Modulation Of miRNAs Research Articles

Effect of Necrosis on the miRNA-mRNA Regulatory Network in CRT-MG Human Astroglioma Cells.

PurposeGlioblastoma multiforme (GBM) is the most common adult primary intracranial tumor. The remarkable features of GBM include central necrosis. MicroRNAs (miRNAs) have been considered as diagnostic/prognostic biomarkers for many cancers, including glioblastoma. However, the effect of necrosis on the miRNA expression profile and predicted miRNA-mRNA regulatory information remain unclear. The purpose of this study is to examine the effect of necrotic cells on the modulation of miRNA and mRNA expression profiles and miRNA-mRNA network in CRT-MG cells.Materials and MethodsWe used human astroglioma cells, CRT-MG, treated with necrotic CRT-MG cells to examine the effect of necrosis on the modulation of miRNA and mRNA by next-generation sequencing. For preparation of necrotic cells, CRT-MGcellswere frozen and thawed through cycle of liquid nitrogen–water bath. The putative miRNA-mRNA regulatory relationshipwas inferred through target information, using miRDB.ResultsThe necrotic cells induced dysregulation of 106 miRNAs and 887 mRNAs. Among them, 11 miRNAs that had a negative correlation value of p < 0.05 by the hypergeometric test were screened, and their target mRNAs were analyzed by Gene Ontology enrichment analysis. Using the Kyoto Encyclopedia of Genes and Genomes database, we also found several necrotic cell treatment-activated pathways that were modulated by relevant gene targets of differentially expressed miRNAs.ConclusionOur result demonstrated that dysregulation of miRNA and mRNA expression profiles occurs when GBM cells are exposed to necrotic cells, suggesting that several miRNAs may have the potential to be used as biomarkers for predicting GBM progression and pathogenesis.

MicroRNAs that affect the Fanconi Anemia/BRCA pathway are downregulated in imatinib-resistant chronic myeloid leukemia patients without detectable BCR-ABL kinase domain mutations

Chronic myeloid leukemia (CML) patients who do not achieve landmark responses following treatment with imatinib mesylate (IM) are considered IM-resistant. Although IM-resistance can be due to BCR-ABL kinase domain (KD) mutations, many IM-resistant patients do not have detectable BCR-ABL KD mutations. MicroRNAs (miRNAs) are short non-coding RNAs that control gene expression. To investigate the role of miRNAs in IM-resistance, we recruited 8 chronic phase CML patients with IM-resistance who tested negative for BCR-ABL KD mutations and 2 healthy normal controls. Using miRNA sequencing, we identified 54 differentially expressed miRNAs; 43 of them downregulated. The 3 most differentially downregulated miRNAs were miR-146a-5p, miR-99b-5p and miR-151a-5p. Using real-time quantitative reverse transcriptase-polymerase chain reaction, the expression patterns of the 3 miRNAs were validated on the same cohort of 8 patients in addition to 3 other IM-resistant CML patients. In-silico analysis showed that the predicted gene targets are ATRIP, ATR, WDR48, RAD51C and FANCA genes which are involved in the Fanconi Anemia/BRCA pathway. This pathway regulates DNA damage response (DDR) and influences disease response to chemotherapy. Thus it is conceivable that DDR constitutes a key component in IM-resistance. Further research is needed to elucidate miRNA modulation of the predicted gene targets.

Interleukin-10 Deficiency Impairs Reparative Properties of Bone Marrow-Derived Endothelial Progenitor Cell Exosomes.

