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Lncrna and Mrna Interaction Study Based on Transcriptome Profiles Reveals Potential Core Genes in the Pathogenesis of Human Thoracic Aortic Dissection

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Lncrna and Mrna Interaction Study Based on Transcriptome Profiles Reveals Potential Core Genes in the Pathogenesis of Human Thoracic Aortic Dissection MOLECULAR MEDICINE REPORTS 18: 3167-3176, 2018 LncRNA and mRNA interaction study based on transcriptome profiles reveals potential core genes in the pathogenesis of human thoracic aortic dissection 1* 2* 1 1 1 YANG LI , NAN YANG , XIANBAO ZHOU , XUEZHI BIAN , GENQIANG QIU , 3 3 3 MO ZHANG , HUAGANG LIN and DINGFENG LI 1Department of General Surgery, Beijing Yuho Rehabilitation Hospital of Integrated Chinese and Western Medicine, Beijing 100039; 2Department of Stomatology, PLA 309th Hospital, Beijing 100091; 3Department of Orthopedics, Beijing Yuho Rehabilitation Hospital of Integrated Chinese and Western Medicine, Beijing 100039, P.R. China Received March 3, 2017; Accepted May 2, 2018 DOI: 10.3892/mmr.2018.9308 Abstract. The aim of the present study was to determine the with an annotated protein-coding gene through the GO term potential core genes in the pathogenesis of human thoracic and scientific literatures. Then a set of significantly expressed aortic dissection (TAD) by analyzing microarray profiles lncRNAs [purinergic receptor P2X7 (P2RX7), hypoxia of long non-coding (lnc)-RNAs between TAD and normal inducing factor (HIF)-1A-AS2, AX746823, RP11-69I8.3 and thoracic aorta (NTA). The differentially expressed lncRNA RP11‑536K7.5) and the corresponding mRNAs (P2RX7, profiles of the aorta tissues between TAD patients (TAD cyclin dependent kinase inhibitor 2B, HIF-1A, runt-related group, n=6) and age-matched donors with aortic diseases transcription factor 1, connective tissue growth factor (NTA group, n=6) were analyzed by lncRNAs microarray. and interleukin 2 receptor a chain] were confirmed using Gene ontology (GO), pathway and network analyses were RT-qPCR. The present study revealed that the expression used to further investigate candidate lncRNAs and mRNAs. profiles of lncRNAs and mRNAs in aorta tissues from TAD Differentially expressed lncRNAs and mRNAs were validated were significantly altered. These results may provide important by reverse transcription-quantitative polymerase chain reaction insights into the pathogenesis of TAD disease. (RT‑qPCR). In total, the present study identified 765 lncRNAs and 619 mRNAs with differential expression between TAD and Introduction NTA (fold change >2.0, P<0.01). GO analysis demonstrated that the differentially upregulated lncRNAs are associated with Thoracic aortic dissection (TAD) is a highly lethal cardio- cell differentiation, homeostasis, cell growth and angiogenesis. vascular disease, which is characterized by the separation of Kyoto Encyclopedia of Gene and Genomes pathway analysis thoracic aortic medial layer along the length of the vessel (1). demonstrated that the differentially downregulated lncRNAs Typical TAD begins with the sudden initial tear in the aortic are mainly associated with arrhythmogenic right ventricular intima, and intraluminal pulsatile blood enters into the medial cardiomyopathy, hypertrophic cardiomyopathy and dilated layer through an intimal tear, resulting in rapid aortic dilation cardiomyopathy. To reduce the lncRNAs for further and rupture. In spite of the improvement of diagnostic and investigation and to enrich those potentially involved in TAD, therapeutic techniques over the years, the overall mortality of a total of 16 candidate lncRNAs with a significant expression TAD remains high (2). The understanding of the pathogen- (fold change >4, P<0.01) were selected, that were associated esis of this serious illness may produce a better outcome in the future. Many studies have been performed to explore the pathogenesis of TAD. Most studies were mainly focused on the genetic diversity, clinical pathology, and hemodynamics. However, the potential molecular mechanism of TAD remains Correspondence to: Dr Yang Li, Department of General Surgery, unclear. Beijing Yuho Rehabilitation Hospital of Integrated Chinese and Long non‑coding RNAs (lncRNAs) are defined as more Western Medicine, 15 Yongding Road, Haidian, Beijing 100039, than 200 nt in length and found to regulate protein-coding gene P. R. Ch i na expression at both the transcriptional and post-transcriptional E-mail: [email protected] levels. Studies showed that lncRNAs play critical roles in *Contributed equally cardiovascular physio-pathological processes (3). For example, lncRNA Braveheart (Bvht) was associated with cardiovascular Key words: long noncoding RNA, messenger RNA, microarray development (4). Upregulated myocardial infarction-associated analysis, thoracic aortic dissection transcript 1 (Mirt1) and Mirt2 can promote cardiac contractile function and decrease left ventricular