Long Noncod ing RNA Exp ression Signatu res o f Abdom inal Ao rtic Aneu rysm Revealed by Mic roarray＊

YANG Yao Guo, LI Mei Xia, KOU Lei, ZHOU You, QIN Yan Wen, LIU Xiao Jun, and CHEN Zhong,#

1. The Department of Vascular Surgery, Beijing Anzhen Hospital, Capital Medical University, Beijing 100029, China; 2. State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; 3. State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences Chinese Academy of Medical Sciences & School of Basic Medicine Peking Union Medical College, Beijing 100005, China

Long Noncod ing RNA Exp ression Signatu res o f Abdom inal Ao rtic Aneu rysm Revealed by Mic roarray＊

YANG Yao Guo1,&, LI Mei Xia2,&, KOU Lei1, ZHOU You1, QIN Yan Wen1, LIU Xiao Jun3,#, and CHEN Zhong1,#

1. The Department of Vascular Surgery, Beijing Anzhen Hospital, Capital Medical University, Beijing 100029, China; 2. State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; 3. State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences Chinese Academy of Medical Sciences & School of Basic Medicine Peking Union Medical College, Beijing 100005, China

ObjectiveThis study is aimed at observing the role of long noncoding RNAs (lncRNAs) in the pathogenesis of abdom inal aortic aneurysm (AAA).

MethodsLncRNA and mRNA expression signatures of AAA tissues and normal abdom inal aortic tissues (NT) were analyzed by m icroarray and further verified by Real-time quantitative reverse-transcription PCR (qRT-PCR). The lncRNAs-mRNAs targeting relationships were identified using computational analysis. The effect of lnc-ARG on 5-lipoxygenase (ALOX5) expression was tested in HeLa cells.

ResultsDifferential expressions of 3,688 lncRNAs and 3,007 mRNAs were identified between AAA and NT tissues. Moreover, 1,284 differentially expressed long intergenic noncoding RNAs and 206 differentially expressed enhancer-like lncRNAs adjacent to protein-coding genes were discerned by bioinformatics analysis. Some differentially expressed lncRNAs and mRNAs between AAA and normal tissue samples were further verified using qRT-PCR. A co-expression network of coding and noncoding genes was constructed based on the correlation analysis between the differentially expressed lncRNAs and mRNAs. In addition, the lnc-ARG located w ithin the upstream of ALOX5 was sorted as a noncoding transcript by analyzing the protein-coding potential using computational analysis. Furthermore, we found that lnc-ARG can decrease the mRNA level of ALOX5 and reactive oxygen species production in HeLa cells.

ConclusionThis study revealed new lncRNA candidates are related to the pathogenesis of AAA.

lncRNA; Abdom inal aortic aneurysm; lnc-ARG; ALOX5

www.besjournal.com (full text)CN: 11-2816/QCopyright ?2016 by China CDC

INTRODUCTION

A bdom inal aortic aneurysm (AAA) is defined as a maximum infrarenal abdom inal aortic aneurysm with a diameter of 3.0 cm or more and was characterized by destruction of elastin and collagen in the aortic media and adventitia[1]. The pathological features of AAA are characterized by extracellular matrix remodeling, loss of integrity of the arterial wall, vascular smooth muscle cell (VSMC) apoptosis, infiltration of inflammatory cells, and oxidative stress[2]. An imbalance between extracellular matrix synthesis and degradation favoring catabolic processes is believed to be critical in AAA pathogenesis. Matrix metalloproteinases (MMPs) have been shown to be the most abundant proteases expressed in AAA wall[3]. The activation of MMPs is tightly regulated by tissue inhibitors of metalloproteinases (TIMPs), and mRNA levels of TIMPs were decreased in AAA tissue[4-5]. Other proteases were also reported to contribute to the initiation and progression of AAA, such as cysteine proteases[6-7].Previousstudiessuggestthat apoptosis and depletion of VSMCs make an important contribution to AAA by elim inating a cell population capable of directing connective tissue repair. Chronic inflammation of the aortic wall plays an important role in the pathogenesis of AAA. Studies of human AAA tissues have shown extensive inflammatory infiltrates containing macrophages, lymphocytes, and mast cells in both the media and adventitia, and increasing aneurysm diameter was associated with a higher density of inflammatory cells in the adventitia[8]. Some mediators, including several proteases, proinflammatory cytokines, grow th factors, and chemokines synthesized and released by infiltrated inflammatory cells, induce adventitial inflammation, apoptosis of VSMCs, activation of MMPs, and neovascularization in the arterial wall[2]. Reactive oxygen species (ROS) production was increased in the aneurysm wall compared with the normal aorta and adjacent non-aneurysmal aortic wall[9]. All of the above mentioned, including infiltrated inflammatory cells, proinflammatory cytokines, mechanical stretch, grow th factors, and lipid mediators, m ight upregulate NADPH oxidase in resident vascular cells, resulting in an increase in the production of ROS and lipid peroxidation products[10]. Overexpressed ROS and nitric oxide increased the expression of MMPs through the activation of nuclear factor-kappa B and induced apoptosis of VSMCs in the aneurysm wall[11].