Endothelial progenitor cell (EPC)-based therapy has immense potential to promote cardiac neovascularization and attenuate ischemic injury. Functional benefits of EPCs and other adult stem cell therapies largely involve paracrine mechanisms and exosomes secreted by stem cells are emerging as pivotal paracrine entity of stem/progenitor cells. However, modest outcomes after EPC-/stem cell-based clinical trials suggest that stem cell/exosome function might be modulated by stimuli they encounter in ischemic tissues, including systemic inflammation. We hypothesized that EPCs under inflammatory stress might produce exosomes of altered and dysfunctional content, which may compromise EPC repair in ischemic heart disease. We have previously shown that EPCs obtained from interleukin-10 knockout (IL-10KO) mice (model mimicking systemic inflammation) display impaired angiogenic functions. Whether IL-10KO-EPC-derived exosomes inherit their parental dysfunctional phenotype and whether inflammatory environment alters the cargo of their secreted exosomes are not known. After cell expansion from IL-10KO and wild-type (WT) mice, we isolated exosomes and compared their functions in terms of effect on cell survival, proliferation, migration, and angiogenic capacity in vitro. WT-EPC-Exo treatment enhanced endothelial cell proliferation and tube formation, and inhibited apoptosis, whereas IL-10KO-Exo exhibited impaired or even detrimental effects, suggesting that the reparative capacity of WT-EPC-Exo is lost in exosomes derived from IL-10-KO-EPCs. Deep RNA sequencing and proteomic analyses to compare WT and IL-10KO-Exo revealed drastically altered exosome cargo. Importantly, IL-10KO-EPC-Exo were highly enriched in microRNAs and proteins that promote inflammation and apoptosis and inhibit angiogenesis. Through modulation of a specific enriched miRNA (miR-375), we partially rescued IL-10KO-EPC-Exo dysfunction. Thus, our study revealed that EPC exosomes display impaired function under inflammatory stimulus through changed exosome contents, and the dysfunction can be rescued by modulation of a specific target packed in exosomes.

Activation of cannabinoid receptors alters the expression of miRNAs in Granulocytic Myeloid Derived Suppressor cells (G-MDSCs) and Splenic Resident Granulocytes (SRG) thereby identifying shared and unique pathways of differentiation

Abstract THC (Δ9-Tetrahydrocannabinol) is the main psychotropic component in marijuana and has been shown to have potent anti-inflammatory properties. Recent studies from our laboratory have shown that THC triggers massive induction of myeloid derived suppressor cells (MDSCs) through modulation of miRNA(miR). In this study, we investigated the effect of THC on the regulation of miRs in Granulocytic MDSCs (G-MDSCs) and splenic resident Granulocytes (SRG) in mice. To this end, mice (C57BL/6) were treated with vehicle or THC (20 mg/kg bw) and 24 hrs post injection, G-MDSCs from peritoneal wash and SRG from spleen were sorted (&amp;gt;95% purity) and microRNA arrays were performed using total RNAs including miRs. There was significant alterations in the expression of about 75 miRs in both G-MDSCs and SRG. Out of the 75 miRs, 31 miRs were upregulated and 9 miRs were downregulated in both G-MDSCs and SRG. There were 21 miRs that were downregulated in G-MDSCs but upregulated in SRG. The remaining 14 miRs were upregulated in G-MDSCs but downregulaed in SRG. Upon analysis of dysregulaed miRs using IPA, there were more than 20 diseases and pathways that were regulated by these miRs. We also observed that dysregulatd miRs played a common role in the development of several diseases including cancer and inflammatory diseases. This study demonstrate for the first time that THC regulates miRs that are both shared and unique to G-MDSCs and SRGs, thereby suggesting that while G-MDSCs are similar in some ways to SRGs, they may be at different stages of differentiation. The present study was supported in part by NIH grants (P20GM103641, P01AT003961, R01AT006888, R01ES019313, and R01MH094755).

