Targeting TGF-ß1/miR-21 Pathway in Keratinocytes Reveals Protective Effects of Silymarin on Imiquimod-Induced Psoriasis Mouse Model.

Epidermal cells integrate multiple signals that activate the signaling pathways involved in skin homeostasis. TGF-ß1 signaling pathway upregulates microRNA (miR)-21-5p in keratinocytes and is often deregulated in skin diseases. To identify the bioactive compounds that enable to modulate the TGF-ß1/miR-21-5p signaling pathway, we screened a library of medicinal plant extracts using our miR-ON RILES luciferase reporter system placed under the control of the miR-21-5p in keratinocytes treated with TGF-ß1. We identified silymarin, a mixture of flavonolignans extracted from Silybum marianum (L.) Gaertn., as the most potent regulator of miR-21-5p expression. Using Argonaute 2 immunoprecipitation and RT-qPCR, we showed that silymarin regulates the expression of miR-21-5p through a noncanonical TGF-ß1 signaling pathway, whereas RNA-sequencing analysis revealed three unexpected transcriptomic signatures associated with keratinocyte differentiation, cell cycle, and lipid metabolism. Mechanistically, we demonstrated that SM blocks cell cycle progression, inhibits keratinocyte differentiation through repression of Notch3 expression, stimulates lipid synthesis via activation of PPARγ signaling and inhibits inflammatory responses by suppressing the transcriptional activity of NF-κB. We finally showed that topical application of silymarin alleviates the development of imiquimod-induced psoriasiform lesions in mice by abrogating the altered expression levels of markers involved in inflammation, proliferation, differentiation, and lipid metabolism.

Intrahippocampal Inoculation of Aß1-42 Peptide in Rat as a Model of Alzheimer's Disease Identified MicroRNA-146a-5p as Blood Marker with Anti-Inflammatory Function in Astrocyte Cells.

Circulating microRNAs (miRNAs) have aroused a lot of interest as reliable blood diagnostic biomarkers of Alzheimer's disease (AD). Here, we investigated the panel of expressed blood miRNAs in response to aggregated Aß1-42 peptides infused in the hippocampus of adult rats to mimic events of the early onset of non-familial AD disorder. Aß1-42 peptides in the hippocampus led to cognitive impairments associated with an astrogliosis and downregulation of circulating miRNA-146a-5p, -29a-3p, -29c-3p, -125b-5p, and-191-5p. We established the kinetics of expression of selected miRNAs and found differences with those detected in the APPswe/PS1dE9 transgenic mouse model. Of note, miRNA-146a-5p was exclusively dysregulated in the Aß-induced AD model. The treatment of primary astrocytes with Aß1-42 peptides led to miRNA-146a-5p upregulation though the activation of the NF-κB signaling pathway, which in turn downregulated IRAK-1 but not TRAF-6 expression. As a consequence, no induction of IL-1ß, IL-6, or TNF-α was detected. Astrocytes treated with a miRNA-146-5p inhibitor rescued IRAK-1 and changed TRAF-6 steady-state levels that correlated with the induction of IL-6, IL-1ß, and CXCL1 production, indicating that miRNA-146a-5p operates anti-inflammatory functions through a NF-κB pathway negative feedback loop. Overall, we report a panel of circulating miRNAs that correlated with Aß1-42 peptides' presence in the hippocampus and provide mechanistic insights into miRNA-146a-5p biological function in the development of the early stage of sporadic AD.

miR-21-3p/IL-22 Axes Are Major Drivers of Psoriasis Pathogenesis by Modulating Keratinocytes Proliferation-Survival Balance and Inflammatory Response.

