Title: Micro <scp>RNA</scp> ‐483 amelioration of experimental pulmonary hypertension
Abstract: Article23 April 2020Open Access Source DataTransparent process MicroRNA-483 amelioration of experimental pulmonary hypertension Jin Zhang Jin Zhang Cardiovascular Research Center, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education of China, Xi'an Jiaotong University, Xian, China Search for more papers by this author Yangyang He Yangyang He State Key Laboratory of Cardiovascular disease & FuWai Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Search for more papers by this author Xiaosong Yan Xiaosong Yan Cardiovascular Research Center, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education of China, Xi'an Jiaotong University, Xian, China Search for more papers by this author Shanshan Chen Shanshan Chen Cardiovascular Research Center, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education of China, Xi'an Jiaotong University, Xian, China Search for more papers by this author Ming He Ming He Department of Medicine, University of California, San Diego, La Jolla, CA, USA Search for more papers by this author Yuyang Lei Yuyang Lei Cardiovascular Research Center, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education of China, Xi'an Jiaotong University, Xian, China Search for more papers by this author Jiao Zhang Jiao Zhang Cardiovascular Research Center, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education of China, Xi'an Jiaotong University, Xian, China Department of Medicine, University of California, San Diego, La Jolla, CA, USA Department of Cardiology, First Affiliated Hospital, Xi'an Jiaotong University, Xian, China Search for more papers by this author Brendan Gongol Brendan Gongol Department of Medicine, University of California, San Diego, La Jolla, CA, USA Search for more papers by this author Mingxia Gu Mingxia Gu Department of Pediatrics (Cardiology), Cardiovascular Institute and Wall Center for Pulmonary Vascular Diseases, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Yifei Miao Yifei Miao Department of Pediatrics (Cardiology), Cardiovascular Institute and Wall Center for Pulmonary Vascular Diseases, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Liang Bai Liang Bai Cardiovascular Research Center, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education of China, Xi'an Jiaotong University, Xian, China Search for more papers by this author Xiaopei Cui Xiaopei Cui Department of Geriatric Medicine, Qilu Hospital of Shandong University, Jinan, China Search for more papers by this author Xiaojian Wang Xiaojian Wang State Key Laboratory of Cardiovascular disease & FuWai Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Search for more papers by this author Yixin Zhang Yixin Zhang State Key Laboratory of Cardiovascular disease & FuWai Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Search for more papers by this author Fenling Fan Fenling Fan Department of Cardiology, First Affiliated Hospital, Xi'an Jiaotong University, Xian, China Search for more papers by this author Zhao Li Zhao Li Cardiovascular Research Center, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education of China, Xi'an Jiaotong University, Xian, China Search for more papers by this author Yuan Shen Yuan Shen Department of Epidemiology and Health Statistics, School of Public Health, Xi'an Jiaotong University, Xian, China Search for more papers by this author Chih-Hung Chou Chih-Hung Chou orcid.org/0000-0001-5714-1718 Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan Search for more papers by this author Hsien-Da Huang Hsien-Da Huang Warshel Institute for Computational Biology, School of Life and Health Sciences, The Chinese University of Hong Kong, Shenzhen, China Search for more papers by this author Atul Malhotra Atul Malhotra Department of Medicine, University of California, San Diego, La Jolla, CA, USA Search for more papers by this author Marlene Rabinovitch Marlene Rabinovitch Department of Pediatrics (Cardiology), Cardiovascular Institute and Wall Center for Pulmonary Vascular Diseases, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Zhi-Cheng Jing Corresponding Author Zhi-Cheng Jing [email protected] orcid.org/0000-0003-0493-0929 Department of Cardiology & Key Lab of Pulmonary Vascular Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China Search for more papers by this author John Y-J Shyy Corresponding Author John Y-J Shyy [email protected] orcid.org/0000-0002-5625-753X Cardiovascular Research Center, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education of China, Xi'an Jiaotong University, Xian, China Department of Medicine, University of California, San Diego, La Jolla, CA, USA Search for more papers by this author Jin Zhang Jin Zhang Cardiovascular Research Center, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education of China, Xi'an Jiaotong University, Xian, China Search for more papers by this author Yangyang He Yangyang He State Key Laboratory of Cardiovascular disease & FuWai Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Search for more papers by this author Xiaosong Yan Xiaosong Yan Cardiovascular Research Center, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education of China, Xi'an Jiaotong University, Xian, China Search for more papers by this author Shanshan Chen Shanshan Chen Cardiovascular Research Center, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education of China, Xi'an Jiaotong University, Xian, China Search for more papers by this author Ming He Ming He Department of Medicine, University of California, San Diego, La Jolla, CA, USA Search for more papers by this author Yuyang Lei Yuyang Lei Cardiovascular Research Center, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education of China, Xi'an Jiaotong University, Xian, China Search for more papers by this author Jiao Zhang Jiao Zhang Cardiovascular Research Center, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education of China, Xi'an Jiaotong University, Xian, China Department of Medicine, University of California, San Diego, La Jolla, CA, USA Department of Cardiology, First Affiliated Hospital, Xi'an Jiaotong University, Xian, China Search for more papers by this author Brendan Gongol Brendan Gongol Department of Medicine, University of California, San Diego, La Jolla, CA, USA Search for more papers by this author Mingxia Gu Mingxia Gu Department of Pediatrics (Cardiology), Cardiovascular Institute and Wall Center for Pulmonary Vascular Diseases, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Yifei Miao Yifei Miao Department of Pediatrics (Cardiology), Cardiovascular Institute and Wall Center for Pulmonary Vascular Diseases, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Liang Bai Liang Bai Cardiovascular Research Center, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education of China, Xi'an Jiaotong University, Xian, China Search for more papers by this author Xiaopei Cui Xiaopei Cui Department of Geriatric Medicine, Qilu Hospital of Shandong University, Jinan, China Search for more papers by this author Xiaojian Wang Xiaojian Wang State Key Laboratory of Cardiovascular disease & FuWai Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Search for more papers by this author Yixin Zhang Yixin Zhang State Key Laboratory of Cardiovascular disease & FuWai Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Search for more papers by this author Fenling Fan Fenling Fan Department of Cardiology, First Affiliated Hospital, Xi'an Jiaotong University, Xian, China Search for more papers by this author Zhao Li Zhao Li Cardiovascular Research Center, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education of China, Xi'an Jiaotong University, Xian, China Search for more papers by this author Yuan Shen Yuan Shen Department of Epidemiology and Health Statistics, School of Public Health, Xi'an Jiaotong University, Xian, China Search for more papers by this author Chih-Hung Chou Chih-Hung Chou orcid.org/0000-0001-5714-1718 Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan Search for more papers by this author Hsien-Da Huang Hsien-Da Huang Warshel Institute for Computational Biology, School of Life and Health Sciences, The Chinese University of Hong Kong, Shenzhen, China Search for more papers by this author Atul Malhotra Atul Malhotra Department of Medicine, University of California, San Diego, La Jolla, CA, USA Search for more papers by this author Marlene Rabinovitch Marlene Rabinovitch Department of Pediatrics (Cardiology), Cardiovascular Institute and Wall Center for Pulmonary Vascular Diseases, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Zhi-Cheng Jing Corresponding Author Zhi-Cheng Jing [email protected] orcid.org/0000-0003-0493-0929 Department of Cardiology & Key Lab of Pulmonary Vascular Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China