Common micro RNAs (miRNAs) target complement factor H (CFH) regulation in Alzheimer’s disease (AD) and in age-related macular degeneration (AMD)

Alzheimer’s disease (AD) and age-related macular degeneration (AMD) are complex and progressive inflammatory degenerations of the human neocortex and retina. Recent molecular, genetic and epigenetic evidence indicate that at least 4 micro RNAs (miRNAs) - including the NF-кB-regulated miRNA-9, miRNA-125b, miRNA-146a and miRNA-155 - are progressively up-regulated in both AD and AMD. This quartet of up-regulated miRNAs in turn down-regulate a small brain- and retinal-cell-relevant family of target mRNAs, including that encoding complement factor H (CFH), a major negative regulator of the innate immune and inflammatory response. Together miRNA-146a and miRNA-155 recognize an overlapping miRNA regulatory control (MiRC) region in the CFH 3’-untranslated region (3’- UTR; 5’-TTTAGTATTAA-3’) to which either of these miRNAs may interact. Progressive, pathogenic increases in specific miRNA binding to the entire 232 nucleotide CFH 3’-UTR appears to be a major regulator of CFH expression down-regulation, and the inflammatory pathology that characterizes both AMD and AD. The data presented in this report provides evidence that up-regulation of brain- and retinal- abundant miRNAs, including miRNA-9, miRNA-125b, miRNA-146a and miRNA-155, are common to the pathogenetic mechanism of CFH deficiency that drives inflammatory neurodegeneration, and for the first time indicates multiple, independent miRNA-mediated regulation of the CFH mRNA 3’-UTR.     

microRNA (miRNA) speciation in Alzheimer’s disease (AD) cerebrospinal fluid (CSF) and extracellular fluid (ECF)

Human cerebrospinal fluid (CSF), produced by the choroid plexus and secreted into the brain ventricles and subarachnoid space, plays critical roles in intra-cerebral transport and the biophysical and immune protection of the brain. CSF composition provides valuable insight into soluble pathogenic bio-markers that may be diagnostic for brain disease. In these experiments we analyzed amyloid beta (Aβ) peptide and micro RNA (miRNA) abundance in CSF and in short post-mortem interval (PMI <2.1 hr) brain tissue-derived extracellular fluid (ECF) from Alzheimer’s disease (AD) and age-matched control neocortex. There was a trend for decreased abundance of Aβ42 in the CSF and ECF in AD but it did not reach statistical significance (mean age ~72 yr; N=12; p~0.06, ANOVA). The most abundant nucleic acids in AD CSF and ECF were miRNAs, and their speciation and inducibility were studied further. Fluorescent miRNA-array-based analysis indicated significant increases in miRNA-9, miRNA-125b, miRNA-146a, miRNA-155 in AD CSF and ECF (N=12; p<0.01, ANOVA). Primary human neuronal-glial (HNG) cell co-cultures stressed with AD-derived ECF also displayed an up-regulation of these miRNAs, an effect that was quenched using the anti-NF-кB agents caffeic acid phenethyl ester (CAPE) or 1-fluoro-2-[2-(4-methoxy-phenyl)-ethenyl]-benzene (CAY10512). Increases in miRNAs were confirmed independently using a highly sensitive LED-Northern dot-blot assay. Several of these NF-кB-sensitive miRNAs are known to be up-regulated in AD brain, and associate with the progressive spreading of inflammatory neurodegeneration. The results indicate that miRNA-9, miRNA-125b, miRNA-146a and miRNA-155 are CSF- and ECF-abundant, NF-кB-sensitive pro-inflammatory miRNAs, and their enrichment in circulating CSF and ECF suggest that they may be involved in the modulation or proliferation of miRNA-triggered pathogenic signaling throughout the brain and central nervous system (CNS).     

血清miRNA-93、miRNA-223联合miRNA-324-3p可作为诊断PCOS的生物标记物

本研究拟基于最近的miRNA报道,结合前期关于肌肉生长抑素(myostatin, MSTN)的相关研究,探究多囊卵巢综合症(ploycystic ovary syndrome, PCOS)患者血清miRNA和生长抑素的表达及其相关临床病理特征。本研究选取160例在湘南学院附属医院妇科就诊的女性为研究对象,通过PCR、ELISA等方法比较PCOS患者血清miRNA和生长抑素的表达情况,并且分析其相关临床病理特征。结果显示,通过PCR的方法发现mi RNA-93、mi RNA-223在PCOS患者的血清中显著升高,而mi RNA-4522、mi RNA-6767-5p和mi RNA-324-3p则表达下降。本研究建立了3个miRNA的预测模型,并证实预测模型在筛选组和验证组中有很好的敏感性和特异性,可以有效区分PCOS患者和正常人群,但研究结果也发现,miRNA模型结合MSTN没有更好的诊断价值。     

MicroRNAs up-regulated by CagA of Helicobacter pylori induce intestinal metaplasia of gastric epithelial cells