Psoriasis is a chronic inflammatory skin disease that is mediated by complex crosstalk between immune cells and keratinocytes (KCs). Emerging studies have showed a specific psoriatic microRNAs signature, in which miR-21 is one of the most upregulated and dynamic miRNAs. In this study, we focused our investigations on the passenger miR-21-3p strand, which is poorly studied in skin and in psoriasis pathogenesis. Here, we showed the upregulation of miR-21-3p in an IMQ-induced psoriasiform mouse model. This upregulation was correlated with IL-22 expression and functionality, both in vitro and in vivo, and it occurred via STAT3 and NF-κB signaling. We identified a network of differentially expressed genes involved in abnormal proliferation control and immune regulatory genes implicated in the molecular pathogenesis of psoriasis in response to miR-21-3p overexpression in KCs. These results were confirmed by functional assays that validated the proliferative potential of miR-21-3p. All these findings highlight the importance of miR-21-3p, an underestimated miRNA, in psoriasis and provide novel molecular targets for therapeutic purposes.

LentiRILES, a miRNA-ON sensor system for monitoring the functionality of miRNA in cancer biology and therapy.

A major unresolved challenge in miRNA biology is the capacity to monitor the spatiotemporal activity of miRNAs expressed in animal disease models. We recently reported that the miRNA-ON monitoring system called RILES (RNAi-inducible expression Luciferase system) implanted in lentivirus expression system (LentiRILES) offers unique opportunity to decipher the kinetics of miRNA activity in vitro, in relation with their intracellular trafficking in glioblastoma cells. In this study, we describe in detail the method for the production of LentiRILES stable cell lines and employed it in several applications in the field of miRNA biology and therapy. We show that LentiRILES is a robust, highly specific and sensitive miRNA sensor system that can be used in vitro as a single-cell miRNA monitoring method, cell-based screening platform for miRNA therapeutics and as a tool to analyse the structure-function relationship of the miRNA duplex. Furthermore, we report the kinetics of miRNA activity upon the intracranial delivery of miRNA mimics in an orthotopic animal model of glioblastoma. This information is exploited to evaluate the tumour suppressive function of miRNA-200c as locoregional therapeutic modality to treat glioblastoma. Our data provide evidence that LentiRILES is a robust system, well suited to resolve the activity of endogenous and exogenously expressed miRNAs from basic research to gene and cell therapy.

Intracellular trafficking and functional monitoring of miRNA delivery in glioblastoma using lipopolyplexes and the miRNA-ON RILES reporter system.

MicroRNA (miRNA) oligonucleotides therapeutics are potent and attractive drugs for cancer treatment, but the kinetics of their intracellular trafficking, RISC processing and interaction with their mRNA targets in the cells are still not well understood. Moreover, the absence of efficient carriers impairs their translation into the clinic. Here, we compare the kinetics of miRNA-133a activity after transfection of U87MG glioblastoma cells with either a home-made lipopolyplexes (LPRi) or with the RNAiMax transfection reagent. For this purpose, we combined miRNA intracellular trafficking studies by confocal microscopy with our previously described RILES miRNA-ON reporter system subcloned here in a lentivirus expression vector (LentiRILES) for longitudinal analysis of miRNA activity in transfected cells. Using the LentiRILES system, we report significant differences in terms of miRNA delivery kinetics performed by these two transfection regents. We decipher the mechanisms of miRNA delivery by LPRi and investigate the main steps of miRNA internalization and cytosolic processing. We demonstrate that LPRi preferentially uses caveolae-mediated endocytosis as the main internalization pathway, releases miRNA into the cytosol after the first 3 h of incubation, and addresses the cytosolic miRNAs to P-bodies, while a fraction of miRNAs are exported to the extracellular space through exosomes which were found fully capable to re-transfect the cells. We implanted the LentiRILES cells in the brain of mice and infused the tumours with LPRi.miRNA using the convection-enhanced delivery method. Bioluminescence imaging of the live mice revealed efficient delivery of miRNAs in glioblastoma tumours, attesting successful miRNA uptake, internalization and RISC activation in vivo. Overall, our study provides a comprehensive overview of miRNA intracellular trafficking and processing in a glioblastoma context and highlights the potential use of LPRi for miRNA-based therapy.

Protective Role of Spirulina platensis Against Bifenthrin-Induced Reprotoxicity in Adult Male Mice by Reversing Expression of Altered Histological, Biochemical, and Molecular Markers Including MicroRNAs.

