MicroRNA-34a regulation of endothelial senescence

Endothelial senescence is thought to play a role in cardiovascular diseases such as atherosclerosis. We hypothesized that endothelial microRNAs (miRNAs) regulate endothelial survival and senescence. We found that miR-34a is highly expressed in primary endothelial cells. We observed that miR-34a expression increases in senescent human umbilical cord vein endothelial cells (HUVEC) and in heart and spleen of older mice. MiR-34a over-expression induces endothelial cell senescence and also suppresses cell proliferation by inhibiting cell cycle progression. Searching for how miR-34a affects senescence, we discovered that SIRT1 is a target of miR-34a. Over-expressing miR-34a inhibits SIRT1 protein expression, and knocking down miR-34a enhances SIRT1 expression. MiR-34a triggers endothelial senescence in part through SIRT1, since forced expression of SIRT1 blocks the ability of miR-34a to induce senescence. Our data suggest that miR-34a contributes to endothelial senescence through suppression of SIRT1.     

TNFα induces the expression of genes associated with endothelial dysfunction through p38MAPK-mediated down-regulation of miR-149

MicroRNAs have been proposed as novel regulators of vascular inflammation and dysfunction. This study aimed to evaluate the role of miR-149 in regulating the expression of key molecules associated with TNFα-induced endothelial activation. miR-149 was selected by in silico analysis and microRNA target prediction. Endothelial dysfunction was induced by TNFα treatment in Eahy926 endothelial cells and HUVEC. miR-149 level was evaluated by quantitative real time-polymerase chain reaction (RT-qPCR). Metalloproteinase-9 (MMP-9) was measured by zymography, Inducible Nitric Oxide Synthase (iNOS) by immunoblotting, Interleukin-6 (IL-6) and Interleukin-8 (IL-8) by ELISA. miR-149 regulatory effect was evaluated by gain-of-function technique upon miR-149 mimics transfection.     

Pathway analysis of senescence-associated miRNA targets reveals common processes to different senescence induction mechanisms

Multiple mechanisms of senescence induction exist including telomere attrition, oxidative stress, oncogene expression and DNA damage signalling. The regulation of the cellular changes required to respond to these stimuli and create the complex senescent cell phenotype has many different mechanisms. MiRNAs present one mechanism by which genes with diverse functions on multiple pathways can be simultaneously regulated. In this study we investigated 12 miRNAs previously identified as senescence regulators. Using pathway analysis of their target genes we tested the relevance of miRNA regulation in the induction of senescence. Our analysis highlighted the potential of these senescence-associated miRNAs (SA-miRNAs) to regulate the cell cycle, cytoskeletal remodelling and proliferation signalling logically required to create a senescent cell. The reanalysis of publicly available gene expression data from studies exploring different senescence stimuli also revealed their potential to regulate core senescence processes, regardless of stimuli. We also identified stimulus specific apoptosis survival pathways theoretically regulated by the SA-miRNAs. Furthermore the observation that miR-499 and miR-34c had the potential to regulate all 4 of the senescence induction types we studied highlights their future potential as novel drug targets for senescence induction.     

miR-223促进小鼠胚胎成纤维细胞复制性衰老

微小RNA（MicroRNAs（或miRNAs）是作为强大的基因表达调控子,广泛参与多种生命过程,在细胞衰老进程中的作用也日益受到关注。miR-223是一个典型的抑癌基因,可显著抑制细胞增殖能力。此外,miR-223与阿尔茨海默症、心血管疾病以及类风湿性关节炎等衰老相关疾病的发生发展密切相关。尽管如此,miR-223在细胞衰老进程中的作用及其分子机制尚未见报道。本研究通过连续传代建立了小鼠胚胎成纤维细胞（MEF细胞）的复制性衰老模型,并利用荧光定量qRT-PCR检测发现,miR-223在衰老MEF细胞中的表达水平显著上调。随后,通过转染miR-223模拟物Agomir-223在MEF细胞中过表达miR-223,结果显示过表达miR-223可显著促进MEF细胞的衰老表型并抑制其增殖能力,而抑制miR-223的表达可延缓MEF细胞的复制性衰老进程。进一步利用生物信息学方法预测获得多个miR-223的候选衰老相关靶基因,包括Rasa1、Ddit4和Smad1等。然而双萤光素酶报告系统结果显示,miR-223并不显著影响其萤光强度,表明它们很可能并不是miR-223的下游靶基因。综上所述,miR-223可显著促进MEF细胞复制性衰老,然而其调节细胞衰老进程的分子机制依然有待深入研究。     

