The Role of miR-342 in Vascular Health. Study in Subclinical Cardiovascular Disease in Mononuclear Cells, Plasma, Inflammatory Cytokines and PANX2.

Cardiovascular disease (CVD) correlates with inflammation and a reduction in circulating endothelial progenitor cells (cEPCs). Recently, CVD was shown to be the main cause of mortality in individuals with type 1 diabetes (T1DM). In animals, miR-342 was shown to exert an anti-inflammatory effect in CVD. Hypothesis: miR-342-3p/-5p are downregulated in subclinical CVD (T1DM), whereas inflammatory cytokines are upregulated. We studied miR -342 -3p/5p in plasma/peripheral blood mononuclear cells (PBMCs) in 29 T1DM and 20 controls (HC). Vascular health was measured by fibronectin adhesion assay (FAA), cEPCs (CD45dimCD34+133+ cells) and by assessing inflammation and tissue inhibition of metalloproteases (TIMP-1). In T1DM IL-7, IL-8, TNFα and VEGF-C were increased in plasma. MiR-342-3p/-5p were downregulated in PBMCs in T1DM, but not in plasma. PANX2, chemokine receptors CXCR1/2 mRNAs, were increased in PBMCs in T1DM. MiR-342-3p was negatively correlated with TIMP-1, IL-6, IL-8, TNF-α, HbA1c and CXCR2, whilst miR-342-5p was negatively correlated with TIMP-1, IL-6, IL-8 and HbA1c. There was a positive correlation among miR-342-3p, FAA and cEPCs, and between miR-342-5p and cEPCs. ROC curve analyses showed significant downregulation of miR-342-3p/-5p at HbA1c > 46.45 mmol/mol, indicating their potential as biomarkers for subclinical CVD. Our findings validated animal studies and confirmed the proangiogenic properties of miR-342-3p/-5p. MiR-342-3p/-5p-based intervention or monitoring may prove to be beneficial in managing CVD.

miR-199a-3p/5p regulate tumorgenesis via targeting Rheb in non-small cell lung cancer.

Lung cancer is one of the deadliest cancers, in which non-small cell lung cancer (NSCLC) accounting for 85% and has a low survival rate of 5 years. Dysregulation of microRNAs (miRNAs) can participate in tumor regulation and many major diseases. In this study, we found that miR-199a-3p/5p were down-expressed in NSCLC tissue samples, cell lines, and the patient sample database. MiR-199a-3p/5p overexpression could significantly suppress cell proliferation, migration ability and promote apoptosis. Through software prediction, ras homolog enriched in brain (Rheb) was identified as a common target of miR-199a-3p and miR-199a-5p, which participated in regulating mTOR signaling pathway. The same effect of inhibiting NSCLC appeared after down-regulating the expression of Rheb. Furthermore, our findings revealed that miR-199a can significantly inhibit tumor growth and metastasis in vivo, which fully demonstrates that miR-199a plays a tumor suppressive role in NSCLC. In addition, miR-199a-3p/5p has been shown to enhance the sensitivity of gefitinib to EGFR-T790M in NSCLC. Collectively, these results prove that miR-199a-3p/5p can act as cancer suppressor genes to inhibit the mTOR signaling pathway by targeting Rheb, which in turn inhibits the regulatory process of NSCLC. Thus, to investigate the anti-cancer effect of pre-miR-199a/Rheb/mTOR axis in NSCLC, miR-199a-3p and miR-199a-5p have the potential to become an early diagnostic marker or therapeutic target for NSCLC.

New insight into the molecular mechanism of miR482/2118 during plant resistance to pathogens.

MicroRNAs (miRNAs), a group of small noncoding RNAs (approximately 20-24 nucleotides), act as essential regulators affecting endogenous gene expression in plants. MiR482/2118 is a unique miRNA superfamily in plants and represses NUCLEOTIDE BINDING SITE-LEUCINE-RICH REPEAT (NBS-LRR) genes to function in plant resistance to pathogens. In addition, over the past several years, it has been found that miR482/2118 not only targets NBS-LRRs but also acts on other molecular mechanisms to affect plant resistance. miR482/2118-5ps, phased small interfering RNAs (phasiRNAs) and long noncoding RNAs (lncRNAs) play important roles in plant disease resistance. This review summarizes the current knowledge of the interactions and links between miR482/2118 and its new interacting molecules, miR482/2118-5p, phasiRNAs and lncRNAs, in plant disease resistance. Here, we aim to provide a comprehensive view describing the new molecular mechanism associated with miR482/2118 in the plant immune system.

A high performance liquid chromatography fluorescence method for the analysis of both pyridoxal-5-phosphate and thiamine pyrophosphate in whole blood.