Noncoding RNAs can be arbitrarily divided into small noncoding RNAs and long noncoding RNAs (lncRNAs) according to their length. LncRNAs are defined as noncoding RNAs longer than 200 nucleotides in length, transcribed by RNA polymerase II (RNA pol II), spliced, capped, and polyadenylated, lacking significant open reading frames, and cannot encode proteins[12]. Collective evidenceindicatesthatlncRNAshave comprehensive biological functions through various mechanisms and are correlated w ith both normal development progress and diseases, such as cancer[13]. Studies on the involvement of lncRNAs in cardiology and vascular biology are scarce to date. However, some prom ising studies indicate that some lncRNAs have been found to be differentially regulated in the developing or diseased heart and can provide a strong indication for their involvement in cardiac (patho)physiology[14]. Previous studies showed that m icroRNAs have been confirmed to be involved in AAA development[15-18]. However, the role of lncRNAs in AAA remains to be clarified.

In this study, we analyzed the expression profiles of lncRNAs and mRNAs in human AAA tissues and normal tissues collected from different subjects by m icroarray. Bioinformatics analysis was performed to predict the target mRNAs of lncRNAs by combining differentially expressed mRNAs and lncRNAs. Among them, we found that lncRNA-ARG inhibited the mRNA level of 5-lipoxygenase (ALOX5), a key enzyme involved in the synthesis of proinflammatory leukotrienes (LTs), and reduced ROS production.

MATERIALS AND METHODS

Patients and Tissue Samples

Three AAA patients and three control subjects (aged 55-65 years) were recruited for m icroarray analysis of lncRNAs. The AAA tissues were acquired by surgery and normal abdom inal aortic tissues (NT) were obtained from subjects w ith physical traumas. AAA tissues and histologically matched NT from each subject were snap-frozen in liquid nitrogen immediately after resection. All participating subjects provided a w ritten informed consent. The study was approved by the Ethics Comm ittee of the Medical Faculty of Beijing Anzhen Hospital, Capital Medical University, Beijing, China and was conducted in accordance w ith the World MedicalAssociation Declaration of Helsinki.

RNA Extraction

Total RNA was extracted from AAA and matched normal tissues using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol.

M icroarray

Arraystar Human LncRNA M icroarray V3.0 is designed for the global profiling of human lncRNAs and protein-coding transcripts, which is updated from the previous M icroarray V2.0. About 30,586 lncRNAs and 26,109 coding transcripts can be detected by V3.0 m icroarray. Sample labeling and array hybridization were performed according to the AgilentOne-ColorMicroarray-BasedGene Expression Analysis protocol (Agilent Technology) w ith m inor modifications. Briefly, mRNA was purified from total RNA after removal of rRNA (mRNA-ONLY?Eukaryotic mRNA Isolation Kit, Epicentre). Then, each sample was amplified and transcribed into fluorescent cRNA along the entire length of the transcripts without 3' bias utilizing a random prim ing method. The labeled cRNAs were purified by RNeasy M ini Kit (Qiagen). The concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000. One m icrogram of each labeled cRNA was fragmented by adding 5 μL of 10 × blocking agent and 1 μL of 25 × fragmentation buffers, and then the m ixture was heated at 60 °C for 30 m in and 25 μL of 2 × GE hybridization buffer was added to dilute the labeled cRNA. Then, 50 μL of hybridization solution was dispensed into the gasket slide and assembled onto the lncRNA expression m icroarray slide. The slides were incubated for 17 h at 65 °C in an Agilent Hybridization Oven. The hybridized arrays were washed, fixed, and scanned using the Agilent DNA M icroarray Scanner (part number G2505C).