Thiourea priming enhances salt tolerance through co-ordinated regulation of microRNAs and hormones in Brassica juncea

Activation of stress tolerance mechanisms demands transcriptional reprogramming. Salt stress, a major threat to plant growth, enhances ROS production and affects transcription through modulation of miRNAs and hormones. The present study delineates salt stress ameliorating action of thiourea (TU, a ROS scavenger) in Brassica juncea and provides mechanistic link between redox, microRNA and hormones. The ameliorative potential of TU towards NaCl stress was related with its ability to decrease ROS accumulation in roots and increase Na+ accumulation in shoots. Small RNA sequencing revealed enrichment of down-regulated miRNAs in NaCl + TU treated roots, indicating transcriptional activation. Ranking analysis identified three key genes including BRX4, CBL10 and PHO1, showing inverse relationship with corresponding miRNA expression, which were responsible for TU mediated stress mitigation. Additionally, ABA level was consistently higher till 24 h in NaCl, while NaCl + TU treated roots showed only transient increase at 4 h suggesting an effective stress management. Jasmonate and auxin levels were also increased, which prioritized defence and facilitated root growth, respectively. Thus, the study highlights redox as one of the “core” components regulating miRNA and hormone levels, and also strengthens the use of TU as a redox priming agent for imparting crop resilience to salt stress.

MiRNAs: New Biomarkers and Therapeutic Targets in Dementia.

Dementia is a complex pathological state that affects millions of individuals worldwide and is responsible for a huge socioeconomic burden, making it a major health concern of current times. Given the impact of dementia in both patients and caregivers, it is crucial to fully clarify the molecular mechanisms underlying dementia-associated disorders, since without this knowledge our ability to correctly diagnose and treat these diseases is severely hampered. Epigenetic mechanisms, such as miRNA-mediated post-transcriptional regulation, have been reported to play a role in dementia pathogenesis. Given their ability to bind complementary mRNA sequences, miRNAs are able to induce temporary or permanent translation repression of their mRNA targets. Consequently, changes in miRNA levels may contribute to alterations in the expression of dementiarelated proteins, impacting the course of the disease. Conversely, studies have also reported that some of these proteins are able to regulate the biogenesis and transport of miRNAs, hinting at novel disease-related mechanisms that are now beginning to be explored. These findings have made miRNAs both interesting tools and promising targets in the design of novel therapeutic strategies. Moreover, the discovery of circulating miRNAs, which are released by cells of various tissues, including the brain, and travel in membrane-bound vesicles found in most biofluids, opened new possibilities concerning the usefulness of miRNAs as biomarkers of disease. In this context, the major aim of this review is to discuss the relevance of these small non-coding RNAs in dementia, focusing on their role as gene expression regulators, their potential as biomarkers of dementia subtype and stage, and the hypothesis of using miRNA modulation as an innovative therapeutic approach to treat dementia-related disorders.

Development of bioluminescent chick chorioallantoic membrane (CAM) models for primary pancreatic cancer cells: a platform for drug testing

The aim of the present study was to develop chick-embryo chorioallantoic membrane (CAM) bioluminescent tumor models employing low passage cell cultures obtained from primary pancreatic ductal adenocarcinoma (PDAC) cells. Primary PDAC cells transduced with lentivirus expressing Firefly-luciferase (Fluc) were established and inoculated onto the CAM membrane, with >80% engraftment. Fluc signal reliably correlated with tumor growth. Tumor features were evaluated by immunohistochemistry and genetic analyses, including analysis of mutations and mRNA expression of PDAC pivotal genes, as well as microRNA (miRNA) profiling. These studies showed that CAM tumors had histopathological and genetic characteristic comparable to the original tumors. We subsequently tested the modulation of key miRNAs and the activity of gemcitabine and crizotinib on CAM tumors, showing that combination treatment resulted in 63% inhibition of tumor growth as compared to control (p < 0.01). These results were associated with reduced expression of miR-21 and increased expression of miR-155. Our study provides the first evidence that transduced primary PDAC cells can form tumors on the CAM, retaining several histopathological and (epi)genetic characteristics of original tumors. Moreover, our results support the use of these models for drug testing, providing insights on molecular mechanisms underlying antitumor activity of new drugs/combinations.

New insights into vitamin D anticancer properties: focus on miRNA modulation.

