Gengze Wu

LincRNA-p21 Regulates Neointima Formation, Vascular Smooth Muscle Cell Proliferation, Apoptosis and Atherosclerosis by Enhancing p53 Activity

Background— Long noncoding RNAs (lncRNAs) have recently been implicated in many biological processes and diseases. Atherosclerosis is a major risk factor for cardiovascular disease. However, the functional role of lncRNAs in atherosclerosis is largely unknown. Methods and Results— We identified lincRNA-p21 as a key regulator of cell proliferation and apoptosis during atherosclerosis. The expression of lincRNA-p21 was dramatically downregulated in atherosclerotic plaques of ApoE−/− mice, an animal model for atherosclerosis. Through loss- and gain-of-function approaches, we showed that lincRNA-p21 represses cell proliferation and induces apoptosis in vascular smooth muscle cells and mouse mononuclear macrophage cells in vitro. Moreover, we found that inhibition of lincRNA-p21 results in neointimal hyperplasia in vivo in a carotid artery injury model. Genome-wide analysis revealed that lincRNA-p21 inhibition dysregulated many p53 targets. Furthermore, lincRNA-p21, a transcriptional target of p53, feeds back to enhance p53 transcriptional activity, at least in part, via binding to mouse double minute 2 (MDM2), an E3 ubiquitin-protein ligase. The association of lincRNA-p21 and MDM2 releases MDM2 repression of p53, enabling p53 to interact with p300 and to bind to the promoters/enhancers of its target genes. Finally, we show that lincRNA-p21 expression is decreased in patients with coronary artery disease. Conclusions— Our studies identify lincRNA-p21 as a novel regulator of cell proliferation and apoptosis and suggest that this lncRNA could serve as a therapeutic target to treat atherosclerosis and related cardiovascular disorders.

Plasma long non-coding RNA, CoroMarker, a novel biomarker for diagnosis of coronary artery disease.

Long non-coding RNAs (lncRNAs) have been reported to be involved in the pathogenesis of cardiovascular disease (CVD), but whether circulating lncRNAs can serve as a coronary artery disease (CAD), biomarker is not known. The present study screened lncRNAs by microarray analysis in the plasma from CAD patients and control individuals and found that 265 lncRNAs were differentially expressed. To find specific lncRNAs as possible CAD biomarker candidates, we used the following criteria for 174 up-regulated lncRNAs: signal intensity ≥8, fold change >2.5 and P<0.005. According to these criteria, five intergenic lncRNAs were identified. After validation by quantitative PCR (qPCR), one lncRNA was excluded from the candidate list. The remaining four lncRNAs were independently validated in another population of 20 CAD patients and 20 control individuals. Receiver operating characteristic (ROC) curve analysis showed that lncRNA AC100865.1 (referred to as CoroMarker) was the best of these lncRNAs. CoroMarker levels were also stable in plasma. The predictive value of CoroMarker was further assessed in a larger cohort with 221 CAD patients and 187 control individuals. Using a diagnostic model with Fishers criteria, taking the risk factors into account, the optimal sensitivity of CoroMarker for CAD increased from 68.29% to 78.05%, whereas the specificity decreased slightly from 91.89% to 86.49%. CoroMarker was stable in plasma because it was mainly in the extracellular vesicles (EVs), probably from monocytes. We conclude that CoroMarker is a stable, sensitive and specific biomarker for CAD.

MicroRNA-613 represses lipogenesis in HepG2 cells by downregulating LXRα

BackgroundMicroRNAs (miRNAs) emerge as new important regulators of lipid homeostasis by regulating corresponding genes. MiR-613 is a newly discovered microRNA, of which the biological function is unknown. A recent report has shown that miR-613 downregulates liver X receptor α (LXRα), a ligand-activated nuclear receptor playing an important role in the regulation of lipid metabolism. The purpose of this study is to explore the effect and the molecular basis of miR-613 on lipogenesis in HepG2 cells.MethodsHepG2 cells were transiently transfected with miR-613 mimic or control microRNA. Real time PCR, Western blot, Luciferase reporter assay and Oil Red O staining were employed to examine the expression of LXRα and its target genes involved in lipogenesis, binding site for miR-613 in 3′-untranslated region (3′-UTR) of LXRα mRNA and lipid droplet accumulation in the cells.ResultsMiR-613 dramatically suppressed the expression of LXRα and its target genes including sterol-regulatory element binding protein 1c (SREBP-1c), fatty acid synthase (FAS), carbohydrate responsive element-binding protein (ChREBP) and acetyl-CoA carboxylase (ACC). Reporter assay showed that miR-613 directly bound to 3′-UTR of LXRα mRNA. Moreover, miR-613 significantly repressed LXRα-induced lipid droplet accumulation in HepG2 cells. Ectopic expression of LXRα without 3′-UTR markedly attenuated the miR-613-mediated downregulation of LXRα’s target genes and LXRα-induced lipid droplet accumulation.ConclusionsMiR-613 suppresses lipogenesis by directly targeting LXRα in HepG2 cells, suggesting that miR-613 may serve as a novel target for regulating lipid homeostasis.