BACKGROUND AND AIMS: The active coenzymes of the water soluble vitamins B1 and B6 (thiamine pyrophosphate (TPP) and pyridoxal-5-phosphate (P5P) respectively) play an important role in numerous bodily functions. The simultaneous analysis of both these analytes is limited to either mass spectrometry based methods or commercial kit suppliers. In this study we developed a novel method for analysis of both TPP and P5P by fluorescence detection. METHODS: Briefly, whole blood samples are precipitated by trichloroacetic acid, and P5P and TPP are both derivatised before separation on a C18-PFP column. The new assay's performance was compared against a recent cycle from an external quality assurance program (RCPAQAP) and the current only existing commercial kit (n = 76). RESULTS: Linearity for both analytes was above 0.99 (r2) up to a concentration range of: 4000 nmol/L (P5P) and 2000 nmol/L (TPP). Precision of the method (intra-day and inter-day) compared against commercial quality control material was below 6% (coefficient of variation). Recovery of both compounds exceeded 90%. Accuracy of the protocol displayed satisfactory results in proficiency testing and had an acceptable level of agreement with the existing current kit method. CONCLUSIONS: Overall, this method provides an economical alternative in routine clinical diagnostic laboratories wishing to perform P5P and TPP analysis.

BCL-6 suppresses miR-142-3p/5p expression in SLE CD4+ T cells by modulating histone methylation and acetylation of the miR-142 promoter.

The reduced expression of miR-142-3p/5p in CD4+ T cells of SLE patients caused T cell hyperactivity and B cell hyperstimulation. This study aimed to investigate the mechanisms of regulating miR-142-3p/5p expression in SLE CD4+ T cells. The BCL-6 expression was significantly increased in SLE CD4+ T cells compared with normal controls, and the BCL-6 expression was inversely correlated with miR-142-3p/5p expression. BCL-6 suppresses the expression of miR-142-3p/5p by increasing H3K27me3 level and reducing H3K9/K14ac levels in SLE CD4+ T cells. BCL-6 regulates histone modifications in miR-142 promoter by recruiting EZH2 and HDAC5. Furthermore, we observed significantly decreased CD40L, ICOS, and IL-21 expression levels in SLE CD4+ T cells with BCL-6 interference, and obviously reduced autoantibody IgG production in autologous B cells co-cultured with BCL-6 inhibited SLE CD4+ T cells. Our study found that increased BCL-6 up-regulates H3K27me3 and down-regulates H3K9/14ac at miR-142 promoter in SLE CD4+ T cells. These factors induce a declination in miR-142-3p/5p expression, consequently resulting in CD4+ T cell hyperactivity.

miR-142-3p Regulates BDNF Expression in Activated Rodent Microglia Through Its Target CAMK2A.

Microglia, the innate immune effector cells of the mammalian central nervous system (CNS), are involved in the development, homeostasis, and pathology of CNS. Microglia become activated in response to various insults and injuries and protect the CNS by phagocytosing the invading pathogens, dead neurons, and other cellular debris. Recent studies have demonstrated that the epigenetic mechanisms ensure the coordinated regulation of genes involved in microglial activation. In this study, we performed a microRNA (miRNA) microarray in activated primary microglia derived from rat pup's brain and identified differentially expressed miRNAs targeting key genes involved in cell survival, apoptosis, and inflammatory responses. Interestingly, miR-142-3p, one of the highly up-regulated miRNAs in microglia upon lipopolysaccharide (LPS)-mediated activation, compared to untreated primary microglia cells was predicted to target Ca2+/calmodulin dependent kinase 2a (CAMK2A). Further, luciferase reporter assay confirmed that miR-142-3p targets the 3'UTR of Camk2a. CAMK2A has been implicated in regulating the expression of brain-derived neurotrophic factor (BDNF) and long-term potentiation (LTP), a cellular mechanism underlying memory and learning. Given this, this study further focused on understanding the miR-142-3p mediated regulation of the CAMK2A-BDNF pathway via Cyclic AMP-responsive element-binding protein (CREB) in activated microglia. The results revealed that CAMK2A was downregulated in activated microglia, suggesting an inverse relationship between miR-142-3p and Camk2a in activated microglia. Overexpression of miR-142-3p in microglia was found to decrease the expression of CAMK2A and subsequently BDNF through regulation of CREB phosphorylation. Functional analysis through shRNA-mediated stable knockdown of CAMK2A in microglia confirmed that the regulation of BDNF by miR-142-3p is via CAMK2A. Overall, this study provides a database of differentially expressed miRNAs in activated primary microglia and reveals that microglial miR-142-3p regulates the CAMK2A-CREB-BDNF pathway which is involved in synaptic plasticity.

A novel identification approach for discovery of 5-HydroxyTriptamine 2A antagonists: combination of 2D/3D similarity screening, molecular docking and molecular dynamics.