: The potential reprotoxicity of bifenthrin remains unclear if only the common clinical indicators of reproductive disease are examined. The present study aimed to investigate the efficacy of Spirulina platensis, a microalga rich in antioxidant compounds, against bifenthrin-induced testicular oxidative damage in male mice. At the first, we demonstrate that administration of bifenthrin resulted in a decline of testosterone level and in deterioration of sperm quality that was correlated with significant transcription changes of some specific mRNA and microRNA involved in cholesterol transport, testosterone synthesis, and spermatogenesis. At the biochemical level, we found that oxidative stress was obvious in the bifenthrin group, as evidenced by increase in malondialdehyde (MDA), protein carbonyls (PCO), reactive oxygen species (ROS), and nitrite oxide (NO) that was correlated with activation of genes related to mitochondrial apoptotic signal pathways. We then brought, for the first time to our knowledge, solid and complete experimental evidences that administration of mice with Spirulina extract was sufficient to protect against deleterious effects BF in testicular tissues by abrogating the change in antioxidant enzyme activities; the increase in MDA, PCO, and NO concentrations; and the altered expression level of miRNA and mRNA involved in spermatogenesis. We finally demonstrate that Spirulina restores the production of testosterone in mice as well as epididymal sperm viability and motility. These results suggest a potential antitoxic activity of Tunisian Spirulina deserving further attention.

Deciphering the Biological Activities of Dunaliella sp. Aqueous Extract from Stressed Conditions on Breast Cancer: from in Vitro to in Vivo Investigations.

In order to harness local resources to improve well-being and human health, we aim in this study to investigate if the microalgae Dunaliella sp. isolated from the Tunisian coastal zone possesses any anticancer activity. Dunaliella sp. was cultured under normal (DSC) or stressed (DSS) conditions and extracted using different procedures. The biological activity assessment was performed on the Triple Negative Breast Cancer (TNBC) using 4T1 murine cells as a model. Results indicate that: (i) aqueous extract was the most cytotoxic compared to ethanolic and hydroalcoholic extracts; (ii) DSS activity was superior to that of DSC. DSS extracts induced apoptosis rather than necrosis, as evidenced by DNA fragmentation, PARP-1 cleavage and caspase-3 activation. Evaluation in an orthotopic TNBC model validated the anticancer activity in vivo. Intratumoral injection of DSS extract resulted in reduced tumor growth and an enhanced immune system activation. On the transcriptional side, the expression level of the immunosuppressive enzyme Arg-1 was decreased, as well as those of NOS-2 and COX-2 genes. These results suggest a potential anticancer activity of Tunisian Dunaliella sp. deserving further attention.

Development of an LC-MS multivariate nontargeted methodology for differential analysis of the peptide profile of Asian hornet venom (Vespa velutina nigrithorax): application to the investigation of the impact of collection period variation.

Insect venom is a highly complex mixture of bioactive compounds, containing proteins, peptides, and small molecules. Environmental factors can alter the venom composition and lead to intraspecific variation in its bioactivity properties. The investigation of discriminating compounds caused by variation impacts can be a key to manage sampling and explore the bioactive compounds. The present study reports the development of a peptidomic methodology based on UHPLC-ESI-QTOF-HRMS analysis followed by a nontargeted multivariate analysis to reveal the profile variance of Vespa velutina venom collected in different conditions. The reliability of the approach was enhanced by optimizing certain XCMS data processing parameters and determining the sample peak threshold to eliminate the interfering features. This approach demonstrated a good repeatability and a criterion coefficient of variation (CV) > 30% was set for deleting nonrepeatable features from the matrix. The methodology was then applied to investigate the impact of collection period variation. PCA and PLS-DA models were used and validated by cross-validation and permutation tests. A slight discrimination was found between winter and summer hornet venom in two successive years with 10 common discriminating compounds. Graphical abstract.

Asian hornet Vespa velutina nigrithorax venom: Evaluation and identification of the bioactive compound responsible for human keratinocyte protection against oxidative stress.