Vitamin D anticancer properties are well known and have been demonstrated in many in vitro and in vivo studies. Mechanistic insights have given an explanation on how vitamin D exerts antineoplastic functions, which are mainly conducted via the canonical vitamin D receptor (VDR)-vitamin D response elements (VDRE) pathway. Numerous findings indicate that dietary components, including vitamin D, could exert chemopreventive effects through alterations of microRNA (miRNA) expression. As miRNAs have important roles in regulating diverse and vital cellular processes, it has been speculated that vitamin D's non-classical effects, including anticancer effects, could be mediated through alterations of miRNA expression level. The current review focuses on up-to-date experimental data on modulation of miRNA expression by vitamin D treatment in cancer, obtained in a cell culture system, animal models and human cohorts. Reported findings in the review show that vitamin D modulates expression of numerous and diverse miRNAs specific for cancer types. Even in its early phases, with many questions remaining to be answered, dissecting the molecular pathways of vitamin D miRNA modulation is an emerging area of science. The complete unraveling of vitamin D molecular mechanisms will emphasize the vitamin D dietary component as a potential chemopreventive agent in cancer and personalized nutrition.

Identification and Characterization of MicroRNA Differentially Expressed in Macrophages Exposed to Porphyromonas gingivalis Infection.

MicroRNAs (miRNAs) are short, noncoding RNAs involved in the regulation of several processes associated with inflammatory diseases and infection. Bacterial infection modulates miRNA expression to subvert any innate immune response. In this study we analyzed, using microarray analysis, the bacterial modulation of miRNAs in bone marrow-derived macrophages (BMMs) in which activity was induced by infection with Porphyromonas gingivalis The expression of several miRNAs was modulated 3 h postinfection (at a multiplicity of infection of 25). A bioinformatic analysis was performed to further identify pathways related to the innate immune host response under the influence of selected miRNAs. To assess the effects of the miRNAs identified on cytokine secretion (tumor necrosis factor alpha [TNF-α] and interleukin-10 [IL-10]), BMMs were transfected with selected miRNA mimics and inhibitors. Transfection with mmu-miR-155 and mmu-miR-2137 did not modify TNF-α secretion, while their inhibitors increased it. Inhibitors of mmu-miR-2137 and mmu-miR-7674 increased the secretion of the anti-inflammatory factor IL-10. In P. gingivalis-infected BMMs, mmu-miR-155-5p significantly decreased TNF-α secretion while inhibitor of mmu-miR-2137 increased IL-10 secretion. In vivo, in a mouse model of P. gingivalis-induced calvarial bone resorption, injection of mmu-miR-155-5p or anti-mmu-miR-2137 reduced the size of the lesion significantly. Furthermore, anti-mmu-miR-2137 significantly reduced inflammatory cell infiltration, osteoclast activity, and bone loss. Bioinformatic analysis demonstrated that pathways related to cytokine- and chemokine-related pathways but also osteoclast differentiation may be involved in the effects observed. This study contributes further to our understanding of P. gingivalis-induced modulation of miRNAs and their physiological effects. It highlights the potential therapeutic merits of targeting mmu-miR-155-5p and mmu-miR-2137 to control inflammation induced by P. gingivalis infection.

The Efficacy of Cardiac Anti-miR-208a Therapy Is Stress Dependent

MicroRNAs (miRNAs) are important regulators of biology and disease. Recent animal efficacy studies validate the therapeutic benefit of miRNA modulation and underscore the therapeutic value of miRNA-targeting oligonucleotides. However, whether disease conditions (stress) influence the pharmacological effects of an anti-miR is currently unknown. To study the effect of disease on target regulation after anti-miR treatment, we injected animals with anti-miR-208a, a synthetic oligonucleotide that inhibits the cardiomyocyte-specific miR-208a. Our data indicate that the presence of stress increases the number of regulated miR-208a targets, and that higher stress levels correlate with stronger target derepression. Additionally, the type of stress also influences which targets are regulated upon miR-208a inhibition. Studies in a large animal model indicate a similar stress-dependent anti-miR effect. Subsequent in vitro studies suggest that the influence of stress on anti-miR efficacy depends at least in part on increased cellular anti-miR uptake. These data indicate that the pharmacological effect of anti-miRs is stronger under disease conditions, and that both the type and severity of disease determine the therapeutic outcome. These facts will be important for assessing the therapeutic dose and predicting the therapeutic outcome when applying anti-miRs in a clinical setting.