Real-time Quantitative Reverse-transcription PCR (qRT-PCR )

About 2 μg of total RNA was reverse-transcribed into first-strand cDNA pool using SuperScriptTMreverse transcriptase and random primers (ABI). Q-PCR was performed using the SYBR Green I Q-PCR kit (Life Sciences Solutions Group, Thermo Fisher Scientific) on a Bio-Rad CFX system. Specific primers are shown in Table S1 (in the website of BES, www.besjournal.com).

Construction of the Coding-noncoding Gene Co-expression Network

The network construction procedures include (i) preprocessing data: the same coding gene w ith different transcripts takes the median value representing the gene expression values, w ithout special treatment of lncRNA expression value; (ii) screening data: removing the subset of data according to the lists that show the differential expression of lncRNAs and mRNAs; (iii) calculation of Pearson correlation coefficient (PCC) and using R value to calculate PCC between lncRNAs and coding genes; and (iv) screening by PCC, selecting the part that has PCC ≥0.99 as significant, and draw ing the coding-noncoding gene co-expression network by Cytoscape.

Prediction of Coding Potential

The coding potential of lncRNAs was evaluated by Coding Potential Calculator (CPC) with higher accuracy and calculation speed[19]. The web-based interface of CPC is at http://cpc.cbi.pku.edu.cn, the exam ined sequences can be pasted directly into the input box, the CPC server runs and results will be shown in the browser once the computation is finished, and the output score is termed as support vector machine (SVM) score. The transcripts w ith scores between ?1 and 1 are marked as ‘weak noncoding' and ‘weak coding' respectively. The farther away the score is from zero, the more reliable the prediction is.

Plasm ids, Cell Culture, and Transfection

pcDNA3.1-ARG was constructed through restriction digestion and ligation-mediated cloning and was verified by restriction digestion and sequencing prior to use. The PCR primers were AAGCTTCTGTGTGCACT (Hind III) and GAATTCTTACTGTAGTAA (EcoRI). HeLa cells were purchased from China infrastructure of cell line resources and grown at 37 °C, with 5% CO2, in Dulbecco's modified Eagle's medium supplemented w ith 10% fetal bovine serum and 1% (v/v) penicillin-streptomycin.Celltransfectionwas performed according to the manufacturer's protocol (Vigorous).

Measurement of Intracellular Superoxide Levels

Superoxide production was detected using the fluorescent 2',7'-dichlorofluorescein (DCF) obtained from Vigorous according to a previous study[20]. HeLa cells were cultured in six-well plates, transfectedw ith the indicated plasm ids, and 48 h later washed w ith PBS and labeled w ith 10 μmol/L DCF in the culture plates at 37 °C for 15 m in in PBS. The culture plates were placed on ice to stop the labeling, trypsinized, and resuspended in ice-cold PBS. Samples were analyzed using a flow cytometer. Data analysis was performed using c-flow software, and the mean fluorescence intensity was used to quantify the responses. A m inimum of 10,000 cells were acquired for each sample.

Data Analysis

Agilent Feature Extraction software (version 11.0.1.1) was used to analyze the acquired array images. Quantile normalization and subsequent data processing were performed using the GeneSpring GX v12.0 software package (Agilent Technologies). After quantile normalization of the raw data, lncRNAs and mRNAs w ith at least three out of six samples having flags in Present or Marginal (‘All Targets Value') were chosen for further data analysis. Differentially expressed lncRNAs and mRNAs with statistical significance between the two groups were identified through volcano plot filtering. Hierarchical clustering was performed using the Agilent GeneSpring GX software (version 12.0). GO analysis and pathway analysis were performed in the standard enrichment computational method. All data were expressed as mean±SD. P value of ＜0.05 was considered as statistically significant.