The present study aimed to explore the potential antioxidant molecules of the Asian hornet venom (Vespa velutina nigrithorax) responsible for radical scavenging activity and human keratinocyte protection against oxidative stress. We developed a first technical platform that combined a DPPH radical scavenging chemical assay and cytotoxicity and ROS (reactive oxygen species) production in HaCaT keratinocyte cells exposed to UVB to evaluate the antioxidant property of V. velutina venom. We further employed Thin Layer Chromatography (TLC) combined with the DPPH assay as a targeted separation approach to isolate the antioxidant compounds responsible for the free radical scavenging property of V. velutina venom. In parallel, the latter was fractionated by a HPLC-DAD non-targeted separation approach. From this experiment, nine fractions were generated which were again evaluated separately for their antioxidant properties using DPPH assays. Results showed that only one fraction exhibited significant antioxidant activity in which serotonin was identified as the major compound by a UHPLC-ESI-QTOF HRMS/MS approach. We finally demonstrated, using purified serotonin molecule that this bioactive structure is mostly responsible for the free radical scavenging property of the crude venom as evidenced by DPPH and ROS assays in HaCaT cells exposed to UVB.

Dendritic Cell Targeting mRNA Lipopolyplexes Combine Strong Antitumor T-Cell Immunity with Improved Inflammatory Safety.

In vitro transcribed mRNA constitutes a versatile platform to encode antigens and to evoke CD8 T-cell responses. Systemic delivery of mRNA packaged into cationic liposomes (lipoplexes) has proven particularly powerful in achieving effective antitumor immunity in animal models. Yet, T-cell responses to mRNA lipoplexes critically depend on the induction of type I interferons (IFN), potent pro-inflammatory cytokines, which inflict dose-limiting toxicities. Here, we explored an advanced hybrid lipid polymer shell mRNA nanoparticle (lipopolyplex) endowed with a trimannose sugar tree as an alternative delivery vehicle for systemic mRNA vaccination. Like mRNA lipoplexes, mRNA lipopolyplexes were extremely effective in conferring antitumor T-cell immunity upon systemic administration. Conversely to mRNA lipoplexes, mRNA lipopolyplexes did not rely on type I IFN for effective T-cell immunity. This differential mode of action of mRNA lipopolyplexes enabled the incorporation of N1 methyl pseudouridine nucleoside modified mRNA to reduce inflammatory responses without hampering T-cell immunity. This feature was attributed to mRNA lipopolyplexes, as the incorporation of thus modified mRNA into lipoplexes resulted in strongly weakened T-cell immunity. Taken together, we have identified lipopolyplexes containing N1 methyl pseudouridine nucleoside modified mRNA as potent yet low-inflammatory alternatives to the mRNA lipoplexes currently explored in early phase clinical trials.

MicroRNA-Based Drugs for Brain Tumors.

MicroRNAs (miRNAs) are key regulatory elements encoded by the genome. A single miRNA can downregulate the expression of multiple genes involved in diverse functions. Because cancer is a disease with multiple gene aberrations, developing novel approaches to identify and modulate miRNA pathways may result in a breakthrough for cancer treatment. With a special focus on glioblastoma (GBM), this review provides an up-to-date summary of miRNA biogenesis, the role of miRNA in cancer resistance, and essential tools for modulating miRNA expression, as well as of clinically promising RNAi delivery systems and how they can be adapted for therapy.

Positive radionuclide imaging of miRNA expression using RILES and the human sodium iodide symporter as reporter gene is feasible and supports a protective role of miRNA-23a in response to muscular atrophy.

MicroRNAs (miRNAs) are key players in many biological processes and are considered as an emerging class of pharmacology drugs for diagnosis and therapy. However to fully exploit the therapeutic potential of miRNAs, it is becoming crucial to monitor their expression pattern using medical imaging modalities. Recently, we developed a method called RILES, for RNAi-Inducible Luciferase Expression System that relies on an engineered regulatable expression system to switch-ON the expression of the luciferase gene when a miRNA of interest is expressed in cells. Here we investigated whether replacing the luciferase reporter gene with the human sodium iodide symporter (hNIS) reporter gene will be also suited to monitor the expression of miRNAs in a clinical setting context. We provide evidence that radionuclide imaging of miRNA expression using hNIS is feasible although it is not as robust as when the luciferase reporter gene is used. However, under appropriate conditions, we monitored the expression of several miRNAs in cells, in the liver and in the tibialis anterior muscle of mice undergoing muscular atrophy. We demonstrated that radiotracer accumulation in transfected cells correlated with the induction of hNIS and with the expression of miRNAs detected by real time PCR. We established the kinetic of miRNA-23a expression in mice and demonstrated that this miRNA follows a biphasic expression pattern characterized by a loss of expression at a late time point of muscular atrophy. At autopsy, we found an opposite expression pattern between miRNA-23a and one of the main transcriptional target of this miRNA, APAF-1, and as downstream target, Caspase 9. Our results report the first positive monitoring of endogenously expressed miRNAs in a nuclear medicine imaging context and support the development of additional work to establish the potential therapeutic value of miRNA-23 to prevent the damaging effects of muscular atrophy.

