Guo-Kun Wang

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

AIMSnmicroRNA (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).nnnMETHODS AND RESULTSnBy 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.nnnCONCLUSIONnElevated cardiac-specific miR-208a in plasma may be a novel biomarker for early detection of myocardial injury in humans.

Detection of Differentially Expressed microRNAs in Serum of Pancreatic Ductal Adenocarcinoma Patients: miR-196a Could Be a Potential Marker for Poor Prognosis

BackgroundMicroRNAs (miRNAs) have long been established to remain stable in circulation, and dysregulated miRNAs in serum of tumor patients could potentially serve as novel biomarkers.AimsTo determine whether certain serum miRNAs could represent potential diagnostic and prognostic biomarkers for pancreatic ductal adenocarcinoma (PDAC).MethodsAbout 35 patients diagnosed with PDAC at different stages between August 2007 and January 2009 were enrolled in this study. Sera from 15 chronic pancreatitis (CP) patients and 15 healthy individuals were treated as controls. Quantitative real-time polymerase chain reaction assays specific to mature miRNAs were used to quantify the relative levels of those PDAC-associated serum miRNAs.ResultsOf the seven miRNAs detected, three were identified as differentially expressed in PDAC and control groups. miR-21 was able to distinguish PDAC patients from CP (pxa0=xa00.033) and healthy subjects (pxa0=xa00.001), whereas miR-155 and miR-196a were able to differentiate sera with sick pancreas (PDAC/CP) from normal pancreas (pxa0=xa00.0002 and 0.010, respectively). Serum miR-196a expression levels in unresectable PDAC (stages III and IV) patients were significantly higher than those in resectable (stages I and II) patients (pxa0=xa00.001). Furthermore, serum miR-196a expression level was found to have a potential value in predicting median survival time of PDAC patients (high-level miR-196a, 6.1xa0months, (95% CI, 4.49–7.72) versus low-level miR-196a, 12.00xa0months, (95% CI, 5.92–18.08), pxa0=xa00.007).ConclusionsSerum miR-196a could be a potential noninvasive marker for PDAC prognosis and selection of laparotomy.

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.

Attenuation of MicroRNA‐22 derepressed PTEN to effectively protect rat cardiomyocytes from hypertrophy

Cardiac hypertrophy, which is characterized by the enlargement of cell size, reactivation of fetal genes, remains one of the most important triggers to heart failure. Increasing evidence shows that microRNA (miRNA) is extensively involved in the pathogenesis of cardiac hypertrophy. But the effects of miRNAs on cardiomyocyte hypertrophy have not been completely solved yet. Here, we showed that a collection of miRNAs was aberrantly expressed in hypertrophic cardiomyocytes induced by phenylephrine (PE) or angiotensin II (Ang II). Among them, miR‐22 was the most strikingly up‐regulated miRNA. To investigate the role of miR‐22 in hypertrophy, both over‐expression and knock‐down assays were performed on cardiomyocytes. The results showed that up‐regulation of miR‐22 significantly increased the cell size and markedly influenced the expression of hypertrophic markers, including induction of nppa and reduction of myh6. In contrast, reduction of miR‐22 level attenuated either PE‐ or Ang II‐induced hypertrophic reaction. Furthermore, several genes, including PTEN, were identified as potential targets of miR‐22 by bioinformatic algorithms. Using luciferase analysis, miR‐22 could significantly suppress the luciferase activity of reporter fused with 3′ untranslated region of PTEN mRNA. Furthermore, up‐regulation of miR‐22 could suppress the protein level of PTEN and reduction of miR‐22 level markedly increased the protein level of PTEN in cardiomyocytes by Western blot analysis, suggesting that the contribution of miR‐22 to cardiomyocyte hypertrophy may be partially through targeting PTEN. Taken together, miRNAs were dynamically regulated in cardiomyocyte hypertrophy and attenuation of miR‐22 in rat cardiomyocytes efficiently protected from hypertrophic effects through derepressing PTEN. J. Cell. Physiol. 227: 1391–1398, 2012.

Lamin B1 Is a Novel Therapeutic Target of Betulinic Acid in Pancreatic Cancer

Purpose: Betulinic acid, a naturally occurring pentacyclic triterpenoid, exhibits potent antitumor activities, whereas the underlying mechanisms remain unclear. In the current study, we sought to determine the role and regulation of lamin B1 expression in human pancreatic cancer pathogenesis and betulinic acid–based therapy. Experimental Design: We used cDNA microarray to identify betulinic acid target genes and used tissue microarray to determine the expression levels of lamin B1 in pancreatic cancer tissues and to define their relationship with the clinicopathologic characteristics of pancreatic cancer. We also used in vitro and in vivo models to determine the biologic impacts of altered lamin B1 expression on and mechanisms underlying lamin B1 overexpression in human pancreatic cancer. Results: We found that lamin B1 was significantly downregulated by betulinic acid treatment in pancreatic cancer in both in vitro culture and xenograft models. Overexpression of lamin B1 was pronounced in human pancreatic cancer, and increased lamin B1 expression was directly associated with low-grade differentiation, increased incidence of distant metastasis, and poor prognosis of patients with pancreatic cancer. Furthermore, knockdown of lamin B1 significantly attenuated the proliferation, invasion, and tumorigenicity of pancreatic cancer cells. Conclusions: Lamin B1 plays an important role in pancreatic cancer pathogenesis and is a novel therapeutic target of betulinic acid treatment. Clin Cancer Res; 19(17); 4651–61. ©2013 AACR.

