Hsa-miRNA-23a-3p promotes atherogenesis in a novel mouse model of atherosclerosis
2020; Elsevier BV; Volume: 61; Issue: 12 Linguagem: Inglês
10.1194/jlr.ra120001121
ISSN1539-7262
AutoresJiayan Guo, Hanbing Mei, Zhen Sheng, Qingyuan Meng, Murielle M. Véniant, Hong Yin,
Tópico(s)Circular RNAs in diseases
ResumoOf the known regulators of atherosclerosis, miRNAs have been demonstrated to play critical roles in lipoprotein homeostasis and plaque formation. Here, we generated a novel animal model of atherosclerosis by knocking in LDLRW483X in C57BL/6 mice, as the W483X mutation in LDLR is considered the most common newly identified pathogenic mutation in Chinese familial hypercholesterolemia (FH) individuals. Using the new in vivo mouse model combined with a well-established atherosclerotic in vitro human cell model, we identified a novel atherosclerosis-related miRNA, miR-23a-3p, by microarray analysis of mouse aortic tissue specimens and human aortic endothelial cells (HAECs). miR-23a-3p was consistently downregulated in both models, which was confirmed by qPCR. Bioinformatics analysis and further validation experiments revealed that the TNFα-induced protein 3 (TNFAIP3) gene was the key target of miR-23a-3p. The miR-23a-3p-related functional pathways were then analyzed in HAECs. Collectively, the present results suggest that miR-23a-3p regulates inflammatory and apoptotic pathways in atherogenesis by targeting TNFAIP3 through the NF-κB and p38/MAPK signaling pathways. Of the known regulators of atherosclerosis, miRNAs have been demonstrated to play critical roles in lipoprotein homeostasis and plaque formation. Here, we generated a novel animal model of atherosclerosis by knocking in LDLRW483X in C57BL/6 mice, as the W483X mutation in LDLR is considered the most common newly identified pathogenic mutation in Chinese familial hypercholesterolemia (FH) individuals. Using the new in vivo mouse model combined with a well-established atherosclerotic in vitro human cell model, we identified a novel atherosclerosis-related miRNA, miR-23a-3p, by microarray analysis of mouse aortic tissue specimens and human aortic endothelial cells (HAECs). miR-23a-3p was consistently downregulated in both models, which was confirmed by qPCR. Bioinformatics analysis and further validation experiments revealed that the TNFα-induced protein 3 (TNFAIP3) gene was the key target of miR-23a-3p. The miR-23a-3p-related functional pathways were then analyzed in HAECs. Collectively, the present results suggest that miR-23a-3p regulates inflammatory and apoptotic pathways in atherogenesis by targeting TNFAIP3 through the NF-κB and p38/MAPK signaling pathways. Atherosclerosis is a chronic inflammatory disease of the arteries characterized by plaques built up in the vessels (1Hansson G.K. Libby P. Tabas I. Inflammation and plaque vulnerability.J. Intern. Med. 2015; 278: 483-493Crossref PubMed Scopus (484) Google Scholar, 2Sakao S. Taraseviciene-Stewart L. Lee J.D. Wood K. Cool C.D. Voelkel N.F. Initial apoptosis is followed by increased proliferation of apoptosis-resistant endothelial cells.FASEB J. 2005; 19: 1178-1180Crossref PubMed Scopus (234) Google Scholar, 3Tardy Y. Resnick N. Nagel T. Gimbrone Jr., M.A. Dewey Jr., C.F. Shear stress gradients remodel endothelial monolayers in vitro via a cell proliferation-migration-loss cycle.Arterioscler. Thromb. Vasc. Biol. 1997; 17: 3102-3106Crossref PubMed Scopus (223) Google Scholar). Endothelial dysfunction in the vessels represents one of the atherogenic processes in the early stage that leads to the formation of atherosclerotic lesions; at a later stage, plaques are built up inside the arteries leading to a narrowed and restricted blood flow, which increases the influx of LDL into the arterial wall (4Cancel L.M. Tarbell J.M. The role of apoptosis in LDL transport through cultured endothelial cell monolayers.Atherosclerosis. 