Pharmacomodulation of microRNA Expression in Neurocognitive Diseases: Obstacles and Future Opportunities.

Given the importance of microRNAs (miRNAs) in modulating brain functions and their implications in neurocognitive disorders there are currently significant efforts devoted in the field of miRNA-based therapeutics to correct and/or to treat these brain diseases. The observation that miRNA 29a/b-1 cluster, miRNA 10b and miRNA 7, for instance, are frequently deregulated in the brains of patients with neurocognitive diseases and in animal models of Alzheimer, Huntington's and Parkinson's diseases, suggest that correction of miRNA expression using agonist or antagonist miRNA oligonucleotides might be a promising approach to correct or even to cure such diseases. The encouraging results from recent clinical trials allow envisioning that pharmacological approaches based on miRNAs might, in a near future, reach the requirements for successful therapeutic outcomes and will improve the healthcare of patients with brain injuries or disorders. This review will focus on the current strategies used to modulate pharmacological function of miRNA using chemically modified oligonucleotides. We will then review the recent literature on strategies to improve nucleic acid delivery across the blood-brain barrier which remains a severe obstacle to the widespread application of miRNA therapeutics to treat brain diseases. Finally, we provide a state-of-art of current preclinical research performed in animal models for the treatment of neurocognitive disorders using miRNA as therapeutic agents and discuss future developments of miRNA therapeutics.

MicroRNAs in Neurocognitive Dysfunctions: New Molecular Targets for Pharmacological Treatments?

BACKGROUND: Neurodegenerative and cognitive disorders are multifactorial diseases (i.e., involving neurodevelopmental, genetic, age or environmental factors) characterized by an abnormal development that affects neuronal function and integrity. Recently, an increasing number of studies revealed that the dysregulation of microRNAs (miRNAs) may be involved in the etiology of cognitive disorders as Alzheimer, Parkinson, and Huntington's diseases, Schizophrenia and Autism spectrum disorders. METHODS: From an extensive search in bibliographic databases of peer-reviewed research literature, we identified relevant published studies related to specific key words such as memory, cognition, neurodegenerative disorders, neurogenesis and miRNA. We then analysed, evaluated and summerized scientific evidences derived from these studies. RESULTS: We first briefly summarize the basic molecular events involved in memory, a process inherent to cognitive disease, and then describe the role of miRNAs in neurodevelopment, synaptic plasticity and memory. Secondly, we provide an overview of the impact of miRNA dysregulation in the pathogenesis of different neurocognitive disorders, and lastly discuss the feasibility of miRNA-based therapeutics in the treatment of these disorders. CONCLUSION: This review highlights the molecular basis of neurodegenerative and cognitive disorders by focusing on the impact of miRNAs dysregulation in these pathological phenotypes. Altogether, the published reports suggest that miRNAs-based therapy could be a viable therapeutic alternative to current treatment options in the future.

Production of DNA minicircles less than 250 base pairs through a novel concentrated DNA circularization assay enabling minicircle design with NF-κB inhibition activity.