Search for more papers by this author John Y-J Shyy Corresponding Author John Y-J Shyy [email protected] orcid.org/0000-0002-5625-753X Cardiovascular Research Center, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education of China, Xi'an Jiaotong University, Xian, China Department of Medicine, University of California, San Diego, La Jolla, CA, USA Search for more papers by this author Author Information Jin Zhang1,‡, Yangyang He2,‡, Xiaosong Yan1, Shanshan Chen1, Ming He3, Yuyang Lei1, Jiao Zhang1,3,4, Brendan Gongol3, Mingxia Gu5, Yifei Miao5, Liang Bai1, Xiaopei Cui6, Xiaojian Wang2, Yixin Zhang2, Fenling Fan4, Zhao Li1, Yuan Shen7, Chih-Hung Chou8, Hsien-Da Huang9, Atul Malhotra3, Marlene Rabinovitch5, Zhi-Cheng Jing *,10 and John Y-J Shyy *,1,3 1Cardiovascular Research Center, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education of China, Xi'an Jiaotong University, Xian, China 2State Key Laboratory of Cardiovascular disease & FuWai Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China 3Department of Medicine, University of California, San Diego, La Jolla, CA, USA 4Department of Cardiology, First Affiliated Hospital, Xi'an Jiaotong University, Xian, China 5Department of Pediatrics (Cardiology), Cardiovascular Institute and Wall Center for Pulmonary Vascular Diseases, Stanford University School of Medicine, Stanford, CA, USA 6Department of Geriatric Medicine, Qilu Hospital of Shandong University, Jinan, China 7Department of Epidemiology and Health Statistics, School of Public Health, Xi'an Jiaotong University, Xian, China 8Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan 9Warshel Institute for Computational Biology, School of Life and Health Sciences, The Chinese University of Hong Kong, Shenzhen, China 10Department of Cardiology & Key Lab of Pulmonary Vascular Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China ‡These authors contributed equally to this work as first authors *Corresponding author. Tel: +86 1069 155023; E-mail: [email protected] *Corresponding author. Tel: +1 858 534 3736; E-mail: [email protected] EMBO Mol Med (2020)12:e11303https://doi.org/10.15252/emmm.201911303 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Endothelial dysfunction is critically involved in the pathogenesis of pulmonary arterial hypertension (PAH) and that exogenously administered microRNA may be of therapeutic benefit. Lower levels of miR-483 were found in serum from patients with idiopathic pulmonary arterial hypertension (IPAH), particularly those with more severe disease. RNA-seq and bioinformatics analyses showed that miR-483 targets several PAH-related genes, including transforming growth factor-β (TGF-β), TGF-β receptor 2 (TGFBR2), β-catenin, connective tissue growth factor (CTGF), interleukin-1β (IL-1β), and endothelin-1 (ET-1). Overexpression of miR-483 in ECs inhibited inflammatory and fibrogenic responses, revealed by the decreased expression of TGF-β, TGFBR2, β-catenin, CTGF, IL-1β, and ET-1. In contrast, inhibition of miR-483 increased these genes in ECs. Rats with EC-specific miR-483 overexpression exhibited ameliorated pulmonary hypertension (PH) and reduced right ventricular hypertrophy on challenge with monocrotaline (MCT) or Sugen + hypoxia. A reversal effect was observed in rats that received MCT with inhaled lentivirus overexpressing miR-483. These results indicate that PAH is associated with a reduced level of miR-483 and that miR-483 might reduce experimental PH by inhibition of multiple adverse responses. Synopsis This study reveals a protective role for miR-483 in the development of pulmonary arterial hypertension (PAH). Overexpression of miR-483 in endothelial cells (ECs) inhibits inflammatory and fibrogenic responses and leads to ameliorated pulmonary hypertension phenotypes in rats. Lower levels of miR-483 were found in the serum from patients with idiopathic pulmonary arterial hypertension, particularly in those with more severe disease. MiR-483 targets multiple genes that are involved in the pathogenesis of PAH. Overexpression of miR-483 in ECs inhibited inflammatory and fibrogenic responses. Intratracheal administration or EC-specific overexpression of miR-483 alleviated pulmonary hypertension in rats. The paper explained Problem PH is characterized by increased pulmonary arterial pressure and small pulmonary vascular remodeling, resulting in right ventricular hypertrophy and finally heart failure. Mounting evidence