2010; 208: 335-341Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), induces the migration of monocytes by activating endothelial cells, and results in lipid accumulation and foam cell formation (5Li D. Chen H. Romeo F. Sawamura T. Saldeen T. Mehta J.L. Statins modulate oxidized low-density lipoprotein-mediated adhesion molecule expression in human coronary artery endothelial cells: role of LOX-1.J. Pharmacol. Exp. Ther. 2002; 302: 601-605Crossref PubMed Scopus (163) Google Scholar, 6Pirillo A. Norata G.D. Catapano A.L. LOX-1, OxLDL, and atherosclerosis.Mediators Inflamm. 2013; 2013152786 Crossref PubMed Scopus (470) Google Scholar). Among atherosclerosis regulators, miRNAs have been shown to play critical roles in processes such as lipoprotein homeostasis (e.g., miR-122, miR-223, and miR-148a) (7Esau C. Davis S. Murray S.F. Yu X.X. Pandey S.K. Pear M. Watts L. Booten S.L. Graham M. McKay R. et al.miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting.Cell Metab. 2006; 3: 87-98Abstract Full Text Full Text PDF PubMed Scopus (1771) Google Scholar, 8Vickers K.C. Shoucri B.M. Levin M.G. Wu H. Pearson D.S. Osei-Hwedieh D. Collins F.S. Remaley A.T. Sethupathy P. MicroRNA-27b is a regulatory hub in lipid metabolism and is altered in dyslipidemia.Hepatology. 2013; 57: 533-542Crossref PubMed Scopus (176) Google Scholar, 9Vickers K.C. Landstreet S.R. Levin M.G. Shoucri B.M. Toth C.L. Taylor R.C. Palmisano B.T. Tabet F. Cui H.L. Rye K.A. et al.MicroRNA-223 coordinates cholesterol homeostasis.Proc. Natl. Acad. Sci. USA. 2014; 111: 14518-14523Crossref PubMed Scopus (190) Google Scholar, 10Goedeke L. Rotllan N. Canfran-Duque A. Aranda J.F. Ramirez C.M. Araldi E. Lin C.S. Anderson N.N. Wagschal A. de Cabo R. et al.MicroRNA-148a regulates LDL receptor and ABCA1 expression to control circulating lipoprotein levels.Nat. Med. 2015; 21: 1280-1289Crossref PubMed Scopus (172) Google Scholar), endothelial cell inflammation and plaque progression (e.g., miR-17-3p, miR-31, miR-92a, and miR-146a) (11Zernecke A. Bidzhekov K. Noels H. Shagdarsuren E. Gan L. Denecke B. Hristov M. Koppel T. Jahantigh M.N. Lutgens E. et al.Delivery of microRNA-126 by apoptotic bodies induces CXCL12-dependent vascular protection.Sci. Signal. 2009; 2: ra81Crossref PubMed Scopus (1079) Google Scholar, 12Suárez Y. Wang C. Manes T.D. Pober J.S. Cutting edge: TNF-induced microRNAs regulate TNF-induced expression of E-selectin and intercellular adhesion molecule-1 on human endothelial cells: feedback control of inflammation.J. Immunol. 2010; 184: 21-25Crossref PubMed Scopus (265) Google Scholar, 13Sun D. Zhang J. Xie J. Wei W. Chen M. Zhao X. MiR-26 controls LXR-dependent cholesterol efflux by targeting ABCA1 and ARL7.FEBS Lett. 2012; 586: 1472-1479Crossref PubMed Scopus (112) Google Scholar, 14Loyer X. Potteaux S. Vion A.C. Guerin C.L. Boulkroun S. Rautou P.E. Ramkhelawon B. Esposito B. Dalloz M. Paul J.L. et al.Inhibition of microRNA-92a prevents endothelial dysfunction and atherosclerosis in mice.Circ. Res. 2014; 114: 434-443Crossref PubMed Scopus (289) Google Scholar, 15Chen Z. Wen L. Martin M. Hsu C.Y. Fang L. Lin F.M. Lin T.Y. Geary M.J. Geary G.G. Zhao Y. et al.Oxidative stress activates endothelial innate immunity via sterol regulatory element binding protein 2 (SREBP2) transactivation of microRNA-92a.Circulation. 