Abstract WP110: Identification and Validation of a Unique Mirna Target Mir-141-3p in Post Stroke Socially Isolated Aged Mice

Introduction: MicroRNAs (miRNAs) are a class of short non-coding RNAs that have been identified as a powerful interventional tool for many diseases, including stroke. Very recent studies have found that microRNAs also mediate aspects of social interaction. Social environments can directly influence miRNA expression, which then triggers a plethora of downstream gene changes. Social isolation (SI) impairs stroke recovery and leads to inflammation. We hypothesize that miRNAs are involved in the detrimental effects of post-stroke social isolation. Methods: Eighteen-month-old male C57BL/6 mice were pair housed (PH) for two weeks prior to stroke and randomly assigned to various housing conditions (ST-ISO or ST-PH) immediately after stroke (ST). Mice were sacrificed either at 3, 7 or 14 days after 60-minute right MCAO or sham surgery (n=4-6/group) and perilesional frontal cortex was isolated for miRNA analysis. Total RNA was isolated using either Qiagen miRNeasy® Mini Kit/ miRVANA miRNA isolation kit (Ambion, Life technologies). Whole miRNOme analysis of total RNA isolated from brain tissue was performed using miRCURY LNA TM Universal RT microRNA. Post-treatment with an ‘in vivo ready’ antagomir of miR-141-3p (7mg/kg i.v/day x 3 days; n=4/group) was given through lateral tail veins. Results: Using whole miRNOme analysis of approx. 800 miRNA, we found miR-141-3p was a unique miRNA whose expression was significantly upregulated in a time dependent manner up to day 14 after stroke. The post treatment with an ‘in vivo ready’ antogomir of miR-141-3p reduced the isolation-induced increase in miR-141-3p to levels almost equal to that of pair-housed controls. Post- treatment significantly reduced mortality (by 21% as compared to -ve control) and sensory motor deficits after stroke. mRNA targets analysis study using qPCR confirmed the significant (p&lt;0.05 vs Neg Control ) upregulation of target several genes like arg-1, ccl22 and TGFbr1, markers of M2 type microglial activation, after antogomir treatment. Summary: The present data suggests the unique role of miR-141-3p in post stroke isolation. Temporal expression profiling studies suggest its validation as a potential targets. Post treatment data confirms the in vivo feasibility of miRNA modulation after stroke.

Two-Step Assembling of Near-Infrared "OFF-ON" Fluorescent Nanohybrids for Synchronous Tumor Imaging and MicroRNA Modulation-Based Therapy.

Theranostic nanoparticles with combined imaging and therapy functions show great promise in cancer precision medicine. In this study, we constructed near-infrared (NIR) "OFF-ON" fluorescent nanohybrids (F-PNDs) for synchronous tumor imaging and microRNA (miRNA) modulation therapy against esophageal cancer. Nanodiamond clusters (NDs) were first functionalized for protamine sulfate immobilization (PNDs) on their surfaces via a noncovalent self-assembling approach and simultaneous encapsulation of NIR emitting fluorescence dye cyanine 5 (Cy-5) (F-PNDs). Tumor suppressor miRNA-203 (miR-203) was then adsorbed onto the surface of F-PNDs to form miR-203/F-PNDs via electrostatic interactions. The size, morphology, photophysical and stability properties of miR-203/F-PNDs were analyzed. We found that the NIR fluorescence of miR-203/F-PNDs could be activated to the "ON" state in intracellular environment while remaining in the "OFF" state in extracellular or blood environment. Furthermore, in vivo live imaging experiments showed that miR-203/F-PNDs could be predominantly accumulated in tumor tissues and image the tumor sites 24 h postintravenous injection. In addition, intravenous and intratumoral injection of miR-203/F-PNDs could efficiently inhibit tumor growth through down-regulation of the expressions of oncogenes Ran and Δp63. Our study indicated that miRNA/F-PNDs could serve as a promising theranostic platform for synchronous tumor imaging and miRNA-based modulation therapy against cancer.