MicroRNA-127 inhibits lung inflammation by targeting IgG Fcγ receptor I

The molecular mechanisms of acute lung injury are incompletely understood. MicroRNAs (miRNAs) are crucial biological regulators that act by suppressing their target genes and are involved in a variety of pathophysiologic processes. miR-127 appears to be downregulated during lung injury. We set out to investigate the role of miR-127 in lung injury and inflammation. Expression of miR-127 significantly reduced cytokine release by macrophages. Looking into the mechanisms of regulation of inflammation by miR-127, we found that IgG FcγRI (CD64) was a target of miR-127, as evidenced by reduced CD64 protein expression in macrophages overexpressing miR-127. Furthermore, miR-127 significantly reduced the luciferase activity with a reporter construct containing the native 3' untranslated region of CD64. Importantly, we demonstrated that miR-127 attenuated lung inflammation in an IgG immune complex model in vivo. Collectively, these data show that miR-127 targets macrophage CD64 expression and promotes the reduction of lung inflammation. Understanding how miRNAs regulate lung inflammation may represent an attractive way to control inflammation induced by infectious or noninfectious lung injury.     

MicroRNA-210 modulates endothelial cell response to hypoxia and inhibits the receptor tyrosine kinase ligand Ephrin-A3

MicroRNAs (miRNAs) are small non-protein-coding RNAs that function as negative gene expression regulators. In the present study, we investigated miRNAs role in endothelial cell response to hypoxia. We found that the expression of miR-210 progressively increased upon exposure to hypoxia. miR-210 overexpression in normoxic endothelial cells stimulated the formation of capillary-like structures on Matrigel and vascular endothelial growth factor-driven cell migration. Conversely, miR-210 blockade via anti-miRNA transfection inhibited the formation of capillary-like structures stimulated by hypoxia and decreased cell migration in response to vascular endothelial growth factor. miR-210 overexpression did not affect endothelial cell growth in both normoxia and hypoxia. However, anti-miR-210 transfection inhibited cell growth and induced apoptosis, in both normoxia and hypoxia. We determined that one relevant target of miR-210 in hypoxia was Ephrin-A3 since miR-210 was necessary and sufficient to down-modulate its expression. Moreover, luciferase reporter assays showed that Ephrin-A3 was a direct target of miR-210. Ephrin-A3 modulation by miR-210 had significant functional consequences; indeed, the expression of an Ephrin-A3 allele that is not targeted by miR-210 prevented miR-210-mediated stimulation of both tubulogenesis and chemotaxis. We conclude that miR-210 up-regulation is a crucial element of endothelial cell response to hypoxia, affecting cell survival, migration, and differentiation.     

miR-17 and -20a Target the Neuron-Derived Orphan Receptor-1 (NOR-1) in Vascular Endothelial Cells

Neuron-derived orphan receptor-1 (NOR-1) plays a major role in vascular biology by controlling fibroproliferative and inflammatory responses. Because microRNAs (miRNAs) have recently emerged as key players in the regulation of gene expression in the vasculature, here we have investigated the regulation of NOR-1 by miRNAs in endothelial cells. Computational algorithms suggest that NOR-1 could be targeted by members of the miR-17 family. Accordingly, ectopic over-expression of miR-17 or miR-20a in endothelial cells using synthetic premiRNAs attenuated the up-regulation of NOR-1 expression induced by VEGF (as evidenced by real time PCR, Western blot and immunocitochemistry). Conversely, the antagonism of these miRNAs by specific antagomirs prevented the down-regulation of NOR-1 promoted by miR-17 or miR-20a in VEGF-stimulated cells. Disruption of the miRNA-NOR-1 mRNA interaction using a custom designed target protector evidenced the selectivity of these responses. Further, luciferase reporter assays and seed-sequence mutagenesis confirmed that miR-17 and -20a bind to NOR-1 3’-UTR. Finally, miR-17 and -20a ameliorated the up-regulation of VCAM-1 mediated by NOR-1 in VEGF-stimulated cells. Therefore, miR-17 and -20a target NOR-1 thereby regulating NOR-1-dependent gene expression.     