RESULTS

LncRNA Expression Profiles in AAA

To analyze the potential biological functions of lncRNAs in AAA, we determ ined the lncRNA expression profile in human AAA aortic tissues through m icroarray technology (Figure 1). From the lncRNA expression profile, tens of thousands of lncRNAs (26,946) collected from human lncRNA databases, including RefSeq_NR, UCSC_knowngene, Ensembl, H-invDB, Fantom, Fantom_stringent, NRED, RNAdb, m isc_lncRNA, UCR, and lncRNA, could be exam ined in normal and AAA aortic tissues (Table S2 in the website of BES, www.besjournal.com). Furthermore, we identified 3,688 lncRNAs that were significantly differentially expressed between the three AAA patients and three normal subjects (≥3-fold, P＜0.05, Table S3 in the website of BES, www.besjournal.com). Among these candidates, 1,582 lncRNAs were upregulated more than 3-fold in the AAA group compared to that in the normal aortic tissuegroup,while2,106lncRNAswere downregulated more than 3-fold (P＜0.05, Table S3 in thewebsiteofBES,www.besjournal.com). ENST00000566575 (log2-fold change T/N=306.7137) was the most significantly downregulated lncRNA, while ENST00000512263 (log2-fold change T/No= 148.8989) was the most significantly upregulated lncRNA (Table 1).

mRNA Expression Profiles in AAA

The mRNA expression profile in human AAA tissues was also exam ined through m icroarray analysis (Figure 2). Up to 22,266 coding transcripts could be detected in the three AAA and three normal aortic tissue samples through 30,215 coding transcripts probes (Table S4 in the website of BES, www.besjournal.com). Among the three pairs of samples, 1,833 mRNAs were upregulated in AAA patients compared to those in the matched normal tissues, while 1,174 mRNAs were downregulated (Table S5 in the website of BES, www.besjournal.com).

Table 1. A Collection of Differential lncRNAs Detected by Microarray in AAA and Control Tissues

GO and pathway analyses showed that the differentially expressed mRNAs m ight be involved in different signaling pathways of biological processes, cellular components, and molecular functions. The upregulated mRNAs primarily correlated w ith the immune system processes and immune response in biological processes and w ith cytokine and chemokine activity involved in molecular functions (Table 2). We also found that some upregulated differentially expressed mRNAs correlated w ith the extracellular space and region in the cellular components (Table 2), and the downregulated mRNAs were primarily associated w ith muscle system process and contraction, muscle structure and tissue development in biological processes, contractile fibers and myofibrils in the cellular components, and cytoskeletal protein binding involved in molecular functions (Table 2). These signaling pathways may be involved in the pathogenesis of AAA. In addition, our results showed that uncoupling protein 1 (UCP-1) was the most significantly downregulated molecule in AAA and MMPs, including MMP12, MMP13, MMP7, and MMP3, were obviously upregulated in AAA (Table 3).

Table 2. Functional Classification of Target Genes by GO

Table 3. A Collection of Differential mRNAs Detected by Microarray in AAA and Control Tissues

Confirmation of Some Differentially Expressed lncRNAs and mRNAs

To further determ ine the accuracy of the m icroarray results, we exam ined the expression of some lncRNAs and mRNAs in AAA and control tissues using qRT-PCR. Compared w ith normal tissue samples, the lncRNAs of ENST00000457602 and ENST00000424851 were upregulated and the lncRNAs of NR_034145 and ENST00000448600 were downregulated, while the mRNAs of BCL-2-like protein 1 (BCL2L11) and ALOX5 were upregulated, and the mRNAs of anthrax toxin receptor 1 isoform 1 (ANTXR1) and pulmonary surfactant-associated protein D (SFTPD) were downregulated in AAA tissue samples (Figure 3), which are consistent with the m icroarray results. These data support a strong consistency between the qRT-PCR result and m icroarray data.

Potential Targets of the Differentially Expressed lncRNAs are Predicted w ith the Differentially Expressed mRNAs

Although the vast majority of lncRNAs described in the literature remain to be studied in a mechanistic detail, previous studies have indicated that lncRNAs participate in biological processes by regulating the expression of their target genes[13]. Long intergenic noncoding RNAs (lincRNAs) have been extensively studied for their expression feature and conservation among species[21]. In the current study, 10,608 differentially expressed lincRNAs could be found in normal and AAA aortic tissues (Table S6 in the website of BES, www.besjournal.com). Furthermore, we identified 1,284 differentially expressed lincRNAs and nearby coding gene pairs (distance＜300 kb, P＜0.05, shown in Table S7 in the website of BES, www.besjournal.com). A previous study indicated that a majority of lncRNAs w ith enhancer-like functions are found in human cell lines and depletion of these lncRNAs led to decreased expression of their neighboring protein-coding genes[22]. Deduced from bioinformatics analysis, we predicted1,611lncRNAcandidatesw ith enhancer-like functions in normal and AAA aortic tissues (shown in Table S8 in the website of BES, www.besjournal.com)andidentified206 differentially expressed enhancer-like lncRNAs and their potential target coding gene pairs (distance＜300 kb, P＜0.05, shown in Table S9 in the website of BES, www.besjournal.com).