HHV-6A in vitro infection of thyrocytes and T cells alters the expression of miRNA associated to autoimmune thyroiditis

BackgroundHuman herpesviruses have been hypothesized as environmental triggers in the development of autoimmune thyroid diseases (AITD), and in particular active human herpesvirus 6A (HHV-6A) infection was detected in thyrocytes of Hashimoto’s thyroiditis (HT) patients, who also show specific anti-viral immune responses. On the other hand, AITD patients display modulation of specific miRNAs in thyroid tissue and blood. We wanted to ascertain whether HHV-6A infection might be correlated to the miRNA dysregulation observed in AITD.MethodsHuman thyroid and T-cell lines were infected in vitro with HHV-6A,-6B or −7, and analysed for miRNAs expression, either by microarray or by specific RT-PCR assays detecting miRNAs associated with AITD in vivo.ResultsHHV-6A infection, but not -6B or −7 infections, induced a decrease in miR-155_2 expression and an increase in miR-1238 expression in thyrocytes, as well as an increase in the expression levels of several autoimmunity-associated miRNAs in T lymphocytes, including miR-16_1, miR34a, miR-130a, miR-143_1, miR-202, miR-301b, miR-302c, miR-449b, miR-451_1, and miR-1238_2.ConclusionsHHV-6A infection modulates miRNAs expression in the cell types involved in the development of AITD. Notably, our in vitro findings correlate with what observed in AITD patients, further supporting the association between HHV-6A infection and AITD development. Moreover, these effects are 6A-specific, emphasizing the differences between the two HHV-6 virus species, and suggesting diverse virus mechanisms of action and therapeutic approaches.

Integration of MicroRNA, mRNA, and Protein Expression Data for the Identification of Cancer-Related MicroRNAs.

MicroRNAs (miRNAs) are responsible for the regulation of target genes involved in various biological processes, and may play oncogenic or tumor suppressive roles. Many studies have investigated the relationships between miRNAs and their target genes, using mRNA and miRNA expression data. However, mRNA expression levels do not necessarily represent the exact gene expression profiles, since protein translation may be regulated in several different ways. Despite this, large-scale protein expression data have been integrated rarely when predicting gene-miRNA relationships. This study explores two approaches for the investigation of gene-miRNA relationships by integrating mRNA expression and protein expression data. First, miRNAs were ranked according to their effects on cancer development. We calculated influence scores for each miRNA, based on the number of significant mRNA-miRNA and protein-miRNA correlations. Furthermore, we constructed modules containing mRNAs, proteins, and miRNAs, in which these three molecular types are highly correlated. The regulatory interactions between miRNA and genes in these modules have been validated based on the direct regulations, indirect regulations, and co-regulations through transcription factors. We applied our approaches to glioblastomas (GBMs), ranked miRNAs depending on their effects on GBM, and obtained 52 GBM-related modules. Compared with the miRNA rankings and modules constructed using only mRNA expression data, the rankings and modules constructed using mRNA and protein expression data were shown to have better performance. Additionally, we experimentally verified that miR-504, highly ranked and included in the identified modules, plays a suppressive role in GBM development. We demonstrated that the integration of both expression profiles allows a more precise analysis of gene-miRNA interactions and the identification of a higher number of cancer-related miRNAs and regulatory mechanisms.

