miRNAs derived from milk small extracellular vesicles inhibit porcine epidemic diarrhea virus infection.

Porcine epidemic diarrhea virus (PEDV), a member of the genus Alphacoronavirus in the family Coronaviridae, causes acute diarrhea and/or vomiting, dehydration, and high mortality in neonatal piglets. It has caused huge economic losses to animal husbandry worldwide. Current commercial PEDV vaccines do not provide enough protection against variant and evolved virus strains. No specific drugs are available to treat PEDV infection. The development of more effective therapeutic anti-PEDV agents is urgently needed. Our previous study suggested that porcine milk small extracellular vesicles (sEV) facilitate intestinal tract development and prevent lipopolysaccharide-induced intestinal injury. However, the effects of milk sEV during viral infection remain unclear. Our study found that porcine milk sEV, which was isolated and purified by differential ultracentrifugation, could inhibit PEDV replication in IPEC-J2 and Vero cells. Simultaneously, we constructed a PEDV infection model for piglet intestinal organoids and found that milk sEV also inhibited PEDV infection. Subsequently, in vivo experiments showed that milk sEV pre-feeding exerted robust protection of piglets from PEDV-induced diarrhea and mortality. Strikingly, we found that the miRNAs extracted from milk sEV inhibited PEDV infection. miRNA-seq, bioinformatics analysis, and experimental verification demonstrated that miR-let-7e and miR-27b, which were identified in milk sEV targeted PEDV N and host HMGB1, suppressed viral replication. Taken together, we revealed the biological function of milk sEV in resisting PEDV infection and proved its cargo miRNAs, miR-let-7e and miR-27b, possess antiviral functions. This study is the first description of the novel function of porcine milk sEV in regulating PEDV infection. It provides a better understanding of milk sEV resistance to coronavirus infection, warranting further studies to develop sEV as an attractive antiviral.

Functional assessment of miR­1291 in colon cancer cells.

miR­1291 exerts an anti­tumor effect in a subset of human carcinomas, including pancreatic cancer. However, its role in colorectal cancer (CRC) is largely unknown. In the present study, the expression and effect of miR­1291 in CRC cells was investigated. It was identified that miR­1291 significantly suppressed the proliferation, invasion, cell mobility and colony formation of CRC cells. Additionally, miR­1291 induced cell apoptosis. A luciferase reporter assay revealed that miR­1291 directly bound the 3'­untranslated region sequence of doublecortin­like kinase 1 (DCLK1). miR­1291 also suppressed DCLK1 mRNA and protein expression in HCT116 cells that expressed DCLK1. Furthermore, miR­1291 suppressed cancer stem cell markers BMI1 and CD133, and inhibited sphere formation. The inhibitory effects on sphere formation, invasion and mobility in HCT116 cells were also explored and verified using DCLK1 siRNAs. Furthermore, miR­1291 induced CDK inhibitors p21WAF1/CIP1 and p27KIP1 in three CRC cell lines, and the overexpression of DCLK1 in HCT116 cells led to a decrease of p21WAF1/CIP1 and p27KIP1. Intravenous administration of miR­1291 loaded on the super carbonate apatite delivery system significantly inhibited tumor growth in the DLD­1 xenograft mouse model. Additionally, the resultant tumors exhibited significant upregulation of the p21WAF1/CIP1 and p27KIP1 protein with treatment of miR­1291. Taken together, the results indicated that miR­1291 served an anti­tumor effect by modulating multiple functions, including cancer stemness and cell cycle regulation. The current data suggested that miR­1291 may be a promising nucleic acid medicine against CRC.

MiRNAs in Gestational Diabetes Mellitus: Potential Mechanisms and Clinical Applications.

Gestational diabetes mellitus (GDM) is a common pregnancy complication which is normally diagnosed in the second trimester of gestation. With an increasing incidence, GDM poses a significant threat to maternal and offspring health. Therefore, we need a deeper understanding of GDM pathophysiology and novel investigation on the diagnosis and treatment for GDM. MicroRNAs (miRNAs), a class of endogenic small noncoding RNAs with a length of approximately 19-24 nucleotides, have been reported to exert their function in gene expression by binding to proteins or being enclosed in membranous vesicles, such as exosomes. Studies have investigated the roles of miRNAs in the pathophysiological mechanism of GDM and their potential as noninvasive biological candidates for the management of GDM, including diagnosis and treatment. This review is aimed at summarizing the pathophysiological significance of miRNAs in GDM development and their potential function in GDM clinical diagnosis and therapeutic approach. In this review, we summarized an integrated expressional profile and the pathophysiological significance of placental exosomes and associated miRNAs, as well as other plasma miRNAs such as exo-AT. Furthermore, we also discussed the practical application of exosomes in GDM postpartum outcomes and the potential function of several miRNAs as therapeutic target in the GDM pathological pathway, thus providing a novel clinical insight of these biological signatures into GDM therapeutic approach.