Double-stranded DNA minicircles of less than 1000 bp in length have great interest in both fundamental research and therapeutic applications. Although minicircles have shown promising activity in gene therapy thanks to their good biostability and better intracellular trafficking, minicircles down to 250 bp in size have not yet been investigated from the test tube to the cell for lack of an efficient production method. Herein, we report a novel versatile plasmid-free method for the production of DNA minicircles comprising fewer than 250 bp. We designed a linear nicked DNA double-stranded oligonucleotide blunt-ended substrate for efficient minicircle production in a ligase-mediated and bending protein-assisted circularization reaction at high DNA concentration of 2 µM. This one pot multi-step reaction based-method yields hundreds of micrograms of minicircle with sequences of any base composition and position and containing or not a variety of site-specifically chemical modifications or physiological supercoiling. Biochemical and cellular studies were then conducted to design a 95 bp minicircle capable of binding in vitro two NF-κB transcription factors per minicircle and to efficiently inhibiting NF-κB-dependent transcriptional activity in human cells. Therefore, our production method could pave the way for the design of minicircles as new decoy nucleic acids.

Regulating the expression of therapeutic transgenes by controlled intake of dietary essential amino acids.

Widespread application of gene therapy will depend on the development of simple methods to regulate the expression of therapeutic genes. Here we harness an endogenous signaling pathway to regulate therapeutic gene expression through diet. The GCN2-eIF2α signaling pathway is specifically activated by deficiencies in any essential amino acid (EAA); EAA deficiency leads to rapid expression of genes regulated by ATF4-binding cis elements. We found that therapeutic genes under the control of optimized amino acid response elements (AAREs) had low basal expression and high induced expression. We applied our system to regulate the expression of TNFSF10 (TRAIL) in the context of glioma therapy and found that intermittent activation of this gene by EEA-deficient meals retained its therapeutic efficacy while abrogating its toxic effects on normal tissue. The GCN2-eIF2α pathway is expressed in many tissues, including the brain, and is highly specific to EAA deficiency. Our system may be particularly well suited for intermittent regulation of therapeutic transgenes over short or long time periods.

The Promise of Gene Therapy for Pancreatic Cancer.

Unlike for other digestive cancer entities, chemotherapy, radiotherapy, and targeted therapies have, so far, largely failed to improve patient survival in pancreatic adenocarcinoma (PDAC), which remains the fourth leading cause of cancer-related death in Europe and the United States. In this context, gene therapy may offer a new avenue for patients with PDAC. In this review, we explore the research currently ongoing in French laboratories aimed at defeating PDAC using nonviral therapeutic gene delivery, targeted transgene expression, or oncolytic virotherapy that recently or will soon bridge the gap between experimental models of cancer and clinical trials. These studies are likely to change clinical practice or thinking about PDAC management, as they represent a major advance not only for PDAC but may also significantly influence the field of gene-based molecular treatment of cancer.

Positive Bioluminescence Imaging of MicroRNA Expression in Small Animal Models Using an Engineered Genetic-Switch Expression System, RILES.

MicroRNAs (miRNAs) are a class of small, noncoding RNAs which regulate gene expression by directing their target mRNA for degradation or translational repression. Since their discovery in the early 1990s, miRNAs have emerged as key components in the posttranscriptional regulation of gene networks, shaping many biological processes from development, morphogenesis, differentiation, proliferation and apoptosis. Although understanding of the molecular basis of miRNA biology is improving, methods to monitor the dynamic and the spatiotemporal aspects of miRNA expression under physiopathological conditions are required. However, monitoring of miRNAs is difficult due to their small size, low abundance, high degree of sequence similarity, and their dynamic expression pattern which is subjected to tight transcriptional and post-transcriptional controls. Recently, we developed a miRNA monitoring system called RILES, standing for RNAi-inducible expression system, which relies on an engineered regulatable expression system, to switch on the expression of the luciferase gene when the targeted miRNA is expressed in cells. We demonstrated that RILES is a specific, sensitive, and robust method to determine the fine-tuning of miRNA expression during the development of an experimental pathological process in mice. Because RILES offers the possibility for longitudinal studies on individual subjects, sharper insights into miRNA regulation can be generated, with applications in physiology, pathophysiology and development of RNAi-based therapies. This chapter describes methods and protocols to monitor the expression of myomiR-206, -1, and -133 in the tibialis anterior muscle of mice. These protocols can be used and adapted to monitor the expression of other miRNAs in other biological processes.