Role of phytochemicals in the modulation of miRNA expression in cancer.

MicroRNAs (miRNAs) are single-stranded non-coding endogenous RNAs linked with normal developmental and physiological processes. Recent studies have demonstrated that the dysregulation of miRNAs plays an important role in the beginning, progression, and metastasis of many cancers. Additionally, they are involved in the regulation of the epithelial-to-mesenchymal transition (EMT) in many cancer cells. Several studies have suggested that phytochemicals play an important role in the modulation of miRNA expression, which is related to changes in oncogenes, tumour suppressors, and cancer-related protein expression. Hence, phytonutrients can suppress tumour development, prevent metastasis, reverse the EMT, and improve drug sensitivity via the modulation of miRNA expression. In this review, we discuss tumour-suppressing and oncogenic miRNAs and the mechanism of action for miRNAs as well as the role of phytochemicals in the modulation of miRNAs in various cancer cells.

Cytokines and MicroRNA in Coronary Artery Disease.

Cardiovascular disease (CVD) is a major health problem globally. The high incidence and case fatality of CVD are, to a large extent, a consequence of its late diagnosis and lack of highly sensitive and specific markers. Only a very small number of biomarkers, such as troponin, detect late disease. There is some evidence of an association and dysregulation between specific cytokines in the pathogenesis of CVD. These molecules are involved in inflammatory and immune mechanisms associated with atherogenesis. Several molecular/cellular pathways that include STAT, MAPK, and SMAD are modulated by cytokines. Against this background, microRNAs (miRNAs) are a class of noncoding RNAs with important roles in pathological events, leading to atherosclerotic CVD. It has been shown that the latter could affect cytokine production and contribute to progression of atherosclerotic CVD. Moreover, modulation of miRNAs appears to inhibit cardiomyocyte apoptosis, attenuate infarct size, and reduce cardiac dysfunction. This review highlights several recent preclinical and clinical studies on the role of cytokines in CVD, novel miRNA-based therapeutic approaches for therapeutic intervention, and potential circulating cytokines that have promise as biomarkers in CVD.

MiRnalyze: an interactive database linking tool to unlock intuitive microRNA regulation of cell signaling pathways.

The various pathophysiological processes occurring in living systems are known to be orchestrated by delicate interplays and cross-talks between different genes and their regulators. Among the various regulators of genes, there is a class of small non-coding RNA molecules known as microRNAs. Although, the relative simplicity of miRNAs and their ability to modulate cellular processes make them attractive therapeutic candidates, their presence in large numbers make it challenging for experimental researchers to interpret the intricacies of the molecular processes they regulate. Most of the existing bioinformatic tools fail to address these challenges. Here, we present a new web resource ‘miRnalyze’ that has been specifically designed to directly identify the putative regulation of cell signaling pathways by miRNAs. The tool integrates miRNA-target predictions with signaling cascade members by utilizing TargetScanHuman 7.1 miRNA-target prediction tool and the KEGG pathway database, and thus provides researchers with in-depth insights into modulation of signal transduction pathways by miRNAs. miRnalyze is capable of identifying common miRNAs targeting more than one gene in the same signaling pathway—a feature that further increases the probability of modulating the pathway and downstream reactions when using miRNA modulators. Additionally, miRnalyze can sort miRNAs according to the seed-match types and TargetScan Context ++ score, thus providing a hierarchical list of most valuable miRNAs. Furthermore, in order to provide users with comprehensive information regarding miRNAs, genes and pathways, miRnalyze also links to expression data of miRNAs (miRmine) and genes (TiGER) and proteome abundance (PaxDb) data. To validate the capability of the tool, we have documented the correlation of miRnalyze’s prediction with experimental confirmation studies. Database URL: http://www.mirnalyze.in

Differentially Expressed MicroRNAs in the Development of Early Diabetic Retinopathy.

