Yongwen Qin

Circulating microRNA: a novel potential biomarker for early diagnosis of acute myocardial infarction in humans

AIMS microRNA (miRNA) is reported to be present in the blood of humans and has been increasingly suggested as a biomarker for diseases. We aim to determine the potential of cardiac-specific miRNAs in circulation to serve as biomarkers for acute myocardial infarction (AMI). METHODS AND RESULTS By verifying their tissue expression patterns with real-time polymerase chain reaction (PCR) analysis, muscle-enriched miRNAs (miR-1, miR-133a, and miR-499) and cardiac-specific miR-208a were selected as candidates for this study. With miRNA microarray and real-time PCR analyses, miR-1, miR-133a, and miR-499 were present with very low abundance, and miR-208a was absent in the plasma from healthy people. In the AMI rats, the plasma levels of these miRNAs were significantly increased. Especially, miR-208a in plasma was undetected at 0 h, but was significantly increased to a detectable level as early as 1 h after coronary artery occlusion. Further evaluation of the miRNA levels in plasma from AMI patients (n = 33) demonstrated that all four miRNA levels were substantially higher than those from healthy people (n = 30, P < 0.01), patients with non-AMI coronary heart disease (n = 16, P < 0.01), or patients with other cardiovascular diseases (n = 17, P < 0.01). Notably, miR-208a remained undetectable in non-AMI patients, but was easily detected in 90.9% AMI patients and in 100% AMI patients within 4 h of the onset of symptoms. By receiver operating characteristic curve analysis, among the four miRNAs investigated, miR-208a revealed the higher sensitivity and specificity for diagnosing AMI. CONCLUSION Elevated cardiac-specific miR-208a in plasma may be a novel biomarker for early detection of myocardial injury in humans.

Endothelial-specific intron-derived miR-126 is down-regulated in human breast cancer and targets both VEGFA and PIK3R2

Endothelial cells are the key components of vascular intima and play pivotal roles in vasculogenesis, angiogenesis, and tumor growth. Using Northern blot and real-time PCR, we confirmed that miR-126 and its host gene EGF-like domain 7 (EGFL7) were widely expressed in rat tissues but strictly expressed in endothelial cells. In mammals, miR-126 gene is embedded in intron7 of EGFL7. To explore the biogenesis of miR-126, plasmid EGFL7(126)-pEGFPc1 containing segment of exon7-intron7-exon8 of EGFL7 was constructed and expressed in 293T. Expression of spliced exon7–8 and excised mature miR-126 was detected by PCR and Northern blot. Knocking-down of endothelial endogenous miR-126 did not affect EGFL7 expression at mRNA or protein level. To investigate the possible roles of miR-126, PicTar, miRBase, miRanda, Bibiserv, and Targetscan were used to screen the targets. VEGFA and PIK3R2 were confirmed as the targets of miR-126 by luciferase reporter assay and Western blot. Interestingly, Northern blot and western blot showed that miR-126 was down-regulated in breast tumors where the VEGF/PI3K/AKT signaling pathway was activated. Introduction of miR-126 mimics into MCF-7 could effectively decrease VEGF/PI3K/AKT signaling activity. In summary, miR-126 was strictly expressed in endothelial cells and excised from EGFL7 pre-mRNA without affecting splicing and expression of its host gene. In addition, miR-126 could target both VEGFA and PIK3R2, and its expression was decreased in human breast cancer, implying that miR-126 may play a role in tumor genesis and growth by regulating the VEGF/PI3K/AKT signaling pathway.

Attenuation of microRNA-1 derepresses the cytoskeleton regulatory protein twinfilin-1 to provoke cardiac hypertrophy