A coding-noncoding gene co-expression network (CNC network) was constructed based on the correlation analysis between the differentially expressed lncRNAs and mRNAs. Seven obviously differentially expressed lncRNAs were selected to draw the network using Cytoscape, based on the PCCs of lncRNAs and mRNAs not less than 0.99. A total of 416 network nodes made associated 573 network pairs of co-expression of lncRNAs and mRNAs, and 371 pairs showed a positive correlation and 202 pairs showed a negative correlation. Among the CNC network, the lncRNAs of NR_024160 and ENST00000457239 correlated w ith 285 and 231 mRNAs, respectively (Figure 4 and Table S10 in the website of BES, www.besjournal.com). ALOX5, an enzyme that catalyzes the first comm itted step in the metabolic pathway leading to the synthesis of proinflammatory LTs[23], correlated w ith the lncRNAs of NR_024160 and ENST00000457239 (Table S10 in the website of BES, www.besjournal.com).

Lnc-ARG Downregulated the Expression of ALOX5 and Reduced ROS Production

Since the possible functions of lncRNAs can often be inferred based on existing examples and their relative genom ic organization[24], we found that ENST00000448600 (termed as ALOX5 regulatory gene, lnc-ARG) is located within the upstream of ALOX5 and prelim inarily investigated the role of lnc-ARG in regulating ALOX5. We analyzed the protein-codingpotentialoflnc-ARGusing computational analysis[19]. The indicative SVM score of lnc-ARG was ?0.918424, which is sorted as a noncoding transcript. Furthermore, our results showed that lnc-ARG overexpression slightly decreased the mRNA level of ALOX5 and ROS production in HeLa cells (Figure 5A and 5B).

DISCUSSION

Exploratory studies have so far identified a few lncRNAs associated with cardiovascular diseases, includingmetastasis-associatedlung adenocarcinoma transcript 1 (MALAT1), lincRNA predicting cardiac remodeling (LIPCAR), cardiac apoptosis-related lncRNA (CARL), and so on[25]. However, specific studies analyzing the lncRNAs in aortic aneurysm are still lacking[25-26]. In this study, we first describe the expression profiles of human lncRNAs in AAA by m icroarray and identify a set of differentially expressed lncRNAs in AAA in comparison with NT, which provides candidates for further insight into the pathogenesis of AAA.

Compared to normal tissues, m icroarray techniques describe a set of differentially expressed lncRNAs, w ith 1,582 upregulated and 2,106 downregulated lncRNAs in AAA tissues. Regarding the mRNA expression profile in human AAA, we also found 1,833 upregulated mRNAs and 1,174 downregulated mRNAs in AAA tissues compared w ith the matched normal tissues by m icroarray analysis. The aim of identifying the putative functions of nearby genes of lncRNAs is to explore the functional roles of lncRNAs. A large number of lincRNAs and lncRNAs w ith enhancer-like functions have been explored, the functions of which are related to cis or trans transcriptional regulation, translational control, splicing regulation, and other post-transcriptional regulation[27-30]. In the current study, according to the genom ic location and functionalprediction,weidentified1,284 differentially expressed lincRNAs and nearby coding gene pairs and 206 differentially expressed enhancer-like lncRNAs located w ithin the regulatory regions of their neighbor protein-coding genes.