Cerium oxide nanoparticle delivery of microRNA-146a for local treatment of acute lung injury.

Acute respiratory distress syndrome (ARDS) is a devastating pulmonary disease with significant in-hospital mortality and is the leading cause of death in COVID-19 patients. Excessive leukocyte recruitment, unregulated inflammation, and resultant fibrosis contribute to poor ARDS outcomes. Nanoparticle technology with cerium oxide nanoparticles (CNP) offers a mechanism by which unstable therapeutics such as the anti-inflammatory microRNA-146a can be locally delivered to the injured lung without systemic uptake. In this study, we evaluated the potential of the radical scavenging CNP conjugated to microRNA-146a (termed CNP-miR146a) in preventing acute lung injury (ALI) following exposure to bleomycin. We have found that intratracheal delivery of CNP-miR146a increases pulmonary levels of miR146a without systemic increases, and prevents ALI by altering leukocyte recruitment, reducing inflammation and oxidative stress, and decreasing collagen deposition, ultimately improving pulmonary biomechanics.

A Transcription Regulatory Sequence in the 5' Untranslated Region of SARS-CoV-2 Is Vital for Virus Replication with an Altered Evolutionary Pattern against Human Inhibitory MicroRNAs.

Our knowledge of the evolution and the role of untranslated region (UTR) in SARS-CoV-2 pathogenicity is very limited. Leader sequence, originated from UTR, is found at the 5' ends of all encoded SARS-CoV-2 transcripts, highlighting its importance. Here, evolution of leader sequence was compared between human pathogenic and non-pathogenic coronaviruses. Then, profiling of microRNAs that can inactivate the key UTR regions of coronaviruses was carried out. A distinguished pattern of evolution in leader sequence of SARS-CoV-2 was found. Mining all available microRNA families against leader sequences of coronaviruses resulted in discovery of 39 microRNAs with a stable thermodynamic binding energy. Notably, SARS-CoV-2 had a lower binding stability against microRNAs. hsa-MIR-5004-3p was the only human microRNA able to target the leader sequence of SARS and to a lesser extent, also SARS-CoV-2. However, its binding stability decreased remarkably in SARS-COV-2. We found some plant microRNAs with low and stable binding energy against SARS-COV-2. Meta-analysis documented a significant (p < 0.01) decline in the expression of MIR-5004-3p after SARS-COV-2 infection in trachea, lung biopsy, and bronchial organoids as well as lung-derived Calu-3 and A549 cells. The paucity of the innate human inhibitory microRNAs to bind to leader sequence of SARS-CoV-2 can contribute to its high replication in infected human cells.

Edible plant-derived exosomal microRNAs: Exploiting a cross-kingdom regulatory mechanism for targeting SARS-CoV-2.

BACKGROUND: The current COVID-19 pandemic is caused by SARS-CoV-2 which belongs to coronaviridae family. Despite the global prevalence, there are currently no vaccines or drugs. Dietary plant derived exosome-like vesicles are known as edible nanoparticles (ENPs). ENPs are filled with microRNAs (miRNAs), in bioavailable form. Recently, cross-kingdom regulation of human transcripts by plant miRNAs have been demonstrated. However, ENP derived miRNAs targeting SARS-CoV-2 has not been described. STUDY DESIGN: Mature ENP-derived miRNA sequences were retrieved from small RNA sequencing datasets available in the literature. In silico target prediction was performed to identify miRNAs that could target SARS-CoV-2. ENPs were isolated from ginger and grapefruit plants and the expression of SARS-CoV-2 targeting miRNAs were confirmed by qRT-PCR. RESULTS: From a total of 260 ENP-derived miRNAs, we identified 22 miRNAs that could potentially target SARS-CoV-2 genome. 11 miRNAs showed absolute target specificity towards SARS-CoV-2 but not SARS-CoV. ENPs from soybean, ginger, hamimelon, grapefruit, tomato and pear possess multiple miRNAs targeting different regions within SARS-CoV-2. Interestingly, osa/cme miR-530b-5p specifically targeted the ribosomal slippage site between ORF1a and ORF1b. We validated the relative expression of six miRNAs (miR-5077, miR-6300, miR-156a, miR-169, miR-5059 and miR-166 m) in ginger and grapefruit ENPs by RT-PCR which showed differential enrichment of specific miRNAs in ginger and grapefruit ENPs. CONCLUSION: Since administration of ENPs leads to their accumulation into lung tissues in vivo, ENP derived miRNAs targeting SARS-CoV-2 genome has the potential to be developed as an alternative therapy.