CagA of Helicobacter pylori is a bacterium-derived oncogenic protein closely associated with the development of gastric cancers. MicroRNAs (miRNAs) are a class of widespread non-coding RNAs, many of which are involved in cell growth, cell differentiation and tumorigenesis. The relationship between CagA protein and miRNAs is unclear. Using mammalian miRNA profile microarrays, we found that miRNA-584 and miRNA-1290 expression was up-regulated in CagA-transformed cells, miRNA-1290 was up-regulated in an Erk1/2-dependent manner, and miRNA-584 was activated by NF-κB. miRNA-584 sustained Erk1/2 activities through inhibition of PPP2a activities, and miRNA-1290 activated NF-κB by knockdown of NKRF. Foxa1 was revealed to be an important target of miRNA-584 and miRNA-1290. Knockdown of Foxa1 promoted the epithelial-mesenchymal transition significantly. Overexpression of miRNA-584 and miRNA-1290 induced intestinal metaplasia of gastric epithelial cells in knock-in mice. These results indicate that miRNA-584 and miRNA-1290 interfere with cell differentiation and remodel the tissues. Thus, the miRNA pathway is a new pathogenic mechanism of CagA.     

Expression differences of miRNAs and genes on NF-κB pathway between the healthy and the mastitis Chinese Holstein cows

In order to discover the variation of microRNAs and genes associated with NF-κB signaling pathway between the healthy and the mastitis Chinese Holstein cows, Illumina Deep Sequencing and qRT-PCR are applied to detect 25 kinds of miRNAs (miR-16, miR-125b, miR-15, miR-29a, miR-23b, miR-146, miR-301a, miR-181b, let-7, miR-30b, miR-21, miR-223, miR-27b, miR-10a, miR-143, etc.) expression levels in blood samples and 14 genes (RelA, RelB, Rel, p105, p100, IκBα, IκBβ, IκBδ, IκBε, IκBζ, Bcl-3, IKKα, IKKβ, IKKγ/NEMO) relative expression levels in nine tissues. The total number of miRNAs is declining, and RelA, Rel, p105, p100, IκBα, IκBβ, IκBδ, IκBζ, Bcl-3, and IKKα expressions are rising in mastitis individuals. So, we suppose that NF-κB pathway is active in mastitis individuals as a result of the decrease inhibition of miRNAs. While in healthy ones, the NF-κB pathway is inactive, because of the miRNAs enhanced inhibition action. However, the specific regulatory mechanism of miRNAs on NF-κB pathway in mastitis Holstein cows needs further investigation. Moreover, due to obvious expression differences, some miRNAs, especially miR-16 and miR-223, may be used as new markers for the dairy mastitis prognosing.     

MiRNA-19a and miRNA-19b regulate proliferation of antler cells by targeting TGFBR2

Transforming growth factor (TGF-β) plays a pivotal role in angiogenesis. The purpose of this study was to explore the microRNA-mediated regulation of TGF-β receptor-II (TGFBR2) expression during rapid antler growth and proliferation of antler cells in sika deer. Deep sequencing–based expression analysis of miRNAs on the antler tip tissue was performed. Then, two bioinformatics software were used to analyze TGFBR2 3′-UTR sequence for predicting the matched and differentially expressed miRNAs in different tissues of the antler. The results indicated that miRNA-19a and miRNA-19b exhibited the highest upregulation among differentially expressed miRNAs. We also found that the TGFBR2 3′-UTR contains a binding site for miRNA-19a and miRNA-19b by transfection of wild-type and mutant dual-luciferase reporter vectors into antler cartilage cells. Meanwhile, overexpression of miRNA-19a and miRNA-19b significantly inhibited the proliferation of cartilage cells in vitro, and decreased the expression level of TGFBR2 protein. Furthermore, the expression levels of insulin-like growth factor 1 (IGF-1) and TGF-β2, which were associated with TGFBR2, reduced after transfection of cartilage cells with miRNA-19a and miRNA-19b. Our results indicate the significant roles of miRNA-19a and miRNA-19b in proliferation of antler cells and its potential application.     

microRNA-342, microRNA-191 and microRNA-510 are differentially expressed in T regulatory cells of type 1 diabetic patients

Regulatory T cells (Tregs) are critical regulators of autoimmune diseases, including type 1 diabetes mellitus. It is hypothesised that Tregs’ function can be influenced by changes in the expression of specific microRNAs (miRNAs). Thus, we performed miRNAs profiling in a population of Tregs separated from peripheral blood of five type 1 diabetic patients and six healthy donors. For more detailed molecular characterisation of Tregs, we additionally compared miRNAs expression profiles of Tregs and conventional T cells. Tregs were isolated according to CD3+, CD4+, CD25^hi+ and CD127− by flow cytometry, and miRNA expression profiling was performed using TaqMan Array Human MicroRNA Panel-1 (384-well low density array). In Tregs of diabetic patients we found significantly increased expression of miRNA-510 (p = 0.05) and decreased expression of both miRNA-342 (p < 0.0001) and miRNA-191 (p = 0.0079). When comparing Tregs and T cells, we revealed that Tregs had significant higher expression of miRNA-146a and lower expression of eight specific miRNAs (20b, 31, 99a, 100, 125b, 151, 335, and 365). To our knowledge, this is the first study demonstrating changes in miRNA expression profiles occurring in Tregs of T1D patients and a miRNAs signature of adult Tregs.     

