miRNome profiling of extracellular vesicles in severe COVID-19 patients and identification of predictors of mortality.

BACKGROUND: Extracellular vesicles (EVs), containing microRNAs (miRNAs) and other molecules, play a central role in intercellular communication, especially in viral infections caused by SARS-CoV-2. This study explores the miRNA profiles in plasma-derived EVs from severe COVID-19 patients referred to controls, identifying potential mortality miRNA predictors. METHODS: A prospective study was carried out, including 36 severe COVID-19 patients and 33 non-COVID-19 controls. EVs-derived miRNAs were sequenced, and bioinformatics and differential expression analysis between groups were performed. The plasma miRNA profile of an additional cohort of severe COVID-19 patients (n=32) and non-COVID-19 controls (n=12) was used to compare with our data. Survival analysis was used to identify potential mortality predictors among the SDE miRNAs in EVs. RESULTS: Severe COVID-19 patients showed 50 significantly differentially expressed (SDE) miRNAs in plasma-derived EVs. These miRNAs were associated with pathways related to inflammation and cell adhesion. Fifteen of these plasma-derived EVs miRNAs were also SDE in the plasma of severe patients vs controls. Two miRNAs, hsa-miR-1469 and hsa-miR-6124, were identified as strong mortality predictors with an área under the ROC Curve (AUC) of 0.938. CONCLUSION: : This research provides insights into the role of miRNAs found within EVs in severe COVID-19 and their potential as clinical biomarkers for mortality.

Protein kinase C family evolution in jawed vertebrates.

Protein kinase C (PKC) was one of the first kinases identified in human cells. It is now known to constitute a family of kinases that respond to diacylglycerol, phosphatidylserine and for some family members, Ca2+. They have a plethora of different functions, such as cell cycle regulation, immune response and memory formation. In mammals, 12 PKC family members have been described, usually divided into 4 different subfamilies. We present here a comprehensive evolutionary analysis of the PKC genes in jawed vertebrates with special focus on the impact of the two tetraploidizations (1R and 2R) before the radiation of jawed vertebrates and the teleost tetraploidization (3R), as illuminated by synteny and paralogon analysis including many neighboring gene families. We conclude that the vertebrate predecessor had five PKC genes, as tunicates and lancelets still do, and that the PKC family should therefore ideally be organized into five subfamilies. The 1R and 2R events led to a total of 12 genes distributed among these five subfamilies. All 12 genes are still present in some of the major lineages of jawed vertebrates, including mammals, whereas birds and cartilaginous fishes have lost one member. The 3R event added another nine genes in teleosts, bringing the total to 21 genes. The zebrafish, a common experimental model animal, has retained 19. We have found no independent gene duplications. Thus, the genome doublings completely account for the complexity of this gene family in jawed vertebrates and have thereby had a huge impact on their evolution.

Opioid and Notch signaling pathways are reciprocally regulated through miR- 29a and miR-212 expression.

BACKGROUND: The abuse of opioids, such as morphine and phentanyl or other drugs as heroin is a social and health problem that affects an increasing number of people each year. The activation of the mu opioid receptor triggers several molecular changes that alter the expression of diverse genes, including miRNAs. The dysregulation of these molecules could explain some of the developmental alterations that are induced after drug intake. In addition, the Notch signaling cascade has also been related to alterations on these processes. METHODS: Zebrafish embryos and SH-SY5Y cells were used to assess the effects of opioid and Notch signaling on the expression on miR-29a and miR-212/132 by qPCR and ChIP-qPCR. Notch1 expression was analyzed using in situ hybridization on 24 hpf zebrafish embryos. In addition, OPRM1 and NICD levels were measured using western blot on the cultured cells to determine the cross-talk between the two pathways. RESULTS: We have observed changes in the levels of miR-212/132 after administrating DAPT to zebrafish embryos indicating that this pathway could be regulating mu opioid receptor expression. In addition, the ISH experiment showed changes in Notch1 expression after morphine and DAPT administration. Moreover, morphine affects the expression of miR-29a through NF-κB, therefore controlling the cleavage and activation of Notch through ADAM12 expression. CONCLUSIONS: This study shows that these two pathways are closely related, and could explain the alterations triggered in the early stages of the development of addiction. GENERAL SIGNIFICANCE: Opioid and Notch pathway are reciprocally regulated by the miRNAs 212/132 and 29a.

Morphine delays neural stem cells differentiation by facilitating Nestin overexpression.