Functional enrichment analysis showed that the upregulateddifferentiallyexpressedmRNAs primarily correlated with the immune system processes and immune response in biological processes and w ith cytokine and chemokine activity involved in molecular functions. Studies on human AAA tissues have shown extensive inflammatory infiltrates containing macrophages, lymphocytes, and mast cells in both the media and adventitia, and increasing aneurysm diameter was associated with a higher density of inflammatory cells in the adventitia[8,31]. Previous studies indicate that UCPs play a major role in thermoregulation, heat generation, and maintenance of basal metabolic rate and participate in the antioxidant protection of the body[32]. Moreover, energy metabolism and oxidation stress are related to the pathology of AAA. In the present study, our results showed that UCP-1 is the most significantly downregulated molecule in AAA and indicated that UCP-1 may have important functions in the pathogenesis of AAA.

Increasing evidence has indicated that lncRNAs have important biological functions such as controlling the expression of protein-coding genes and some lncRNAs could act as cofactors to modulate transcription factor activity to induce expression of adjacent protein-coding genes[33], while some lncRNAs could interact w ith the initiation complex to repress the expression of downstream genes[28-29]. Collective studies indicate that lncRNAs are involved in human diseases such as tumors, Alzheimer'sdisease,andcardiovascular diseases[25,34-36]. Recently, Huang et al. in 2016 demonstrated that the expression of several lncRNAs is dynam ically regulated in ischem ic cardiomyopathy. Furthermore, lncRNAs regulated the expression and function of the extracellular matrix and cardiac fibrosis during the development of ischem ic cardiomyopathy[37]. In addition, a previous study showed that HIF1A-AS1 mediates the pro-apoptosis and antiproliferative responses induced by the Brahma-related gene 1 (BRG1) in VSMCs, indicating that the lncRNA HIF1A-AS1 plays an important role in the pathophysiology of VSMCs[38]. Furthermore, the expression of several lncRNAs was found to be regulated by Ang II in VSMCs and the lncRNA AngII362 mediated the proliferation of VSMCs by acting on m iR221/222[39]. There are some candidate lncRNAs that may play a role in inflammation and extracellular matrix remodeling and fibrosis[14]. In this study, bioinformatics analysis was performed to predict the targeted mRNAs of lncRNAs by combining differentially expressed mRNAs and lncRNAs and to construct the co-expression networks of lncRNAs and mRNAs in AAA. We found that lnc-ARG overexpression slightly inhibited the mRNA level of its downstream gene, ALOX5. ALOX5 was found to be present in human atherosclerotic aorta and the coronary and carotid arteries and may play a significant role in modifying the pathogenesis of atherosclerosis[40]. Atherosclerosis represents an important chronic inflammatory process associated w ith several pathophysiological reactions in the vascular wall[41]. LTs can induce proinflammatory signaling and plays an important role in atherosclerosis through the activation of specific BLT and CysLT receptors, further causing vascular wall morphological alterations such as early lipid retention and accumulation of foam cells, development of intimal hyperplasia, and advancing atherosclerotic lesions, eventually, resulting in the rupture of atherosclerotic plaque[40]. A previous study showed that ALOX5 plays a critical role in 4-hydroxynonenal-inducedROSgenerationin murine macrophages through the activation of NADPH oxidase[42]. In the present study also, lnc-ARG overexpression slightly reduced ROS production.

In summary, the differentially expressed lncRNAs identified in our study may play important roles in the pathology and formation of AAA. Analyzing the co-expression networks of lncRNAs and mRNAs in AAA will help provide further insight into the pathogenesis of AAA. Because of the difficulty in obtaining tissue samples, the small sample size is a lim itation of this study and a further study w ith large sample size is planned to verify the relevant results and to clarify the detailed molecular mechanism of lncRNA-mediated regulation of their potential coding genes in AAA.

ACKNOWLEDGMENTS

We thank all the donors who participated in this program. We also thank all those who devoted to the M icroarray service at KangChen Bio-technology Company at Shanghai.

CONFLICTS OF INTEREST

The authors declare that they have no potential conflicts of interest.

Accepted: October 2, 2016

REFERENCES

1. Lederle FA, Johnson GR, Wilson SE, et al. The aneurysm detection and management study screening program: validation cohort and final results. Aneurysm Detection and Management Veterans Affairs Cooperative Study Investigators. Arch Intern Med, 2000; 160, 1425-30.

2. M iyake T, Morishita R. Pharmacological treatment of abdom inal aortic aneurysm. Cardiovasc Res, 2009; 83, 436-43.

3. Morris DR, Biros E, Cronin O, et al. The association of genetic variants of matrix metalloproteinases w ith abdom inal aortic aneurysm: a systematic review and meta-analysis. Heart, 2014; 100, 295-302.