Activation of receptor for advanced glycation end products contributes to aortic remodeling and endothelial dysfunction in sinoaortic denervated rats

OBJECTIVEnThe aim of present study was to test the hypothesis that activation of receptor for advanced glycation end products (RAGE) pathway contributes to aortic remodeling and endothelial dysfunction in sinoaortic denervated (SAD) rats.nnnMETHODS AND RESULTSnExperiment 1: 8 weeks after sinoaortic denervation, aortas were removed for measurement of AGE/RAGE pathway. Sinoaortic denervation in rats resulted in enhanced activity of aldose reductase, reduced activity of glyoxalase 1, accumulation of methylglyoxal and AGE, and upregulated expression of RAGE in aortas. Experiment 2: 5 weeks after sinoaortic denervation, the rats received intraperitoneal injections of 500 μg soluble RAGE (sRAGE) daily for 3 weeks. Treatment of SAD rats with sRAGE attenuated aortic remodeling, marked by reduction in AW/length, wall thickness, proliferation of SMC, and collagen deposition, and improvement of endothelial function. Treatment of SAD rats with sRAGE abated aortic oxidative stress, marked by reduction in formation of malondialdehyde, reactive oxygen species, superoxide, peroxynitrite and 3-nitrotyrosine, and enhancement of ratio of GSH/GSSG. Treatment of SAD rats with sRAGE attenuated aortic mitochondrial dysfunction. Treatment of SAD rats with sRAGE suppressed aortic NFκB nuclear translocation and inflammation. Treatment of SAD rats with sRAGE restored aortic NO formation through upregulating eNOS and dimethylarginine dimethylaminohydrolase-2 and downregulating protein arginine methyltransferase-1.nnnCONCLUSIONnActivated RAGE contributed to aortic remodeling and endothelial dysfunction in SAD rats, possibly via induction of oxidative stress and inflammation, impairment of mitochondrial function, and reduction in NO bioavailability.

Effects of renal sympathetic denervation and angiotensin-converting enzyme inhibitor on left ventricular hypertrophy

ObjectivesThe aim of this study was to investigate whether renal sympathetic denervation (RSD) is more effective on myocardial hypertrophy than the angiotensin-converting enzyme inhibitor (ACEI) perindopril in spontaneously hypertensive rats (SHRs).MethodsAfter bilateral renal denervation blood pressure (BP) was measured every 10xa0days. On day 50 the heart was (histo)pathologically examined. The ventricular weight to body weight ratios (VW/BW), myocardial diameter and collagen volume fraction (CVF) were calculated, and cardiac hypertrophy marker genes were analyzed by RT-PCR.ResultsAt the baseline evaluation all groups had comparable BP. After treatment the BP of the RSD group was significantly reduced (pu2009<u20090.05). The BP of the RSD group was lower than that of the perindopril group on days 10, 20 and 30th (pu2009<u20090.05) but on day 50 systolic BP of the RSD group was significantly higher (pu2009<u20090.05) whereas there were no significant differences in diastolic BP. The VW/BW decreased in the RSD group, whereas the value did not change significantly in the perindopril group. The myocardial diameter of the left ventricular cardiomyocytes was also significantly lower in the RSD group and stayed the same in the perindopril group. Collagen volume fraction (CVF) in the RSD group was significantly lower than in the perindopril group (pu2009<u20090.05). Significant changes in the expression levels of NPPA, MYH7, and MYH6 (Pu2009<u20090.05) were observed in the RD-SHR groups (pu2009<u20090.05). There was a significant difference in the expression level of MYH6 (pu2009<u20090.05) between the RSD group and the perindopril group but the expression levels of NPPA and MYH7 were not significantly different.ConclusionIn this study, RSD had a significant antihypertensive effect and inhibited hypertensive-induced cardiac hypertrophy in SHRs and showed advantages compared with ACEI in decreasing BP in the early stage and in inhibiting myocardial fibrosis.ZusammenfassungZielZiel der Studie war zu untersuchen, ob bei Ratten mit spontan aufgetretener Hypertonie (SHR) und Myokardhypertrophie die renale Sympathikusdenervation (RSD) wirksamer als der Angiotensin-converting-Enzym-Inhibitor (ACEI) Perindopril.MethodenNach beidseitiger renaler Denervation wurde der Blutdruck (RR) alle 10 Tage gemessen. Am Tag 50 wurde das Herz histopathologisch untersucht. Das Verhältnis von Ventrikelgewicht zu Körpergewicht (VW/BW), Myokarddurchmesser und Kollagenvolumenfraktion (CVF) wurden ermittelt, Markergene für kardiale Hypertrophie wurden per Echtzeitpolymerasekettenreaktion (RT-PCR) analysiert.ErgebnisseBei der Eingangsuntersuchung wiesen alle Gruppen einen vergleichbaren RR auf. Nach Therapie war der RR der RSD-Gruppe signifikant vermindert (pu2009<u20090,05). Der RR in der RSD-Gruppe war an Tag 10, 20 und 30 niedriger als in der Perindopril-Gruppe (pu2009<u20090,05), aber an Tag 50 war der systolische RR der RSD-Gruppe signifikant höher (pu2009<u20090,05), während es keine signifikanten Unterschiede beim diastolischen RR gab. Der VW/BW-Wert nahm in der RSD-Gruppe zwar ab, doch er änderte sich in der Perindopril-Gruppe nicht signifikant. Der anhand der linksventrikulären Kardiomyozyten bestimmte Myokarddurchmesser war in der RSD-Gruppe ebenfalls signifikant geringer und blieb in der Perindopril-Gruppe gleich. Die CVF war in der RSD-Gruppe signifikant niedriger als in der Perindopril-Gruppe (pu2009<u20090,05). Signifikante Veränderungen des Expressionsgrads von NPPA, MYH7 und MYH6 (pu2009<u20090,05) wurden in der RSD-SHR-Gruppe festgestellt (pu2009<u20090,05). Es bestand ein signifikanter Unterschied beim Expressionsgrad von MYH6 (pu2009<u20090,05) zwischen der RSD-Gruppe und der Perindopril-Gruppe, nicht jedoch beim Expressiongrad von NPPA und MYH7.SchlussfolgerungIn dieser Studie hatte die RSD eine signifikante antihypertensive Wirkung, sie hemmte die hypertonieinduzierte kardiale Hypertrophie bei SHR und wies Vorteile im Vergleich zum ACEI bei der RR-Senkung im Frühstadium sowie bei der Hemmung der Myokardfibrose auf.