BACKGROUND: Morphine is used as an analgesic although it causes important secondary effects. These effects are triggered by several mechanisms leading to the dysregulation of gene expression. Here we aimed to study these alterations on neural stem cells (NSC) during CNS development. METHODS: AB strain and tg nestin:GFP zebrafish embryos, zebrafish primary neuron culture and mouse embryonic stem cells were used to assess the effect of morphine by qPCR, time lapse microscopy and western blot. ChIP-qPCR and bisulfite conversion assay were performed to determine the changes exerted by morphine in a Nestin candidate enhancer. RESULTS: Morphine increases GFP in nestin:GFP embryos and overexpresses the NSC marker Nestin. Morphine also exerts a hyperacetylation effect on H3K27 and decreases DNA methylation within a region located 18 Kb upstream nestin transcription starting site. Here, a binding site for the transcription factor complex Sox2/Oct4/Nanog was predicted. These factors are also upregulated by morphine. Besides, morphine increases the histone acetyl transferase p300. The inhibition of p300 activity decreases Nestin. CONCLUSIONS: Morphine facilitates Nestin increase by several mechanisms which include hyperacetylation of H3K27, decreased DNA methylation and the overexpression of the transcription factors sox2, oct4 and nanog. It has also been demonstrated that nestin levels depend on p300 activity. The facilitated Nestin expression delays the normal differentiation of neural stem cells. GENERAL SIGNIFICANCE: The present work provides novel evidence of the effects induced by morphine in the normal differentiation of NSCs, altering Nestin through changes on p300, H3K27ac, DNA methylation and Oct4, Sox2, and Nanog.

µ Opioid Receptor Expression after Morphine Administration Is Regulated by miR-212/132 Cluster.

Since their discovery, miRNAs have emerged as a promising therapeutical approach in the treatment of several diseases, as demonstrated by miR-212 and its relation to addiction. Here we prove that the miR-212/132 cluster can be regulated by morphine, through the activation of mu opioid receptor (Oprm1). The molecular pathways triggered after morphine administration also induce changes in the levels of expression of oprm1. In addition, miR-212/132 cluster is actively repressing the expression of mu opioid receptor by targeting a sequence in the 3' UTR of its mRNA. These findings suggest that this cluster is closely related to opioid signaling, and function as a post-transcriptional regulator, modulating morphine response in a dose dependent manner. The regulation of miR-212/132 cluster expression is mediated by MAP kinase pathway, CaMKII-CaMKIV and PKA, through the phosphorylation of CREB. Moreover, the regulation of both oprm1 and of the cluster promoter is mediated by MeCP2, acting as a transcriptional repressor on methylated DNA after prolonged morphine administration. This mechanism explains the molecular signaling triggered by morphine as well as the regulation of the expression of the mu opioid receptor mediated by morphine and the implication of miR-212/132 in these processes.

MicroRNA let-7d is a target of cannabinoid CB1 receptor and controls cannabinoid signaling.

Cannabinoid CB1 receptor, the molecular target of endocannabinoids and cannabis active components, is one of the most abundant metabotropic receptors in the brain. Cannabis is widely used for both recreational and medicinal purposes. Despite the ever-growing fundamental roles of microRNAs in the brain, the possible molecular connections between the CB1 receptor and microRNAs are surprisingly unknown. Here, by using reporter gene constructs that express interaction sequences for microRNAs in human SH-SY5Y neuroblastoma cells, we show that CB1 receptor activation enhances the expression of several microRNAs, including let-7d. This was confirmed by measuring hsa-let-7d expression levels. Accordingly, knocking-down CB1 receptor in zebrafish reduced dre-let-7d levels, and knocking-out CB1 receptor in mice decreased mmu-let-7d levels in the cortex, striatum and hippocampus. Conversely, knocking-down let-7d increased CB1 receptor mRNA expression in zebrafish, SH-SY5Y cells and primary striatal neurons. Likewise, in primary striatal neurons chronically exposed to a cannabinoid or opioid agonist, a let-7d-inhibiting sequence facilitated not only cannabinoid or opioid signaling but also cannabinoid/opioid cross-signaling. Taken together, these findings provide the first evidence for a bidirectional link between the CB1 receptor and a microRNA, namely let-7d, and thus unveil a new player in the complex process of cannabinoid action.

Role of morphine, miR-212/132 and mu opioid receptor in the regulation of Bdnf in zebrafish embryos.

BACKGROUND: Morphine is one of the first-line therapies for the treatment of pain despite its secondary effects. It modifies the expression of epigenetic factors like miRNAs. In the present study, we analyzed miR-212 and miR-132 and their implication in morphine effects in the zebrafish Central Nervous System (CNS) through the regulation of Bdnf expression. METHODS: We used control and knock-down zebrafish embryos to assess the effects of morphine in miRNAs 212/132 and mitotic or apoptotic cells by qPCR, immunohistochemistry and TUNEL assay, respectively. Bdnf and TrkB were studied by western blot and through a primary neuron culture. A luciferase assay was performed to confirm the binding of miRNAs 212/132 to mecp2. RESULTS: Morphine exposure decreases miR-212 but upregulates miR-132, as wells as Bdnf and TrkB, and changes the localization of proliferative cells. However, Bdnf expression was downregulated when miRNAs 212/132 and oprm1 were knocked-down. Furthermore, we proved that these miRNAs inhibit mecp2 expression by binding to its mRNA sequence. The described effects were corroborated in a primary neuron culture from zebrafish embryos. CONCLUSIONS: We propose a mechanism in which morphine alters the levels of miRNAs 212/132 increasing Bdnf expression through mecp2 inhibition. oprm1 is also directly involved in this regulation. The present work confirms a relationship between the opioid system and neurotrophins and shows a key role of miR-212 and miR-132 on morphine effects through the regulation of Bdnf pathway. GENERAL SIGNIFICANCE: miRNAs 212/132 are novel regulators of morphine effects on CNS. Oprm1 controls the normal expression of Bdnf.