4. Tamarina NA, McM illan WD, Shively VP, et al. Expression of matrix metalloproteinases and their inhibitors in aneurysms and normal aorta. Surgery, 1997; 122, 264-71; discussion 271-62.

5. Defawe OD, Colige A, Lambert CA, et al. TIMP-2 and PAI-1 mRNA levels are lower in aneurysmal as compared to athero-occlusive abdom inal aortas. Cardiovasc Res, 2003; 60, 205-13.

6. Abisi S, Burnand KG, Waltham M, et al. Cysteine protease activity in the wall of abdom inal aortic aneurysms. J Vasc Surg,2007; 46, 1260-6.

7. Lindholt JS, Erlandsen EJ, Henneberg EW. Cystatin C deficiency is associated w ith the progression of small abdom inal aortic aneurysms. Br J Surg, 2001; 88, 1472-5.

8. Freestone T, Turner RJ, Coady A, et al. Inflammation and matrix metalloproteinases in the enlarging abdom inal aortic aneurysm. Arterioscler Thromb Vasc Biol, 1995; 15, 1145-51.

9. M iller FJ, Jr., Sharp WJ, Fang X, et al. Oxidative stress in human abdom inal aortic aneurysms: a potential mediator of aneurysmal remodeling. Arterioscler Thromb Vasc Biol, 2002; 22, 560-5.

10. McCorm ick ML, Gavrila D, Weintraub NL. Role of oxidative stress in the pathogenesis of abdom inal aortic aneurysms. Arterioscler Thromb Vasc Biol, 2007; 27, 461-9.

11. Zhang J, Schm idt J, Ryschich E, et al. Inducible nitric oxide synthase is present in human abdom inal aortic aneurysm and promotes oxidative vascular injury. J Vasc Surg, 2003; 38, 360-7.

12. Ng SY, Lin L, Soh BS, Stanton LW. Long noncoding RNAs in development and disease of the central nervous system. Trends Genet, 2013; 29, 461-8.

13. Kung JT, Colognori D, Lee JT. Long noncoding RNAs: past, present, and future. Genetics, 2013; 193, 651-69.

14. Peters T, Schroen B. M issing links in cardiology: long non-coding RNAs enter the arena. Pflugers Arch, 2014; 466, 1177-87.

15. Maegdefessel L, Azuma J, Toh R, et al. M icroRNA-21 blocks abdom inalaorticaneurysmdevelopmentand nicotine-augmented expansion. Sci Transl Med, 2012; 4, 122ra122.

16. Leeper NJ, Raiesdana A, Kojima Y, et al. M icroRNA-26a is a novel regulator of vascular smooth muscle cell function. J Cell Physiol, 2011; 226, 1035-43.

17. Maegdefessel L, Azuma J, Toh R, et al. Inhibition of m icroRNA-29b reduces murine abdom inal aortic aneurysm development. J Clin Invest, 2012; 122, 497-506.

18. Kin K, M iyagawa S, Fukushima S, et al. Tissue- and plasma-specific M icroRNA signatures for atherosclerotic abdom inal aortic aneurysm. J Am Heart Assoc, 2012; 1, e000745.

19. Kong L, Zhang Y, Ye ZQ, et al. CPC: assess the protein-coding potential of transcripts using sequence features and support vector machine. Nucleic Acids Res, 2007; 35, W345-9.

20.Eruslanov E, Kusmartsev S. Identification of ROS using oxidized DCFDA and flow-cytometry. Methods Mol Biol, 2010; 594, 57-72.

21. Ma L, Bajic VB, Zhang Z. On the classification of long non-coding RNAs. RNA Biol, 2013; 10, 925-33.

22. Orom UA, Derrien T, Beringer M, et al. Long noncoding RNAs w ith enhancer-like function in human cells. Cell, 2010; 143, 46-58.

23. Anwar Y, Sabir JS, Qureshi M I, et al. 5-lipoxygenase: a prom ising drug target against inflammatory diseasesbiochem ical and pharmacological regulation. Curr Drug Targets, 2014; 15, 410-22.

24. Knauss JL, Sun T. Regulatory mechanisms of long noncoding RNAs in vertebrate central nervous system development and function. Neuroscience, 2013; 235, 200-14.