The pathological mechanisms of diabetic retinopathy (DR), a leading cause of blindness in adults with diabetes mellitus, remain incompletely understood. Because microRNAs (miRNAs) represent effective DR therapeutic targets, we identified aberrantly expressed miRNAs associated with cellular dysfunction in early DR and detected their potential targets. We exposed human retinal endothelial cells (HRECs) and a cell line of retinal pigment epithelial (RPE) cells to high glucose (25 mmol/L, 1–7 days) to mimic DR progression and used streptozotocin-injected rats (4–8 weeks) for an in vivo diabetes model. HREC/RPE viability decreased after 24 h incubation and diminished further over 6 days, and Hoechst staining revealed hyperglycemia-induced HREC/RPE apoptosis. Although miR-124/-125b expression decreased with DR progression in vitro and in vivo, miR-135b/-199a levels decreased in retinal cells under hyperglycemia exposure, but increased in diabetic retinas. Moreover, miR-145/-146a expression decreased gradually in high-glucose-treated HRECs, but increased in hyperglycemia-exposed RPE cells and in diabetic rats. Our findings suggested that aberrant miRNA expression could be involved in hyperglycemia-induced retinal-cell dysfunction, and the identified miRNAs might vary in different retinal layers, with expression changes associated with DR development. Therefore, miRNA modulation and the targeting of miRNA effects on transcription factors could represent novel and effective DR-treatment strategies.

Hyperglycaemia suppresses microRNA expression in platelets to increase P2RY12 and SELP levels in type 2 diabetes mellitus.

Megakaryocyte (MK)-derived miRNAs have been detected in platelets. Here, we analysed the expression of platelet and circulating miR-223, miR-26b, miR-126 and miR-140 that might be altered with their target mRNAs in type 2 diabetes mellitus (DM2). MiRNAs were isolated from leukocyte-depleted platelets and plasma samples obtained from 28 obese DM2, 19 non-DM obese and 23 healthy individuals. The effect of hyperglycaemia on miRNAs was also evaluated in MKs using MEG-01 and K562 cells under hyperglycaemic conditions after 8 hours up to four weeks. Quantitation of mature miRNA, pre-miRNAs and target mRNA levels (P2RY12 and SELP) were measured by RT-qPCR. To prove the association of miR-26b and miR-140 with SELP (P-selectin) mRNA level, overexpression or inhibition of these miRNAs in MEG-01 MKs was performed using mimics or anti-miRNAs, respectively. The contribution of calpain substrate Dicer to modulation of miRNAs was studied by calpain inhibition. Platelet activation was evaluated via surface P-selectin by flow cytometry. Mature and pre-forms of investigated miRNAs were significantly reduced in DM2, and platelet P2RY12 and SELP mRNA levels were elevated by two-fold at increased platelet activation compared to controls. Significantly blunted miRNA expressions were observed by hyperglycaemia in MEG-01 and K562-MK cells versus baseline values, while the manipulation of miR-26b and miR-140 expression affected SELP mRNA level. Calpeptin pretreatment restored miRNA levels in hyperglycaemic MKs. Overall, miR-223, miR-26b, miR-126 and miR-140 are expressed at a lower level in platelets and MKs in DM2 causing upregulation of P2RY12 and SELP mRNAs that may contribute to adverse platelet function.

MiRNA Update: A Review Focus on Clinical Implications of miRNA in Vascular Remodeling

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level. Through specific base pairing with their targets messenger RNAs (mRNA), miRNA can modify cell phenotype and function. Several miRNAs are aberrantly expressed in diseased arteries and may influence different features of vascular remodeling, including neointimal formation and diminished re-endothelialization. This review will discuss the clinical implications of miRNAs in the field of vascular remodeling and their potential role as diagnostic and therapeutic tools. miRNA modulation offers a promising strategy for therapeutic intervention to inhibit smooth muscle cell proliferation and enhance endothelial regeneration after percutaneous coronary intervention (PCI) in order to reduce restenosis and late thrombosis.