indicates that endothelial dysfunction plays an important role in regulating the pulmonary arterial remodeling during the pathogenic process of PH. The current clinical therapy for PH mainly focuses on the dilation of pulmonary vascular by targeting pathways related to ET-1, NO, and prostaglandins. However, these treatments are not curative. MicroRNAs are small molecules that target mRNA transcripts and inhibit their expression, which are implicated in EC health and disease. The current study explores whether miR-483, a protective miR in ECs, could protect against PH pathogenesis. Results Our results showed that miR-483 was decreased in the sera of patients with IPAH, especially those with more severe disease. RNA-sequencing and bioinformatics analyses revealed that miR-483 decreases and targets a panel of PAH-related genes, including TGF-β, TGFBR2, β-catenin, CTGF, IL-1β, and ET-1. Overexpression of miR-483 in PAECs inhibits these PAH-related genes. When challenged with MCT or Sugen + hypoxia, rats with EC-specific miR-483 overexpression exhibited ameliorated PH and reduced right ventricular hypertrophy. Moreover, exogenously delivered miR-483 suppressed PAH-related gene expression and reversed the PH pathogenesis in rats received MCT. Impact Our findings suggest that decreased circulating level of miR-483 is associated with human IPAH subjects and rodent PH models. Overexpression of miR-483 suppresses the PAH-related genes and therefore tends to protect EC function and ameliorate experimental PH. Introduction Pulmonary arterial hypertension (PAH) is a multifaceted vascular disease resulting from functional changes and structural remodeling in small pulmonary arteries (Hoeper et al, 2013). In patients with PAH and various animal models with experimental pulmonary hypertension (PH), endothelial dysfunction is linked to increased production of vasoconstrictors, pro-inflammatory cytokines, and growth factors together with attenuated production of vasodilators (Schermuly et al, 2011). The current drug therapy for PAH, including bosentan, sildenafil, macitentan, riociguat, prostacyclin, and its derivatives, aims to target pathways related to endothelin-1 (ET-1), nitric oxide, and vasoconstrictor prostaglandins (Galie et al, 2016a,b). However, these agents improve quality of life and longevity but are not curative. MicroRNAs (miRNAs) modulate gene expression by inhibiting translation or inducing degradation of target mRNA transcripts. New therapeutic approaches involving miRNA therapeutics are emerging that either mimic or inhibit miRNAs (Janssen et al, 2013). MicroRNA-483 (miR-483), containing both miR-483-3p and miR-483-5p (hereafter referred to as miR-483-3p/-5p), are intronic miRNAs that are encoded together with insulin-like growth factor 2 (IGF2) (Li et al, 2015). The sequences of miR-483-3p/-5p are highly conserved among mammalian species including human, mouse, and rat. MiR-483 targets the 3′UTR of connective tissue growth factor (CTGF), platelet-derived growth factor-β (PDGF-β), tissue inhibitor of metalloproteinase 2, and Smad4, a key component protein in the transforming growth factor β (TGF-β) signaling pathway (Li et al, 2014; Anderson & McAlinden, 2017; He et al, 2017). Additionally, miR-483 suppresses the growth and proliferation of several cell types by downregulating extracellular signal-regulated kinase 1, mitogen-activated protein kinase-activated protein kinase 2, Yes-associated protein 1, and IGF1 (Bertero et al, 2011; Wang et al, 2012; Ni et al, 2013). With respect to endothelial biology, we previously showed that miR-483 enhances endothelial cell (EC) function, in part via its anti-fibrogenic effect and a recent report by others shows that disturbed flow decreases miR-483 level in ECs (He et al, 2017; Fernandez Esmerats et al, 2019). Based on this conceptual framework, we sought to test the hypothesis that miR-483 is pathophysiologically important in PH and that it may be useful as a disease biomarker and a therapeutic target. In the current study, we investigated whether low levels of miR-483 were associated with idiopathic PAH (IPAH). Our results show that the levels of circulating miR-483 were indeed lower in IPAH patients and seemed to correlate inversely with the severity of clinical manifestations. Experimental validation revealed that miR-483 targeted multiple genes including TGF-β, interleukin 1β (IL-1β), and ET-1 in ECs. Rats with miR-483 overexpression