25.Duggirala A, Delogu F, Angelini TG, et al. Non coding RNAs in aortic aneurysmal disease. Front Genet, 2015, 6, 125.

26. Jiang X, Ning Q. The emerging roles of long noncoding RNAs in common cardiovascular diseases. Hypertens Res, 2015; 38, 375-9.

27. Willingham AT, Orth AP, Batalov S, et al. A strategy for probing the function of noncoding RNAs finds a repressor of NFAT. Science, 2005; 309, 1570-3.

28. Martianov I, Ramadass A, Serra Barros A, et al. Repression of the human dihydrofolate reductase gene by a non-coding interfering transcript. Nature, 2007; 445, 666-70.

29. Wang X, Arai S, Song X, et al. Induced ncRNAs allosterically modify RNA-binding proteins in cis to inhibit transcription. Nature, 2008; 454, 126-30.

30. Mercer TR, Dinger ME, Mattick JS. Long non-coding RNAs: insights into functions. Nat Rev Genet, 2009; 10, 155-9.

31. Tsuruda T, Kato J, Hatakeyama K, et al. Adventitial mast cells contribute to pathogenesis in the progression of abdom inal aortic aneurysm. Circ Res, 2008; 102, 1368-77.

32. Mailloux RJ, Harper ME. Uncoupling proteins and the control of m itochondrial reactive oxygen species production. Free Radic Biol Med, 2011; 51, 1106-15.

33. Feng J, Bi C, Clark BS, et al. The Evf-2 noncoding RNA is transcribed from the Dlx-5/6 ultraconserved region and functions as a Dlx-2 transcriptional coactivator. Genes Dev, 2006; 20, 1470-84.

34.Shi X, Sun M, Liu H, et al. Long non-coding RNAs: a new frontier in the study of human diseases. Cancer Lett, 2013; 339, 159-66.

35.Esteller M. Non-coding RNAs in human disease. Nat Rev Genet, 2011; 12, 861-74.

36.Tang JY, Lee JC, Chang YT, et al. Long noncoding RNAs-related diseases, cancers, and drugs. TheScientificWorldJournal, 2013; 2013, 943539.

37.Huang ZP, Ding Y, Chen J, et al. Long non-coding RNAs link extracellularmatrixgeneexpressiontoischem ic cardiomyopathy. Cardiovasc Res, 2016.

38.Wang S, Zhang X, Yuan Y, et al. BRG1 expression is increased in thoracic aortic aneurysms and regulates proliferation and apoptosis of vascular smooth muscle cells through the long non-coding RNA HIF1A-AS1 in vitro. Eur J Cardiothorac Surg, 2015; 47, 439-46.

39.Leung A, Trac C, Jin W, et al. Novel long noncoding RNAs are regulated by angiotensin II in vascular smooth muscle cells. Circ Res, 2013; 113, 266-78.

40. Riccioni G, Zanasi A, Vitulano N, et al. Leukotrienes in atherosclerosis: new target insights and future therapy perspectives. Mediators Inflamm, 2009; 2009, 737282.

41.Ross R. Atherosclerosis--an inflammatory disease. N Engl J Med, 1999; 340, 115-26.

42. Yun MR, Park HM, Seo KW, et al. 5-Lipoxygenase plays an essential role in 4-HNE-enhanced ROS production in murine macrophages via activation of NADPH oxidase. Free Radic Res, 2010; 44, 742-50.

Biomed Environ Sci, 2016; 29(10): 713-72310.3967/bes2016.096ISSN: 0895-3988

*This research was supported by the grants from National Natural Science Foundation of China (No. 81100226, 31300889, 91439127, and 81570435).

&The first two authors should be regarded as joint

s.

#Correspondence should be addressed to LIU Xiao Jun, Tel: 86-10-69156424, E-mail: xiaojunliu@ibms.pumc.edu.cn; CHEN Zhong, Tel: 86-10-64456383, E-mail: chenzhong201402@163.com

Biographical notes of the s: YANG Yao Guo, male, born in 1977, MD, majoring in the mechanism and clinical research of vascular diseases; LI Mei Xia, female, born in 1976, PhD, majoring in the functional and mechanism of long noncoding RNAs in the nervous system.

April 29, 2016;