Platelet Microparticles Enriched in miR-223 Reduce ICAM-1-Dependent Vascular Inflammation in Septic Conditions

In the process of sepsis, activated platelets shed microvesicles containing microRNAs (miRNAs), which can be internalized by distinct recipient cells in circulation, consequently eliciting a potent capability to regulate their cellular functions in different diseases. In the present study, activated human platelets transferring miR-223 into endothelial cells via platelet-derived microparticles (PMPs) was investigated in vitro during septic conditions with a proposed mechanism involving in downregulation of the enhanced expression of intercellular adhesion molecule-1 (ICAM-1). The uptake of PMPs encasing miR-223 and the adhesion of peripheral blood mononuclear cells (PBMCs) on human coronary artery endothelial cells (HCAECs) were observed by immunofluorescence microscopy upon co-culture with PMPs isolated from sepsis or control plasma. The expression of miR-223-3p and its gene target ICAM1 in HCAECs were quantified by RT-qPCR and ELISA after the cells were incubated with septic or control PMPs, whose levels were induced with thrombin-receptor activating peptide (TRAP). Leukocyte-depleted platelets (LDPs) from septic patients showed a decreased miR-223 level, while septic plasma and PMPs revealed an elevated miRNA level compared to control samples. Similarly, TRAP-activated LDPs demonstrated a reduced intracellular miR-223 expression, while increased levels in the supernatant and PMP isolates were observed vs. untreated samples. Furthermore, TNF-α alone resulted in decreased miR-223 and elevated ICAM1 levels in HCAECs, while PMPs raised the miRNA level that was associated with downregulated ICAM1 expression at both mRNA and protein levels under TNF-α treatment. Importantly, miR-223 was turned out not to be newly synthesized as shown in unchanged pre-miR-223 level, and mature miR-223 expression was also elevated in the presence of PMPs in HCAECs after transfection with Dicer1 siRNA. In addition, septic PMPs containing miR-223 decreased ICAM1 with a reduction of PBMC binding to HCAECs. In conclusion, septic platelets released PMPs carrying functional miR-223 lower ICAM1 expression in endothelial cells, which may be a protective role against excessive sepsis-induced vascular inflammation.

miRNA-mediated Drug Repurposing Unveiled Potential Candidate Drugs for Prolactinoma Treatment

Introduction: Prolactinomas, also called lactotroph adenomas, are the most encountered type of hormone-secreting pituitary neuroendocrine tumors (PitNET) in the clinic. The preferred first-line therapy is a medical treatment with dopamine agonists (DA), mainly cabergoline, to reduce serum prolactin levels, tumor volume, and mass effect. However, in some cases, patients have displayed DA-resistance with aggressive tumor behavior or are faced with recurrence after drug withdrawal. Also, currently used therapeutics have notorious side effects and impair the life quality of the patients. Methods: Since the amalgamation of clinical and laboratory data besides tumor histopathogenesis and transcriptional regulatory features of the tumor emerge to exhibit essential roles in the behavior and progression of prolactinomas, in this work, we integrated mRNA and microRNA (miRNA) level transcriptome data that exploit disease-specific signatures in addition to biological and pharmacological data to elucidate a rational prioritization of pathways and drugs in prolactinoma. Results: We identified eight drug candidates through drug repurposing based on mRNA-miRNA level data integration and evaluated their potential through in vitro assays in the MMQ cell line. Seven re-purposed drugs including 5-flourocytosine, nortriptyline, neratinib, puromycin, taxifolin, vorinostat, and zileuton were proposed as potential drug candidates for the treatment of prolactinoma. We further hypothesized possible mechanisms of drug action on MMQ cell viability through analyzing PI3K/Akt signaling pathway and cell cycle arrest via flow cytometry and western blotting. Discussion: We presented the transcriptomic landscape of prolactinoma through miRNA and mRNA level data integration and proposed repurposed drug candidates based on this integration. We validated our findings through testing cell viability, cell cycle phases, and PI3K/Akt protein expressions. Effects of the drugs on cell cycle phases and inhibition of PI3K/Akt pathway by all drugs gave us promising output for further studies using these drugs in the treatment of prolactinoma. This is the first study that reports miRNA-mediated repurposed drugs for prolactinoma treatment via in vitro experiments.

Lipid Droplets Promote Phase Separation of Ago2 to Accelerate Dicer1 Loss and Decelerate miRNA Activity in Lipid Exposed Hepatic Cells

AbstractmiR-122 is a liver specific miRNA that plays an important role in controlling metabolic homeostasis in mammalian liver cells. Interestingly, miR-122 on exposure to lipotoxic stress is reduced in liver cells. To fight stress, miRNA processor Dicer1 is depleted to cause reduced miR-122 production and the lowering of miRNA level ensures a better stress response in hepatocytes under lipotoxic stress. Interestingly, lipid droplets, formed in the liver cells on exposure to high fat, ensure cytoplasmic phase separation of Ago2 and prevent interaction of Ago2 with Dicer1. Lipid droplets bind miRNA and enhance miRNA-Ago2 uncoupling and Ago2 phase separation. Loss of interaction between Ago2 and Dicer1 eventually facilitates export and lowering of cellular Dicer1, a process also dependent on the endosomal maturation controller protein Alix, thereby ceasing pre-miRNA processing by Dicer1 in lipid exposed cells. Depletion of lipid droplets by downregulation of Perilipins with siRNAs resulted in a rescue of cellular Dicer1 level and Ago2-Dicer1 interaction. This is a novel mechanism that liver cells adopt to restrict cellular miRNA levels under stress condition. Thus, lipid droplets prevent cell death upon exposure to high fat by reducing intra and extracellular pool of miR-122 in hepatic tissue.