miR-22 represses cancer progression by inducing cellular senescence

Cellular senescence acts as a barrier to cancer progression, and microRNAs (miRNAs) are thought to be potential senescence regulators. However, whether senescence-associated miRNAs (SA-miRNAs) contribute to tumor suppression remains unknown. Here, we report that miR-22, a novel SA-miRNA, has an impact on tumorigenesis. miR-22 is up-regulated in human senescent fibroblasts and epithelial cells but down-regulated in various cancer cell lines. miR-22 overexpression induces growth suppression and acquisition of a senescent phenotype in human normal and cancer cells. miR-22 knockdown in presenescent fibroblasts decreased cell size, and cells became more compact. miR-22-induced senescence also decreases cell motility and inhibits cell invasion in vitro. Synthetic miR-22 delivery suppresses tumor growth and metastasis in vivo by inducing cellular senescence in a mouse model of breast carcinoma. We confirmed that CDK6, SIRT1, and Sp1, genes involved in the senescence program, are direct targets of miR-22. Our study provides the first evidence that miR-22 restores the cellular senescence program in cancer cells and acts as a tumor suppressor.     

IGF-II is regulated by microRNA-125b in skeletal myogenesis

MicroRNAs (miRNAs) have emerged as key regulators of skeletal myogenesis, but our knowledge of the identity of the myogenic miRNAs and their targets remains limited. In this study, we report the identification and characterization of a novel myogenic miRNA, miR-125b. We find that the levels of miR-125b decline during myogenesis and that miR-125b negatively modulates myoblast differentiation in culture and muscle regeneration in mice. Our results identify IGF-II (insulin-like growth factor 2), a critical regulator of skeletal myogenesis, as a direct and major target of miR-125b in both myocytes and regenerating muscles, revealing for the first time an miRNA mechanism controlling IGF-II expression. In addition, we provide evidence suggesting that miR-125b biogenesis is negatively controlled by kinase-independent mammalian target of rapamycin (mTOR) signaling both in vitro and in vivo as a part of a dual mechanism by which mTOR regulates the production of IGF-II, a master switch governing the initiation of skeletal myogenesis.     

高糖诱导内皮细胞损伤microRNA表达紊乱的体外研究

目的:研究高糖诱导的内皮细胞损伤微小RNA(microRNA,miRNA)的表达变化。方法:常规培养的人冠状动脉内皮细胞,利用不同浓度D-葡萄糖溶液(0 mmol/L、5 mmol/L、15 mmol/L和25 mmol/L),诱导刺激24 h后分别用CCK-8法和流式细胞术检测其生长活力和凋亡水平。收集细胞总RNA,利用实时定量PCR(Quantitative real-time PCR,q RT-PCR)检测miRNA的表达变化,同时利用TargetScan、PicTar等生物信息学预测软件预测可能的靶基因。结果:高糖溶液(25 mmol/L)刺激内皮细胞后,细胞生长活力明显降低,为对照组的67.5%(P0.01),凋亡水平为对照组的4.5倍(P0.01)。QRT-PCR结果显示miRNA的表达出现了明显的紊乱,其中miR-451、miR-504、miR-302d、miR-18b*、miR-198、miR-328和miR-517c明显下调,miR-29c、miR-100*、miR-137、miR-660和miR-217明显上调(P0.05)。靶基因预测发现miR-217和miR-451可能调控内皮细胞功能相关的多个基因的表达。结论:在高糖诱导的内皮细胞损伤中,miRNA表达紊乱提示其可能参与内皮细胞功能。