MicroRNAs are involved in several aspects of cardiac hypertrophy, including cardiac growth, conduction, and fibrosis. However, their effects on the regulation of the cardiomyocyte cytoskeleton in this pathological process are not known. Here, with microRNA microarray and small RNA library sequencing, we show that microRNA-1 (miR-1) is the most abundant microRNA in the human heart. By applying bioinformatic target prediction, a cytoskeleton regulatory protein twinfilin-1 was identified as a potential target of miR-1. Overexpression of miR-1 not only reduced the luciferase activity of the reporter containing the 3′ untranslated region of twinfilin-1 mRNA, but also suppressed the endogenous protein expression of twinfilin-1, indicating that twinfilin-1 is a direct target of miR-1. miR-1 was substantially downregulated in the rat hypertrophic left ventricle and phenylephrine-induced hypertrophic cardiomyocytes, and accordingly, the protein level of twinfilin-1 was increased. Furthermore, overexpression of miR-1 in hypertrophic cardiomyocytes reduced the cell size and attenuated the expression of hypertrophic markers, whereas silencing of miR-1 in cardiomyocytes resulted in the hypertrophic phenotype. In accordance, twinfilin-1 overexpression promoted cardiomyocyte hypertrophy. Taken together, our results demonstrate that the cytoskeleton regulatory protein twinfilin-1 is a novel target of miR-1, and that reduction of miR-1 by hypertrophic stimuli induces the upregulation of twinfilin-1, which in turn evokes hypertrophy through the regulation of cardiac cytoskeleton.

MicroRNAs are dynamically regulated in hypertrophic hearts, and miR-199a is essential for the maintenance of cell size in cardiomyocytes†

Cardiac hypertrophy, which is characterized by an increase in cell size and reactivation of fetal genes, occurs as an adaptive response to diverse forms of stress and often results in heart failure and sudden death. Growing evidence indicates that microRNAs (miRNAs) are involved in cardiac hypertrophy, but the function of these miRNAs remains elusive. Here, using real time PCR analysis, we showed that several miRNAs were dynamically regulated in the rat hypertrophic hearts and miR‐199a was up‐regulated by 10‐fold in hypertrophic hearts after abdominal aorta constriction for 12 weeks. With tissue profiling analysis, we showed that miR‐199a was predominantly expressed in cardiomyocytes, but was also faintly detected in cardiac fibroblasts. To investigate whether miR‐199a was involved in cardiac hypertrophy, both over‐expression and knockdown of miR‐199a were performed in cultured cardiomyocytes. Over‐expression of miR‐199a in cardiomyocytes increased the cell size as measured by cell surface area, and also reduced the mRNA expression level of α‐myosin heavy chain. In accordance, knockdown of endogenous miR‐199a in cardiomyocytes reduced the cell size. Down‐regulation of miR‐199a also attenuated the phenylephrine‐induced increase of cell size. Furthermore, bioinformatic algorithms were used to predict the potential targets of miR‐199a in cardiac hypertrophy, and hypoxia‐inducible factor 1 alpha was confirmed by the luciferase reporter assay to be a potential target of miR‐199a. Taken together, our results demonstrated that miR‐199a, which was predominantly expressed in cardiomyocytes, was essential for the maintenance of cell size of cardiomyocytes and might play a role in the regulation of cardiac hypertrophy. J. Cell. Physiol. 225: 437–443, 2010.

MicroRNA-663 Regulates Human Vascular Smooth Muscle Cell Phenotypic Switch and Vascular Neointimal Formation

Rationale: Abnormal phenotypic switch of vascular smooth muscle cell (VSMC) is a hallmark of vascular disorders such as atherosclerosis and restenosis after angioplasty. MicroRNAs (miRNAs) have emerged as important regulators for VSMC function, and we recently identified miR-663 as critical for controlling human aortic smooth muscle cell proliferation. Objective: To investigate whether miR-663 plays a role in human VSMC phenotypic switch and the development of neointima formation. Methods and Results: By using quantitative reverse-transcription polymerase chain reaction, we found that miR-663 was significantly downregulated in human aortic VSMCs on platelet-derived growth factor treatment, whereas expression was markedly increased during VSMC differentiation. Furthermore, we demonstrated that overexpression of miR-663 increased expression of VSMC differentiation marker genes, such as smooth muscle 22&agr;, smooth muscle &agr;-actin, calponin, and smooth muscle myosin heavy chain, and potently inhibited platelet-derived growth factor–induced VSMC proliferation and migration. We identified the transcription factor JunB and myosin light chain 9 as downstream targets of miR-663 in human VSMCs, because overexpression of miR-663 markedly inhibited expression of JunB and its downstream molecules, such as myosin light chain 9 and matrix metalloproteinase 9. Finally, we showed that adeno-miR-663 markedly suppressed the neointimal lesion formation by ≈50% in mice after vascular injury induced by carotid artery ligation, specifically via decreased JunB expression. Conclusions: These results indicate that miR-663 is a novel modulator of human VSMC phenotypic switch by targeting JunB/myosin light chain 9 expression. These findings suggest that targeting miR-663 or its specific downstream targets in human VSMCs may represent an attractive approach for the treatment of proliferative vascular diseases.