Role of miR-122-5P, miR-107, miR-22-3P, and miR-330-3P microRNAs in regulation of HIF1A expression in high-grade gliomas.

e14550 Background: HIF1-alfa is a powerful regulator of angiogenesis and cell invasion activating a wide range of oncogenes. Control of the HIF1-alpha transcription is strictly regulated by many molecular pathways, including miRNAs which can become targets for gene therapy of cancer. Methods: The study included 30 patients with histologically verified high-grade gliomas. RNAs were isolated from 30 paired (tumor and control) samples using the TRIzol reagent (Thermo Fisher, USA). The cDNA synthesis was performed using the Reverta MMLV kit (Sintol, Russia). Expression of miRNAs and HIF1A was evaluated by RT-PCR using the CFX96 system (Bio-Rad, USA). The 2−ΔΔ Сt method was used to analyze the qPCR data. Statisical processing of the results was performed with the Statistica 10 program (StatSoft Inc., USA). Results: Using analysis of TargetScan and miRTarBase databases and appropriate literature, miRNAs (miR-122-5p, miR-107, miR-22-5p and miR-330-3p) able to regulate HIF1A expression were selected. The HIF1A expression in the tumor was 1.8 times higher, compared to normal tissue (p = 0.0031). The 2−ΔΔ Сt value for miR-122-5p, miR-107, miR-22-3p and miR-330-3p decreased by 40%, 45%, 36%, and 91.5%, respectively, in tumor samples relative to the control point (p < 0.05 in all cases). A correlation was found between the expression of miRNA 122-5p and HIF1A (Spearman’s r = -0.46 at p < 0.05). Conclusions: An increase in the HIF1A miRNA level was found, together with a decrease in the expression of four potentially targeting miRNAs in high-grade gliomas. A negative correlation between the expression of miR-122-5p and HIF1A in high-grade gliomas was revealed, which can be used for the development of new therapy options.

sTAM: An Online Tool for the Discovery of miRNA-set Level Disease Biomarkers

AbstractmicroRNAs (miRNAs) are one class of important small noncoding RNA molecules, which have shown their excellent ability as biomarkers of various diseases. However, current miRNA biomarkers including those comprised of multiple miRNAs work at a single-miRNA level but not at a miRNA set level. Given the rapidly accumulated miRNA omics data, it is believed that miRNA set level analysis could be an important supplement to the single miRNA level analysis. For doing so, here we presented a computational method for single-sample miRNA set enrichment analysis and developed the sTAM tool (http://mir.rnanut.net/stam). Moreover, we demonstrated the usefulness of sTAM scores in discovering miRNA-set level biomarkers through two case studies. We conducted pan-cancer analysis of the sTAM scores of “tumor suppressor miRNA set” on 15 types of cancers from TCGA and 14 types of cancers from GEO, finding that the scores show a good performance in distinguishing the cancers from the controls. Moreover, we revealed that the sTAM score of “brain development” miRNA set can effectively predict cerebrovascular disorder (CVD). Finally, we believe sTAM can be used in discovering disease-related biomarkers at a miRNA-set level.

The Crosstalk of miRNA and Oxidative Stress in the Liver: From Physiology to Pathology and Clinical Implications

The liver is the central metabolic organ of mammals. In humans, most diseases of the liver are primarily caused by an unhealthy lifestyle–high fat diet, drug and alcohol consumption- or due to infections and exposure to toxic substances like aflatoxin or other environmental factors. All these noxae cause changes in the metabolism of functional cells in the liver. In this literature review we focus on the changes at the miRNA level, the formation and impact of reactive oxygen species and the crosstalk between those factors. Both, miRNAs and oxidative stress are involved in the multifactorial development and progression of acute and chronic liver diseases, as well as in viral hepatitis and carcinogenesis, by influencing numerous signaling and metabolic pathways. Furthermore, expression patterns of miRNAs and antioxidants can be used for biomonitoring the course of disease and show potential to serve as possible therapeutic targets.

Serum and Lipoprotein Particle miRNA Profile in Uremia Patients

microRNAs (miRNAs) are post-transcriptional regulators of messenger RNA (mRNA), and transported through the whole organism by—but not limited to—lipoprotein particles. Here, we address the miRNA profile in serum and lipoprotein particles of healthy individuals in comparison with patients with uremia. Moreover, we quantitatively determined the cellular lipoprotein-particle-uptake dependence on the density of lipoprotein particle receptors and present a method for enhancement of the transfer efficiency. We observed a significant increase of the cellular miRNA level using reconstituted high-density lipoprotein (HDL) particles artificially loaded with miRNA, whereas incubation with native HDL particles yielded no measurable effect. Thus, we conclude that no relevant effect of lipoprotein-particle-mediated miRNA-transfer exists under in vivo conditions though the miRNA profile of lipoprotein particles can be used as a diagnostic marker.