Up-regulation of microRNA-155 in macrophages contributes to increased tumor necrosis factor {alpha} (TNF{alpha}) production via increased mRNA half-life in alcoholic liver disease

Activation of Kupffer cells (KCs) by gut-derived lipopolysaccharide (LPS) and Toll-Like Receptors 4 (TLR4)-LPS-mediated increase in TNFα production has a central role in the pathogenesis of alcoholic liver disease. Micro-RNA (miR)-125b, miR-146a, and miR-155 can regulate inflammatory responses to LPS. Here we evaluated the involvement of miRs in alcohol-induced macrophage activation. Chronic alcohol treatment in vitro resulted in a time-dependent increase in miR-155 but not miR-125b or miR-146a levels in RAW 264.7 macrophages. Furthermore, alcohol pretreatment augmented LPS-induced miR-155 expression in macrophages. We found a linear correlation between alcohol-induced increase in miR-155 and TNFα induction. In a mouse model of alcoholic liver disease, we found a significant increase in both miR-155 levels and TNFα production in isolated KCs when compared with pair-fed controls. The mechanistic role of miR-155 in TNFα regulation was indicated by decreased TNFα levels in alcohol-treated macrophages after inhibition of miR-155 and by increased TNFα production after miR-155 overexpression, respectively. We found that miR-155 affected TNFα mRNA stability because miR-155 inhibition decreased whereas miR-155 overexpression increased TNFα mRNA half-life. Using the NF-κB inhibitors, MG-132 or Bay11-7082, we demonstrated that NF-κB activation mediated the up-regulation of miR-155 by alcohol in KCs. In conclusion, our novel data demonstrate that chronic alcohol consumption increases miR-155 in macrophages via NF-κB and the increased miR-155 contributes to alcohol-induced elevation in TNFα production via increased mRNA stability.     

NF-κB is involved in the LPS-mediated proliferation and apoptosis of MAC-T epithelial cells as part of the subacute ruminal acidosis response in cows

Objectives

To determine the effect of NF-κB on cell proliferation and apoptosis, we investigate the expression of inflammation and apoptosis-related factors in the bovine mammary epithelial cell line, MAC-T.

Results

MAC-T cells were cultured in vitro and MTT and LDH assays used to determine the effects of lipopolysaccharide (LPS) on proliferation and cytotoxicity respectively. RT-PCR and western blotting were used to evaluate the effect of LPS and NF-κB inhibition [pyrrolidine dithiocarbamate (PDTC) treatment] on the expression of inflammation and apoptosis-related factors. LPS significantly inhibited MAC-T cell proliferation in a dose- and time-dependent manner. Furthermore, LPS promoted apoptosis while the NF-кB inhibitor PDTC attenuated this effect. After LPS treatment, the NF-кB signaling pathway was activated, and the expression of inflammation and apoptosis-related factors increased. When PDTC blocked NF-кB signaling, the expression of inflammation and apoptosis-related factors were decreased in MAC-T cells.

Conclusions

LPS activates the TLR4/NF-κB signaling pathway, inhibits proliferation and promotes apoptosis in MAC-T cells. NF-кB inhibition attenuates MAC-T cell apoptosis and TLR4/NF-κB signaling pathway. NF-кB inhibitor alleviating MAC-T cell apoptosis is presumably modulated by NF-кB.

     

miRNA-181a在胃癌细胞增殖和迁移中的作用

miRNA与恶性肿瘤患者的诊断和预后密切相关,为了考察miRNA-181a在胃癌细胞增殖和迁移中的作用,本研究检测了miRNA-181a在胃癌组织中的表达,并通过对人胃癌细胞系MGC-803转染miR-181a模拟物或抑制剂来考察miR-181a对细胞迁移和增殖的影响。RT-PCR显示,miRNA-181a在胃癌组织中的表达水平显著高于癌旁组织(p<0.05)。伤口愈合实验和Transwell实验显示,转染miR-181a抑制剂或TGF-β受体2(TGFβR2)过表达的pcDNA3.1质粒均可抑制MGC-803细胞的迁移。EdU实验和CCK-8实验显示,转染miR-181a抑制剂或TGFβR2过表达的pcDNA3.1质粒均可抑制MGC-803细胞的增殖。此外,miR-181a抑制剂处理可使TGFβR2蛋白表达明显升高。然而,miR-181a模拟物或抑制剂处理后TGFβR2mRNA水平没有显著变化。总之,本研究表明高表达的miR-181a通过在转录后抑制TGFβR2蛋白表达来促进胃癌细胞的迁移和增殖。miR-181a有望成为胃癌的潜在治疗靶点。