Differential expression profile of long non-coding RNAs in human thoracic aortic aneurysm

Thoracic aortic aneurysm (TAA) is progressive fatal aortic pathological dilation, which is characterized by increased proteoglycans and loss of elastic fibers. Recent advances in long non‐coding RNAs (lncRNAs), an important regulator in many biological processes, suggested the close correlation between expression patterns and disease progression. In the present study, the ascending aortic tissues were collected from ascending TAA patients (nu2009=u200933) and organ donors (nu2009=u200916). Microarray analysis and real‐time PCR were then applied to detect the lncRNA expression profiles. A total of 147 differentially expressed lncRNAs were determined, including 104 upregulated and 43 downregulated lncRNAs. Bioinformatics analysis showed 51.7% of differentially expressed lncRNAs were sense‐overlapping, and most of the down‐regulated lncRNAs were located on chromosome 1, 7, and 12. Subgroup analysis of TAA patients indicated that the expression of lnc‐HLTF‐5 was significantly higher in hypertension group than non‐hypertension group (Pu2009<u20090.05). Spearman correlation analysis further confirmed that the lnc‐HLTF‐5 level was positively correlated with the expanded ascending aortic diameter (rsu2009=u20090.483, Pu2009=u20090.004) and MMP9 level (rsu2009=u20090.465, Pu2009=u20090.006). Our results expanded the lncRNA expression patterns in aortic disease, and provided experimental basis for future investigation on TAA pathogenesis.

Screening and Function Analysis of MicroRNAs Involved in Exercise Preconditioning-Attenuating Pathological Cardiac Hypertrophy

Exercise preconditioning (EP) attenuates pathological cardiac hypertrophy by increasing the functional capacity of the cardiovascular system; however, the underlying molecular mechanisms remain unclear. MicroRNAs (miRNAs) play important roles in various physiological and pathological processes by regulating the expression of the targeted gene. In this study, we aimed to screen the miRNAs involved in EP-attenuating pathological cardiac hypertrophy. The histological and echocardiographic parameters assessment showed that pathological cardiac hypertrophy induced by transverse aortic constriction (TAC) was significantly alleviated in EP treated rats. The left ventricular tissues (n = 3) from Sham, TAC and EP + TAC groups were subjected to small RNA deep sequencing. A total of 570 known mature miRNAs and 530 putative novel miRNAs were detected. DEGseq analysis showed that there were 37 and 88 differentially expressed miRNAs in the comparisons of TAC versus Sham and EP + TAC versus TAC, respectively. Among them, EP treatment could relieve the expression changes of 32 miRNAs, which were supposed to be involved in EP-attenuating pathological cardiac hypertrophy. After miRNAs target genes prediction by miRDB algorithm, pathway analysis showed that the most frequently represented pathways were involved in Calcium signaling pathway and MAPK signaling pathway. The results would provide valuable clues to finding therapeutic targets for the treatment of pathological cardiac hypertrophy.