Transferred BCR/ABL DNA from K562 Extracellular Vesicles Causes Chronic Myeloid Leukemia in Immunodeficient Mice

Our previous study showed that besides mRNAs and microRNAs, there are DNA fragments within extracellular vesicles (EVs). The BCR/ABL hybrid gene, involved in the pathogenesis of chronic myeloid leukemia (CML), could be transferred from K562 EVs to neutrophils and decrease their phagocytic activity in vitro. Our present study provides evidence that BCR/ABL DNAs transferred from EVs have pathophysiological significance in vivo. Two months after injection of K562 EVs into the tail vein of Sprague-Dawley (SD) rats, they showed some characteristics of CML, e.g., feeble, febrile, and thin, with splenomegaly and neutrophilia but with reduced neutrophil phagocytic activity. These findings were also observed in immunodeficient NOD/SCID mice treated with K562 EVs; BCR/ABL mRNA and protein were found in their neutrophils. The administration of actinomycin D, an inhibitor of de novo mRNA synthesis, prevented the abnormalities caused by K562 EVs in NOD/SCID mice related to CML, including neutrophilia and bone marrow hyperplasia. As a specific inhibitor of tyrosine kinases, imatinib blocked the activity of tyrosine kinases and the expression of phospho-Crkl, induced by the de novo BCR/ABL protein caused by K562 EVs bearing BCR/ABL DNA. Our current study shows the pathophysiological significance of transferred tumor gene from EVs in vivo, which may represent an important mechanism for tumorigenesis, tumor progression, and metastasis.

Long non-coding RNAs link extracellular matrix gene expression to ischemic cardiomyopathy

Aims Ischemic cardiomyopathy (ICM) resulting from myocardial infarction is a major cause of heart failure (HF). Recently, thousands of long non-coding RNAs (lncRNAs) have been discovered and implicated in a variety of biological processes. However, the role of most lncRNAs in HF remains largely unknown. The aim of this study is to test the hypothesis that the expression and function of lncRNAs are differentially regulated in diseased hearts. Methods and results In this study, we performed RNA deep sequencing of protein-coding and non-coding RNAs from cardiac samples of patients with ICM ( n  = 15) and controls ( n  = 15). Genome-wide transcriptome analysis confirmed that many protein-coding genes previously known to be involved in HF were altered in ICM hearts. Among the 145 differentially expressed lncRNAs identified in ICM hearts, we found a set of 35 lncRNAs that display strong positive expression correlation. Expression correlation coefficient analyses of differentially expressed lncRNAs and protein-coding genes revealed a strong association between lncRNAs and extracellular matrix (ECM) protein-coding genes. We overexpressed or knocked down selected lncRNAs in cardiac fibroblasts and our results suggest that lncRNAs are important regulators of fibrosis and the expression of ECM synthesis genes. Moreover, we show that lncRNAs participate in the TGF-β pathway to modulate the expression of ECM genes and myofibroblast differentiation. Conclusion Our studies demonstrate that the expression of many lncRNAs is dynamically regulated in ICM. lncRNAs regulate the expression and function of ECM and cardiac fibrosis during the development of ICM. Our results further indicate that lncRNAs may represent novel regulators of heart function and cardiac disorders, including ICM.

Functional Transferred DNA within Extracellular Vesicles.

Extracellular vesicles (EVs) are small membrane vesicles including exosomes and shedding vesicles that mediated a cell-to-cell communication. EVs are released from almost all cell types under both physiological and pathological conditions and incorporate nuclear and cytoplasmic molecules for intercellular delivery. Besides protein, mRNA, and microRNA of these molecules, as recent studies show, specific DNA are prominently packaged into EVs. It appears likely that some of exosomes or shedding vesicles, bearing nuclear molecules are released upon bubble-like blebs. Specific interaction of EVs with susceptible recipients performs the uptake of EVs into the target cells, discharging their cargo including nuclear and cytoplasmic macromolecules into the cytosol. These findings expand the nucleic acid content of EVs to include increased levels of specific DNA. Thus, EVs contain a repertoire of genetic information available for horizontal gene transfer and potential use as blood biomarkers for cancer and atherosclerosis. In this review, the focus is on the characteristics, biological functions, and roles in diseases of DNA within EVs.

microRNAs in cardiac regeneration and cardiovascular disease

AbstractmicroRNAs (miRNAs) are a class of small non-coding RNAs, which have been shown important to a wide range of biological process by post-transcriptionally regulating the expression of protein-coding genes. miRNAs have been demonstrated essential to normal cardiac development and function. Recently, numerous studies indicate miRNAs are involved in cardiac regeneration and cardiac disease, including cardiac hypertrophy, myocardial infarction and cardiac arrhythmia. These observations suggest miRNAs play important roles in cardiology. In this review, we summarize the recent progress of studying miRNAs in cardiac regeneration and cardiac disease. We also discuss the diagnostic and therapeutic potential of miRNAs in heart disease.