5-HydroxyTriptamine 2A antagonists are potential targets for treatment of various cerebrovascular and cardiovascular disorders. In this study, we have developed and performed a unique screening pipeline for filtering ZINC database compounds on the basis of similarities to known antagonists to determine novel small molecule antagonists of 5-HydroxyTriptamine 2A. The screening pipeline is based on 2D similarity, 3D dissimilarity and a combination of 2D/3D similarity. The shortlisted compounds were docked to a 5-HydroxyTriptamine 2A homology-based model, and complexes with low binding energies (287 complexes) were selected for molecular dynamics (MD) simulations in a lipid bilayer. The MD simulations of the shortlisted compounds in complex with 5-HydroxyTriptamine 2A confirmed the stability of the complexes and revealed novel interaction insights. The receptor residues S239, N343, S242, S159, Y370 and D155 predominantly participate in hydrogen bonding. π-π stacking is observed in F339, F340, F234, W151 and W336, whereas hydrophobic interactions are observed amongst V156, F339, F234, V362, V366, F340, V235, I152 and W151. The known and potential antagonists shortlisted by us have similar overlapping molecular interaction patterns. The 287 potential 5-HydroxyTriptamine 2A antagonists may be experimentally verified.

microRNA-22 can regulate expression of the long non-coding RNA MEG3 in acute myeloid leukemia.

AIM: Acute myeloid leukemia (AML) is the most common blood tumor with poor prognosis. At present, the research found that the pathogenesis of AML is related to many factors, such as recurrent somatic mutations and gene expression and epigenetic changes, however, the molecular mechanism of AML is still unclear. Long non-coding RNA MEG3 is a newly found tumor suppressor and plays a very important role in the regulation of a variety of tumor formation and progression. Studies found that the MEG3 expression was significantly decreased in AML. However, to date, it is not clear the cause of its abnormal expression. Therefore, the molecular mechanism of AML is urgently needed to study the molecular mechanism of AML. METHODS: The different expression level of MEG3, TET2, miR-22-3p, miR-22-5p in AML was detected by real-time quantification PCR. MEG3, TET2, miR-22-3p, miR-22-3p expression cell pools in K562 cells was used to interfering and TET2, MEG3 TET2, relations with miR-22-3p, miR-22-5p. The effect of AML cell on proliferation was evaluated by TET2 lower expression. RESULTS: 1. The lower expression of MEG3 and TET2 in AML cell lines was detected by RT-qPCR. 2. The stable MEG3, TET2 overexpression cell pools in K562 cells was successful established. 3. After transfection, MTT assay revealed that cell growth was significantly increased in AML cell lines transfected with TET2 compared with controls. CONCLUSIONS: Our findings suggested that MEG3 is significantly down regulated in AML cell lines.

Down-regulation of miR-30a-3p/5p promotes esophageal squamous cell carcinoma cell proliferation by activating the Wnt signaling pathway.

AIM: To investigate the potential role of microRNA-30a (miR-30a) in esophageal squamous cell carcinoma (ESCC). METHODS: Expression of miR-30a-3p/5p was analyzed using microarray data and fresh ESCC tissue samples. Both in vitro and in vivo assays were used to investigate the effects of miR-30a-3p/5p on ESCC cell proliferation. Furthermore, Kyoto Encyclopedia of Genes and Genomes analysis was performed to explore underlying mechanisms involved in ESCC, and then, assays were carried out to verify the potential molecular mechanism of miR-30a in ESCC. RESULTS: Low expression of miR-30a-3p/5p was closely associated with advanced ESCC progression and poor prognosis of patients with ESCC. Knock-down of miR-30a-3p/5p promoted ESCC cell proliferation. Increased miR-30a-3p/5p expression inhibited the Wnt signaling pathway by targeting Wnt2 and Fzd2. CONCLUSION: Down-regulation of miR-30a-3p/5p promotes ESCC cell proliferation by activating the Wnt signaling pathway through inhibition of Wnt2 and Fzd2.

Oral supplementations of betaine, choline, creatine and vitamin B6 and their influence on the development of homocysteinaemia in neonatal piglets.

Homocysteine (Hcy) is an intermediary sulphur amino acid recognised for pro-oxidative properties in several species which may weaken immune competence in piglets. In this species, there is an acute 10-fold increase of concentrations of plasma Hcy (pHcy) during the first 2 weeks of life. The present experiment aimed to determine if pHcy in piglets can be regulated by oral supplementations of betaine as a methyl group supplier, creatine for reducing the demand for methyl groups, choline with both previous functions and vitamin B6 as enzymic co-factor for Hcy catabolism. A total of seventeen sows (second parity) were fed gestation and lactation diets supplemented with folic acid (10 mg/kg) and vitamin B12 (150 µg/kg). Eight piglets in each litter received daily one of the eight following oral treatments (mg/kg body weight): (1) control (saline); (2) betaine (50); (3) choline (70); (4) creatine (300); (5) pyridoxine (0·2); (6) treatments 2 and 5; (7) treatments 3 and 4; and (8) treatments 2, 3, 4 and 5. According to age, pHcy increased sharply from 2·48 µm at birth to 17·96 µm at 21 d of age (P < 0·01). Concentrations of pHcy tended to be lower (P = 0·09) in treated than in control piglets but the highest and sole pairwise significant decrease (23 %) was observed between treatments 1 and 8 (P = 0·03). Growth from birth to 21 d of age was not influenced by treatments (P > 0·70). Therefore, it appears possible to reduce pHcy concentrations in suckling piglets but a combination of all chosen nutrients is required.