Activation of p38 Mitogen-Activated Protein Kinase by Oxidized LDL in Vascular Smooth Muscle Cells Mediation via Pertussis Toxin–Sensitive G Proteins and Association With Oxidized LDL-Induced Cytotoxicity

Oxidized low-density lipoproteins (oxLDL) have been shown to play a crucial role in atherosclerosis, but the underlying molecular mechanisms have not been fully understood. The present study showed that oxLDL strongly evoked phosphorylation and activation of p38 mitogen-activated protein kinase (MAPK) in rat vascular smooth muscle cells (VSMCs) in concentration- and time-dependent manners, reaching the maximal activation at 100 microg/mL within 5 minutes. The results from immunofluorescence staining also revealed that p38 MAPK was activated by oxLDL in 5 minutes, and the activated p38 MAPK was translocated from cytoplasm to nucleus of VSMCs in 15 minutes. Activation of p38 MAPK by oxLDL was apparently not mediated by their classical scavenger receptors and was not affected by tyrosine kinase inhibitors. However, activation of p38 MAPK was effectively blocked by pretreatment with pertussis toxin and was significantly reduced by phospholipase C inhibitor U-73122. OxLDL also inhibited forskolin-stimulated cAMP accumulation and increased inositol phosphate formation. More interestingly, inhibition of p38 MAPK by its specific inhibitor SB203580 significantly blocked oxLDL-induced cytotoxicity (increased leakage of cytoplasmic lactate dehydrogenase to the culture medium, reduced [3H]thymidine incorporation, and attenuated mitochondrial metabolism of tetrazolium salt, (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-s ulfophenyl)- 2H-tetrazolium), MTS) in VSMCs, and pretreatment with pertussis toxin also inhibited oxLDL-induced cytotoxicity. Taken together, our data clearly demonstrated that oxLDL effectively activated p38 MAPK in VSMCs, which was likely mediated via pertussis toxin-sensitive G proteins, and the p38 activation was functionally associated with oxLDL-induced cytotoxicity in VSMCs.

Elevated microRNA-155 promotes foam cell formation by targeting HBP1 in atherogenesis

AIM MicroRNAs (miRNAs) play key roles in inflammatory responses of macrophages. However, the function of miRNAs in macrophage-derived foam cell formation is unclear. Here, we investigated the role of miRNAs in macrophage-derived foam cell formation and atherosclerotic development. METHODS AND RESULTS Using quantitative reverse transcription-PCR (qRT-PCR), we found that the level of miR-155 expression was increased significantly in both plasma and macrophages from atherosclerosis (ApoE(-/-)) mice. We identified that oxidized low density lipoprotein (oxLDL) induced the expression and release of miR-155 in macrophages, and that miR-155 was required to mediate oxLDL-induced lipid uptake and reactive oxygen species (ROS) production of macrophages. Furthermore, ectopic overexpression and knockdown experiments identified that HMG box-transcription protein1 (HBP1) is a novel target of miR-155. Knockdown of HBP1 enhanced lipid uptake and ROS production in oxLDL-stimulated macrophages, and overexpression of HBP1 repressed these effects. Furthermore, bioinformatics analysis identified three YY1 binding sites in the promoter region of pri-miR-155 and verified YY1 binding directly to its promoter region. Detailed analysis showed that the YY1/HDAC2/4 complex negatively regulated the expression of miR-155 to suppress oxLDL-induced foam cell formation. Importantly, inhibition of miR-155 by a systemically delivered antagomiR-155 decreased clearly lipid-loading in macrophages and reduced atherosclerotic plaques in ApoE(-/-) mice. Moreover, we observed that the level of miR-155 expression was up-regulated in CD14(+) monocytes from patients with coronary heart disease. CONCLUSION Our findings reveal a new regulatory pathway of YY1/HDACs/miR-155/HBP1 in macrophage-derived foam cell formation during early atherogenesis and suggest that miR-155 is a potential therapeutic target for atherosclerosis.

Lysophosphatidylcholine activates p38 and p42/44 mitogen-activated protein kinases in monocytic THP-1 cells, but only p38 activation is involved in its stimulated chemotaxis.