remodeling (5). Myosin heavy chain-associated RNA transcript (Mhrt) can protect the 3168 LI et al: LONG NON-CODING RNAs IN THORACIC AORTIC DISSECTION heart from pathological cardiac hypertrophy (6). Although an Table I. Clinical characteristics. increasing number of lncRNAs have been characterized, the role of lncRNAs in TAD has not been investigated. A recent study Variables NTA (n=6) TAD (n=6) revealed that the overexpressed lncRNA HIF1 alpha-antisense RNA 1 (HIF1A-AS1) in the thoraco-abdominal aortic Age (years) 49.6±12.6 51.4±13.4 aneurysm (TAAA) promoted the proliferation and apoptosis of Males/females 6/0 6/0 vascular smooth muscle cells (VSMCs), which may contribute Hypertension 2 (33.3%) 3 (50.0%) to the pathogenesis of TAAA (7). Wang et al (8) reported that Atherosclerosis 1 (16.7%) 2 (33.3%) the interaction between BRG1 and HIF1A-AS1 may be involved Smoking 2 (33.3%) 3 (50.0%) in the pathogenesis of thoracic aortic aneurysms. Since TAA Aortic size (mm) 31.4±9.7 63.4±15.2 and TAD possesses similar pathological basis, these results suggested the potential role of lncRNAs in the pathogenesis of Data are presented as the mean ± standard deviation, as the n number TAD. However, until now, there was no study on the expression of individuals or as n (%). NTA, normal thoracic aorta; TAD, thoracic profile of lncRNAs in TAD. aortic dissection. In the present study, we demonstrated expression profile of lncRNAs between TAD and normal thoracic aorta (NTA) using third-generation lncRNA microarray techniques. These results will help provide further insight into the pathogenesis evaluated in triplicate. Gene expression results were analyzed of TAD. with the 2‑∆∆Cq method and normalized to GAPDH expression. Materials and methods Gene ontology (GO) and pathway analysis. The GO analysis (www.geneontology.org) and Kyoto Encyclopedia of Gene and Acquisition of clinical specimens. Ascending aorta specimens Genomes database (KEGG, www.genome.jp/kegg) were used were obtained from TAD patients undergoing surgical repair to analyze the main function and pathway of differentially (TAD group, n=6; mean age, 51.4±13.4 years) at Fuwai Hospital expressed lncRNAs and mRNAs. and organ donors without aortic diseases (NTA group, n=6; mean age, 49.6±12.6 years). No significant difference in the LncRNA and mRNA interaction network analysis. age was found between TAD and NTA (P>0.05). All patients MetaCoreTM software (Auto Expand Algorithm) was used were confirmed to have acute Stanford type I aortic dissec- to analyze the regulatory network of differentially expressed tion within 14 days of the symptom onset before surgery. All genes associated with lncRNAs. subjects have not any history of Marfan syndrome, bicuspid aortic valve, or any other connective tissue disease. The Statistical analysis. All statistical data were analyzed by SPSS clinical characteristics of patients and donors were in Table I. v20.0 software (IBM Corp., Armonk, NY, USA). Data were The study protocol was approved by the international review presented as the mean ± standard deviation of at least 3 inde- board of Beijing Yuho Rehabilitation Hospital, (Beijing, pendent experiments. The results were analyzed by analysis of China). Written informed consent was obtained from each of variance with a Bonferroni post hoc test, or by Student's t-test. the patients. Aortic media tissues were sectioned into smaller P<0.05 was considered to indicate a statistically significant sizes and briefly stored at ‑80˚C until RNA extraction. difference. RNA isolation and lncRNA microarray analysis. Total RNA Results was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) following the manufac- LncRNA microarray profiling. Arraystar lncRNA Microarray turer's instruction. For the global profiling of human lncRNAs (v3.0), which can detect about 30,586 lncRNAs and 26,109 and protein-coding transcripts, we utilized the third-generation coding transcripts, was used for the comprehensive analysis lncRNA microarray (Arraystar v3.0; KangChen, Shanghai, of core genes associated with the pathogenesis of TAD. Using China), which contains more than 30,000 capture probes, third-generation lncRNA microarray, we detected a total of covering all lncRNAs from authoritative databases (UCSC 25,348 lncRNAs and 24,852 mRNAs. The statistical information Knowngenes, RefSeq and Ensembl) and their coding proteins. of lncRNAs was shown in Fig. 1A. Furthermore, the differentially The microarray hybridization was performed based on manu- expressed lncRNAs and mRNAs were screened as per the facturer's instruction. Quantile normalization and subsequent fold-change and P-value. The screening criteria for differentially data analysis was performed using GeneSpring GX 11.5.1 expressed lncRNAs and mRNAs is fold‑change ≥2 and P‑value software (Agilent Technologies, Inc., Santa Clara, CA, USA). <0.01. Subsequently, we have used log2 (fold-change) as the X-axis and log10 (P-value)
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