in the endothelium were PH-protected. Of translational relevance, intratracheal administered miR-483 reversed monocrotaline (MCT)-induced PH in rats, which provides therapeutic potential of miR-483. Results Decreased miR-483 level in human IPAH patients We first examined the circulatory levels of miR-483-3p/-5p in a cohort of IPAH patients (patient demographics and clinical characteristics in Table 1). Compared to age- and sex-matched healthy controls (HCs), the circulating levels of miR-483-3p/-5p were significantly lower in IPAH patients (Fig 1A). The receiver operating characteristic (ROC) curves shown in Fig 1B reveal that patients with miR-483-3p and miR-483-5p levels < 27% and 26% of those in HCs might have PAH, with sensitivity 88.4% and 82.1%; specificity 56.8% and 48.9% (area under the ROC curve, 0.77 and 0.66, respectively). According to the new PAH risk stratification for prognosis and initial therapy (2018 World Symposium on Pulmonary Hypertension; Galie et al, 2019), we divided IPAH patients into low-, intermediate-, and high-risk groups with estimated 1-year mortality risks of < 5%, 5–10%, and > 10%, respectively. Of note, miR-483-3p level was lower in intermediate- and high-risk than low-risk patients (Fig 1C). Table 1. Clinical characteristics of PAH patients and control subjects Serum CD144-EVs Control (n = 95) IPAH (n = 139) Control (n = 34) IPAH (n = 37) Sex [female, n (%)] 77 (81%) 117 (84%) 3 (82%) 5 (86%) Age (years) 33 (21–47) 32 (17–70) 37 (20–46) 35 (23–40) Functional class [n (%)] I – 3 (2%) – 2 (5%) II – 21 (42%) – 16 (43%) III – 23 (49%) – 16 (43%) IV – 4 (7%) – 3 (8%) CI (l/min/m2) – 2.3 ± 0.6 – 2.2 ± 0.5 PVR (Wood U) – 14.1 ± 6.2 – 14.2 ± 6.2 mPAP (mmHg) – 60.0 ± 17.2 – 58.7 ± 15.5 RAP (mmHg) – 8.1 ± 4.8 – 7.9 ± 5.0 SvO2 (%) – 66.5 ± 8.7 – 66.2 ± 8.1 NT-proBNP (ng/l) – 1683 ± 2348 – 1933 ± 2393 6MWD (m) – 444.5 ± 102.7 – 436.9 ± 102.7 Medication [n (%)] ERA – 67 (48.2%) – 22 (59.5%) PDE5 inhibitor – 79 (56.8%) – 27 (73.0%) Epoprostenol – 26 (18.7%) – 7 (18.9%) Figure 1. Lower serum miR-483 level in IPAH patients A. Serum levels of miR-483-3p/-5p in IPAH patients (n = 139) and HC (n = 95) measured by qPCR. The data are fold change normalized to the averaged level of HC. B. ROC curve with sensitivity and specificity of serum levels of miR-483-3p/-5p for differentiating IPAH patients from HCs at diagnosis (IPAH, n = 139; HC, n = 95). C. Levels of miR-483-3p/-5p associated with PAH risk in three groups. IPAH patients were divided into a low-risk group (Low) and an intermediate- plus high-risk group (Inter&high) according to the World Symposium on Pulmonary Hypertension 2018 [14]. D. Levels of miR-483-3p were inversely correlated with serum levels of ET-1 (IPAH, n = 118; HC, n = 95) and IL-6 (IPAH, n = 112; HC, n = 93). Data information: Values are expressed as median ± interquartile range. Statistical test: Mann–Whitney U-test (A), Kruskal–Wallis U-test (C). Source data are available online for this figure. Source Data for Figure 1 [emmm201911303-sup-0002-SDataFig1.xlsx] Download figure Download PowerPoint Patients with various forms of PAH, including IPAH, have higher serum or plasma levels of ET-1 and IL-6 (Schermuly et al, 2011). In the present study, the serum levels of ET-1 and IL-6 in the IPAH cohort were also significantly higher than those in control groups (Appendix Fig S1A and B) with areas under the ROC curve 0.74 and 0.74, respectively (Appendix Fig S1C and D). Interestingly, the serum levels of ET-1 and IL-6 seemed to be inversely correlated with those of miR-483-3p (Fig 1D). miR-483 targets PAH-related genes and pathways in endothelium We postulated that the lower levels of miR-483-3p/-5p in IPAH patient sera were linked to their decreased expression in pulmonary endothelium. To confirm this notion, we isolated CD144 (VE-cadherin, an EC marker)-enriched extracellular vesicles (EVs) from serum of IPAH patients and HCs. As illustrated in Fig 2A, miR-483-3p/-5p content was lower in CD144-enriched EVs from IPAH than HC. The lower levels of miR-483 found in circulation and CD144-enriched EVs of IPAH patients suggested that the expression of miR-483-3p/-5p might affect genes and pathways involved in PAH. Figure 2. MiR-483 targets PAH-related genes and pathways in PAECs CD144-enriched EVs were isolated from serum of IPAH patients (n = 37) and HCs (n = 34). The levels of miR-483-3p/-5p were measured by qPCR. PAECs were transfected with miR-483-3p/-5p