PinX1, a novel target gene of p53, is suppressed by HPV16 E6 in cervical cancer cells.

The aberrant activation of telomerase is critical for the initiation and development of human cervical cancer, which is dependent on the activation of human telomerase reverse transcriptase (hTERT). Recently, Pin2/TRF1-interacting protein X1 (PinX1) has been identified as a suppressor of hTERT. It has been found that the telomerase is activated while the level of PinX1 is decreased in cervical cancer. However, the regulatory mechanism of PinX1 in cervical cancer cells remains unclear. In the present study, we demonstrated that the level of PinX1 is regulated by p53, and p53 functions as a transcriptional factor to directly activate the expression of PinX1 in cervical cancer cells. Moreover, we found that HPV16 E6 suppresses the expression of PinX1 via inhibiting p53 transcriptional activity, resulting in the enhancement of telomerase activity. This study not only for the first time shows that PinX1 is a novel target gene of p53 but also suggests that suppression of p53/PinX1 pathway may be a novel mechanism by which HPV16 E6 enhances the telomerase activity in cervical cancer cells.

Cardiomyocyte-enriched protein CIP protects against pathophysiological stresses and regulates cardiac homeostasis

Cardiomyopathy is a common human disorder that is characterized by contractile dysfunction and cardiac remodeling. Genetic mutations and altered expression of genes encoding many signaling molecules and contractile proteins are associated with cardiomyopathy; however, how cardiomyocytes sense pathophysiological stresses in order to then modulate cardiac remodeling remains poorly understood. Here, we have described a regulator in the heart that harmonizes the progression of cardiac hypertrophy and dilation. We determined that expression of the myocyte-enriched protein cardiac ISL1-interacting protein (CIP, also known as MLIP) is reduced in patients with dilated cardiomyopathy. As CIP is highly conserved between human and mouse, we evaluated the effects of CIP deficiency on cardiac remodeling in mice. Deletion of the CIP-encoding gene accelerated progress from hypertrophy to heart failure in several cardiomyopathy models. Conversely, transgenic and AAV-mediated CIP overexpression prevented pathologic remodeling and preserved cardiac function. CIP deficiency combined with lamin A/C deletion resulted in severe dilated cardiomyopathy and cardiac dysfunction in the absence of stress. Transcriptome analyses of CIP-deficient hearts revealed that the p53- and FOXO1-mediated gene networks related to homeostasis are disturbed upon pressure overload stress. Moreover, FOXO1 overexpression suppressed stress-induced cardiomyocyte hypertrophy in CIP-deficient cardiomyocytes. Our studies identify CIP as a key regulator of cardiomyopathy that has potential as a therapeutic target to attenuate heart failure progression.

cTnI Exacerbates Myocardial Ischemia/Reperfusion Injury by Inducing Adhesion of Monocytes to VECs via TLR4/NF-κB-dependent Pathway.

Cardiac troponin I (cTnI), a biomarker for myocardial damage and risk stratification, may be involved in the pathogenesis of cardiovascular diseases, which was ascribed to the effect of cTnI auto-antibodies. Whether or not cTnI itself has a direct impact on acute myocardial injury is unknown. To exclude the influence of cTnI antibody on the cardiac infarct size, we studied the effect of cTnI shortly after myocardial ischaemia-reperfusion (I/R) injury when cTnI antibodies were not elevated. Pretreatment with cTnI augmented the myocardial infarct size caused by I/R, accompanied by an increase in inflammatory markers in the blood and myocardium. Additional experiments using human umbilical vein endothelial cells (HUVECs) showed that the detrimental effect of cTnI was related to cTnI-induced increase in vascular cell adhesion molecule-1 (VCAM-1) expression and VCAM-1 mediated adhesion of human monocytes (THP-1) to HUVECs, which could be neutralized by VCAM-1 antibody. Both toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) were involved in the signalling pathway, because blockade of either TLR4 or NF-κB inhibited the cTnIs effect on VCAM-1 expression and adhesion of monocytes to endothelial cells. Moreover, TLR4 inhibition reduced cTnI-augmented cardiac injury in rats with I/R injury. We conclude that cTnI exacerbates myocardial I/R injury by inducing the adhesion of monocytes to vascular endothelial cells via activation of the TLR4/NF-κB pathway. Inhibition of TLR4 may be an alternative strategy to reduce cTnI-induced myocardial I/R injury.