Mature microRNAs (miRNAs) are 18-24-nucleotide non-coding RNAs with post-transcriptional regulatory functions and have been documented as an essential cornerstone of the genetic system. Although the traditional idea suggests that RNA molecules cannot be stable in extracellular environments due to ubiquitous ribonuclease, miRNA has now been verified as circulating in various body fluids in a stable, cell-free form. By associating with microvesicles (MVs) or RNA-binding proteins, extracellular miRNAs can be actively secreted and transferred into recipient cells, where they regulate target genes. Importantly, extracellular miRNAs have been demonstrated as participating in various physiological and pathological processes in bodies and have significant roles in fetal-maternal crosstalk and cross-kingdom regulation. Furthermore, the abnormal expression of extracellular miRNAs has been shown to be associated with many diseases, making extracellular miRNAs promising novel noninvasive diagnostic markers. In this review, we summarize the recent literature on the biogenesis, delivery and uptake of extracellular miRNAs, elaborate on the regulating function of extracellular miRNAs between different cells and between individuals and highlight their therapeutic potential in clinical applications.

Infection of H5N1 influenza virus causes the highest mortality among all influenza viruses. The mechanisms underlying such high viral pathogenicity are incompletely understood. Here, we report that the H5N1 influenza virus encodes a microRNA-like small RNA, miR-HA-3p, which is processed from a stem loop-containing viral RNA precursor by Argonaute 2, and plays a role in enhancing cytokine production during H5N1 infection. Mechanistic study shows that miR-HA-3p targets poly(rC)-binding protein 2 (PCBP2) and suppresses its expression. Consistent with PCBP2 being an important negative regulator of RIG-I/MAVS-mediated antiviral innate immunity, suppression of PCBP2 expression by miR-HA-3p promotes cytokine production in human macrophages and mice infected with H5N1 virus. We conclude that miR-HA-3p is the first identified influenza virus-encoded microRNA-like functional RNA fragment and a novel virulence factor contributing to H5N1-induced 'cytokine storm' and mortality.

Hepatic miR-122 can serve as a pro-apoptotic factor to suppress tumorigenesis. The underlying mechanism, however, remains incompletely understood. Here we present the first evidence that miR-122 promotes hepatocellular carcinoma cell apoptosis through directly silencing the biogenesis of cell survival oncomiR miR-21 at posttranscriptional level. We find that miR-122 is strongly expressed in primary liver cell nucleus but its nuclear localization is markedly decreased in transformed cells particularly in chemoresistant tumor cells. MiRNA profiling and RT-qPCR confirm an inverse correlation between miR-122 and miR-21 in hepatocellular carcinoma tissues/cells, and increasing or decreasing nuclear level of miR-122 respectively reduces or increases miR-21 expression. Mechanistically, nuclear miR-122 suppresses miR-21 maturation via binding to a 19-nt UG-containing recognition element in the basal region of pri-miR-21 and preventing the Drosha-DGCR8 microprocessor's conversion of pri-miR-21 into pre-miR-21. Furthermore, both in vitro and in vivo studies demonstrate that nuclear miR-122 participates in the regulation of HCC cell apoptosis through modulating the miR-21-targeted programmed cell death 4 (PDCD4) signal pathway.

Methylation of miRNAs at the 2′-hydroxyl group on the ribose at 3′-end (2′-O-methylation, 2′Ome) is critical for miRNA function in plants and Drosophila. Whether this methylation phenomenon exists for mammalian miRNA remains unknown. Through LC–MS/MS analysis, we discover that majority of miR-21-5p isolated from human non-small cell lung cancer (NSCLC) tissue possesses 3′-terminal 2′Ome. Predominant 3′-terminal 2′Ome of miR-21-5p in cancer tissue is confirmed by qRT-PCR and northern blot after oxidation/β-elimination procedure. Cancerous and the paired non-cancerous lung tissue miRNAs display different pattern of 3′-terminal 2′Ome. We further identify HENMT1 as the methyltransferase responsible for 3′-terminal 2′Ome of mammalian miRNAs. Compared to non-methylated miR-21-5p, methylated miR-21-5p is more resistant to digestion by 3′→5′ exoribonuclease polyribonucleotide nucleotidyltransferase 1 (PNPT1) and has higher affinity to Argonaute-2, which may contribute to its higher stability and stronger inhibition on programmed cell death protein 4 (PDCD4) translation, respectively. Our findings reveal HENMT1-mediated 3′-terminal 2′Ome of mammalian miRNAs and highlight its role in enhancing miRNA’s stability and function.

Background Although using a blockade of programmed death-ligand 1 (PD-L1) to enhance T cell immune responses shows great promise in tumor immunotherapy, the immune-checkpoint inhibition strategy is limited for patients with solid tumors. The mechanism and efficacy of such immune-checkpoint inhibition strategies in solid tumors remains unclear. Results Employing qRT-PCR, Sanger sequencing, and RNA BaseScope analysis, we show that human lung adenocarcinoma (LUAD) all produce a long non-coding RNA isoform of PD-L1 (PD-L1-lnc) by alternative splicing, regardless if the tumor is positive or negative for the protein PD-L1. Similar to PD-L1 mRNA, PD-L1-lnc in various lung adenocarcinoma cells is significantly upregulated by IFNγ. Both in vitro and in vivo studies demonstrate that PD-L1-lnc increases proliferation and invasion but decreases apoptosis of lung adenocarcinoma cells. Mechanistically, PD-L1-lnc promotes lung adenocarcinoma progression through directly binding to c-Myc and enhancing c-Myc transcriptional activity. Conclusions In summary, the PD-L1 gene can generate a long non-coding RNA through alternative splicing to promote lung adenocarcinoma progression by enhancing c-Myc activity. Our results argue in favor of investigating PD-L1-lnc depletion in combination with PD-L1 blockade in lung cancer therapy.