mimic or scramble RNA for 36 hr before RNA isolation and then analyzed by RNA-seq. Data are results from two biological repeats. (B) PAH-related GO enrichment delineated by DAVID for the top 300 upregulated or downregulated genes with the cutoff of P < 0.05. (C) Heat map comparison of log2 fold changes of the indicated genes. Data information: Values are expressed as median ± interquartile range. Statistical test: Mann–Whitney U-test. Source data are available online for this figure. Source Data for Figure 2 [emmm201911303-sup-0003-SDataFig2.xlsx] Download figure Download PowerPoint We conducted RNA-seq analysis to examine the transcriptomes in cultured human pulmonary arterial ECs (PAECs) overexpressing miR-483. As illustrated in Fig 2B and Appendix Fig S2, gene ontology (GO) analysis of differentially regulated pathways showed that miR-483 was correlated with negative regulation of Wnt signaling and inflammatory response, TGF-β receptor signaling, cell adhesion, response to hypoxia, apoptotic processes, oxidation–reduction processes, and negative regulation of cell migration and proliferation. Such changes in transcriptomes suggested that miR-483-3p/-5p could affect the EC phenotype. The heatmap shown in Fig 2C revealed that miR-483 overexpression downregulated genes involved in cell proliferation, migration, inflammation, Wnt and TGF-β receptor signaling, apoptosis process, response to hypoxia, and oxidative stress. In contrast, genes (e.g., COL4A2) involved in cell adhesion (Rhodes et al, 2015) were upregulated. Data illustrated in Fig 2 suggest that elevated miR-483-3p/-5p inhibits PAH-related genes and pathways in the pulmonary endothelium. miR-483 targets TGF-β, TGFBR2, IL-1β, and ET-1 mRNAs We then used RNAhybrid software to predict miR-483-targeted mRNAs. As illustrated in Fig 3A, while miR-483-3p was predicted to target the 3′UTR of TGF-β, TGFβR2, Smad2, ROCK1, β-catenin, and ET-1 mRNAs, miR-483-5p targets the 3′UTR of TGF-β, TGFBR2, Smad2, Smad3, IL-1β, and ET-1 mRNAs. Additionally, miR-483 targeting 3′UTR of TGF-β, TGFBR2, IL-1β, and ET-1 mRNAs was found conserved among human, rat, and mouse (Appendix Table S1). To validate the in silico predictions, pre-miR-483 was overexpressed in human PAECs by lentivirus (Lenti-miR-483, Fig 3B). As predicted, the mRNA and protein levels of TGF-β, TGFBR2, β-catenin, CTGF, IL-1β, and ET-1 were lower in PAECs infected with Lenti-miR-483 (Fig 3C and D). In the complementary approach, miR-483 inhibition by anti-miR-483 resulted in increased mRNA and protein levels of TGF-β, TGFBR2, β-catenin, CTGF, IL-1β, and ET-1 (Appendix Fig S3A–C). We then examined whether miR-483-3p/-5p directly targets these genes by using TGF-β, TGFBR2, IL-1β, and ET-1 3′UTR constructs conjugated with a luciferase reporter. As shown in Fig 3E, miR-483 overexpression decreased the luciferase activity of the wild-type reporters Luc-TGF-β (WT), Luc-TGFBR2 (WT), Luc-IL-1β (WT), and Luc-ET-1 (WT). However, no reduction of luciferase activity was observed in ECs transfected with Luc-TGF-β (mut), Luc-TGFBR2 (mut), Luc-IL-1β (mut), or Luc-ET-1 (mut) in which the miR-483-targeted sequences were mutated. Moreover, miR-483-3p/-5p levels were increased in the miR-induced silencing complexes (miRISC) (i.e., Ago1, Ago2) in PAECs overexpressing miR-483 (Fig 3F). Additionally, levels of TGF-β, TGFBR2, β-catenin, CTGF, IL-1β, and ET-1 mRNAs were also increased in association with Ago1 and Ago2 (Fig 3G), indicating these mRNAs were targeted by miR-483 in the miRISC. Figure 3. MiR-483-targeted genes A. Predicted binding sites for miR-483-3p/-5p on the 3′UTR of mRNAs as indicated. B–D. PAECs were infected with Lenti-pre-miR-483 or Lenti-null for 24 hr. Expression levels of miR-483-3p/-5p, TGF-β, TGFBR2, β-catenin, CTGF, IL-1β, and ET-1 mRNA and protein were measured by qPCR and Western blot, respectively. E. Bovine aortic ECs were transfected with a luciferase reporter fused with the 3′UTR of TGF-β (Luc-TGF-β WT), TGFBR2 (Luc-TGFBR2 WT), IL-1β (Luc-IL-1β WT), or ET-1 (Luc-ET-1 WT) or a binding site mutation (Luc-TGF-β Mut, Luc-TGFBR2 Mut, Luc-IL-1β Mut, ET-1-Mut), then infected with Lenti-pre-miR-483 for additional 36 hr. Luciferase activity was measured. F, G. PAECs were infected with Lenti-pre-miR-483 or Lenti-null for 36 h. The Ago1- or Ago2-associated miRNAs and mRNAs were enriched by immunoprecipitation with anti-Ago1 or anti-Ago2. Levels of miR-483-3p/-5p and TGF-β, TG