Hsa-miR-217 Inhibits the Proliferation, Migration, and Invasion in Non-small Cell Lung Cancer Cells Via Targeting SIRT1 and P53/KAI1 Signaling

Background: Brain metastasis is a major cause of cancer death in patients with lung cancer. Sirtuin 1 and hsa-miR-217 have been identified to mediate the development of non-small cell lung cancer. Aims: To investigate the roles of hsa-miR-217, its target sirtuin 1, and the P53/KAI1 axis in the brain metastasis from non-small cell lung cancer. Study Design: Cell culture study. Methods: Human pulmonary adenocarcinoma brain metastasis cell line PC-14/B were incubated and treated with constructed lentiviral plasmids expressing miR-217 and/or sirtuin 1. BEAS-2B cell line was used as a control. The targeted regulation of miR-217 to sirtuin 1was examined using a dual-luciferase reporter assay. Cell proliferation, migration, invasion, and related protein expression were detected to examine the effect of the miR-217/sirtuin 1 expression on metastasis. Results: PC-14/B cells expressed higher sirtuin 1 and lower P53 and KAI1 compared with BEAS-2B control cells (p<0.05). Sirtuin 1 was a direct target of miR-217. MiR-217 expression suppressed PC-14/B cell invasion (p=0.004), migration (p=0.001), and proliferation (p<0.05), whereas sirtuin 1 overexpression reversed all processes. sirtuin 1 expression inhibited P53, KAI1/CD82, matrix metalloproteinase-9, and ß-catenin but upregulated E-cadherin protein. MiR-217 overexpression induced reverse changes. Conclusion: Hsa-miR-217 and its target sirtuin 1 acted as metastasis suppressor and promoter gene in non-small cell lung cancer, respectively. The hsa-miR-217/sirtuin 1/P53/KAI1 metastasis regulatory pathway showed novel and crucial roles in brain metastasis from non-small cell lung cancer. This axis might be a potential target for the treatment of brain metastasis of lung cancer.

RNA Drugs and RNA Targets for Small Molecules: Principles, Progress, and Challenges.

RNA-based therapies, including RNA molecules as drugs and RNA-targeted small molecules, offer unique opportunities to expand the range of therapeutic targets. Various forms of RNAs may be used to selectively act on proteins, transcripts, and genes that cannot be targeted by conventional small molecules or proteins. Although development of RNA drugs faces unparalleled challenges, many strategies have been developed to improve RNA metabolic stability and intracellular delivery. A number of RNA drugs have been approved for medical use, including aptamers (e.g., pegaptanib) that mechanistically act on protein target and small interfering RNAs (e.g., patisiran and givosiran) and antisense oligonucleotides (e.g., inotersen and golodirsen) that directly interfere with RNA targets. Furthermore, guide RNAs are essential components of novel gene editing modalities, and mRNA therapeutics are under development for protein replacement therapy or vaccination, including those against unprecedented severe acute respiratory syndrome coronavirus pandemic. Moreover, functional RNAs or RNA motifs are highly structured to form binding pockets or clefts that are accessible by small molecules. Many natural, semisynthetic, or synthetic antibiotics (e.g., aminoglycosides, tetracyclines, macrolides, oxazolidinones, and phenicols) can directly bind to ribosomal RNAs to achieve the inhibition of bacterial infections. Therefore, there is growing interest in developing RNA-targeted small-molecule drugs amenable to oral administration, and some (e.g., risdiplam and branaplam) have entered clinical trials. Here, we review the pharmacology of novel RNA drugs and RNA-targeted small-molecule medications, with a focus on recent progresses and strategies. Challenges in the development of novel druggable RNA entities and identification of viable RNA targets and selective small-molecule binders are discussed. SIGNIFICANCE STATEMENT: With the understanding of RNA functions and critical roles in diseases, as well as the development of RNA-related technologies, there is growing interest in developing novel RNA-based therapeutics. This comprehensive review presents pharmacology of both RNA drugs and RNA-targeted small-molecule medications, focusing on novel mechanisms of action, the most recent progress, and existing challenges.