Oxidized LDLs (OxLDLs) have been shown to be involved in recruitment of blood monocytes into the arterial subendothelial space, which is the earliest step in atherogenesis, but the underlying molecular mechanisms are poorly understood. The present study demonstrated that lysophosphatidylcholine (LPC), a major phospholipid component of OxLDL, strongly evoked phosphorylation and activation of p38 and p42/44 mitogen-activated protein kinases in monocytic cells. The stimulation of p38 and p42/44 occurred in a dose- and time-dependent manner, reaching the maximal activation at 25 microg/mL LPC within 5 minutes. Interestingly, inhibition of p38 activation by OxLDL or LPC, using its selective inhibitors (SB203580 and SKF86002), completely blocked OxLDL- or LPC-stimulated chemotaxis of THP-1 cells, which was measured in a transwell chemotaxis assay. In contrast, inhibition of p42/44 activation by its potent inhibitor (PD98059) did not block OxLDL- or LPC-stimulated chemotaxis. Moreover, expression of a p38 dominant-negative mutant (p38AF) reduced cell chemotaxis significantly. In addition, activation of p38 by LPC was apparently mediated neither by scavenger receptors nor by tyrosine kinase receptors. It was, however, effectively blocked by pertussis toxin and substantially reduced by phospholipase C inhibitor (U73122) and phosphatidylinositol 3-kinase inhibitors (wortmannin and LY294002). LPC also inhibited forskolin-stimulated cAMP accumulation in a pertussis toxin-sensitive manner, indicating that Gi/Go proteins likely mediated the effects of LPC. Our results suggested that OxLDL/LPC efficiently activated both p38 and p42/44, but only the activation of p38 was functionally associated with OxLDL-/LPC-induced chemotaxis in THP-1 cells.

MicroRNA degradation and turnover: regulating the regulators.

MicroRNAs (miRNAs) are endogenous, small noncoding RNAs that play important regulatory roles in gene expression. The control of miRNA biogenesis has been well characterized, but their degradation is not fully understood. Recent discoveries indicate that miRNAs have a long life span in general. However, rapid turnover dynamics of miRNAs in a variety of specific cellular contexts has been documented, as well as the requirement of sequence elements for miRNA decay. Furthermore, several ribonucleases that degrade miRNAs have been identified. Here, we discuss the cellular contexts and biochemical mechanisms of miRNA decay, together with several prominent questions in this field. WIREs RNA 2012, 3:593–600. doi: 10.1002/wrna.1114

MicroRNA-638 is highly expressed in human vascular smooth muscle cells and inhibits PDGF-BB-induced cell proliferation and migration through targeting orphan nuclear receptor NOR1

AIMS Aberrant vascular smooth muscle cell (VSMC) proliferation and migration contribute significantly to the development of vascular pathologies, such as atherosclerosis and restenosis. MicroRNAs have recently emerged as critical modulators in cellular processes and the purpose of this study is to identify novel miRNA regulators implicated in human aortic VSMC proliferation and migration. METHODS AND RESULTS To identify miRNAs that are differentially expressed in human VSMCs, we performed miRNA microarray analysis in human aortic smooth muscle cells (SMCs) at different time points after platelet-derived growth factor (PDGF) stimulation. Here, we identified microRNA-638 (miR-638) as a transcript that was one of the most significantly down-regulated in human VSMCs after PDGF stimulation. Furthermore, we confirmed, by Quantitative RT-PCR, that miR-638 is highly expressed in human VSMCs, and its expression is markedly down-regulated in a dose- and time-dependent manner upon PDGF treatment. Consistent with a critical role in SMC proliferation, we found that miR-638 expression was significantly up-regulated in human VSMCs cultured in differentiation medium, a condition that inhibits SMC proliferation. Furthermore, we identified the orphan nuclear receptor NOR1 as a downstream target gene product of miR-638 and down-regulation of NOR1 is critical for miR-638-mediated inhibitory effects on PDGF-induced cyclin D1 expression, cell proliferation, and migration in human aortic SMCs. CONCLUSION These results indicate that miR-638 is a key molecule in regulating human VSMC proliferation and migration by targeting the NOR1/cyclin D pathway and suggest that specific modulation of miR-638 in human VSMCs may represent an attractive approach for the treatment of proliferative vascular diseases.