2 selects strands with 5'-uridine or 5'-adenosine and thermodynamically unstable 5' ends in parallel through its two sensor regions, which contact the 5' nucleobases and 5'-phosphates of prospective guide strands. Hence, miRNA asymmetry shows superposed patterns reflecting 5'-end nucleotide identity ('digital' pattern) and thermodynamic stability ('analog' pattern). Furthermore, we demonstrate that cancer-associated miRNA variations reprogram asymmetric selection. Finally, our study presents a model of this universal principle, to aid in comprehensive understanding of miRNA function and development of new RNA-silencing therapies in precision medicine.