2015; 131: 805-814Crossref PubMed Scopus (118) Google Scholar), cytokine response (e.g., miR-181b) (16Sun X. Icli B. Wara A.K. Belkin N. He S. Kobzik L. Hunninghake G.M. Vera M.P. Registry M. Blackwell T.S. et al.MicroRNA-181b regulates NF-kappaB-mediated vascular inflammation.J. Clin. Invest. 2012; 122: 1973-1990PubMed Google Scholar), and leukocyte recruitment and activation (e.g., miR-26,miR-144, and miR-148a) (10Goedeke L. Rotllan N. Canfran-Duque A. Aranda J.F. Ramirez C.M. Araldi E. Lin C.S. Anderson N.N. Wagschal A. de Cabo R. et al.MicroRNA-148a regulates LDL receptor and ABCA1 expression to control circulating lipoprotein levels.Nat. Med. 2015; 21: 1280-1289Crossref PubMed Scopus (172) Google Scholar, 13Sun D. Zhang J. Xie J. Wei W. Chen M. Zhao X. MiR-26 controls LXR-dependent cholesterol efflux by targeting ABCA1 and ARL7.FEBS Lett. 2012; 586: 1472-1479Crossref PubMed Scopus (112) Google Scholar, 17de Aguiar Vallim T.Q. Tarling E.J. Kim T. Civelek M. Baldan A. Esau C. Edwards P.A. MicroRNA-144 regulates hepatic ATP binding cassette transporter A1 and plasma high-density lipoprotein after activation of the nuclear receptor farnesoid X receptor.Circ. Res. 2013; 112: 1602-1612Crossref PubMed Scopus (145) Google Scholar). The latest miRNA database (miRBase 22) released 2,654 human mature miRNAs (18Kozomara A. Birgaoanu M. Griffiths-Jones S. miRBase: from microRNA sequences to function.Nucleic Acids Res. 2019; 47: D155-D162Crossref PubMed Scopus (1785) Google Scholar). However, it remains unknown whether novel atherosclerosis-regulating miRNAs could be identified in this updated database. To investigate the roles of miRNAs in atherosclerosis, both in vitro and in vivo models have commonly been used (14Loyer X. Potteaux S. Vion A.C. Guerin C.L. Boulkroun S. Rautou P.E. Ramkhelawon B. Esposito B. Dalloz M. Paul J.L. et al.Inhibition of microRNA-92a prevents endothelial dysfunction and atherosclerosis in mice.Circ. Res. 2014; 114: 434-443Crossref PubMed Scopus (289) Google Scholar, 19Soh J. Iqbal J. Queiroz J. Fernandez-Hernando C. Hussain M.M. MicroRNA-30c reduces hyperlipidemia and atherosclerosis in mice by decreasing lipid synthesis and lipoprotein secretion.Nat. Med. 2013; 19: 892-900Crossref PubMed Scopus (217) Google Scholar, 20Bao M.H. Li J.M. Zhou Q.L. Li G.Y. Zeng J. Zhao J. Zhang Y.W. Effects of miR590 on oxLDLinduced endothelial cell apoptosis: Roles of p53 and NFkappaB.Mol. Med. Rep. 2016; 13: 867-873Crossref PubMed Scopus (21) Google Scholar, 21Zhang H. Zheng J. Lin J. Chen J. Yu Z. Chen C. Liu T. miR-758 mediates oxLDL-dependent vascular endothelial cell damage by suppressing the succinate receptor SUCNR1.Gene. 2018; 663: 1-8Crossref PubMed Scopus (17) Google Scholar). For instance, an in vitro model was established by treating human aortic endothelial cells (HAECs) with oxidized LDL (oxLDL), a well-known atherogenic factor (6Pirillo A. Norata G.D. Catapano A.L. LOX-1, OxLDL, and atherosclerosis.Mediators Inflamm. 2013; 2013152786 Crossref PubMed Scopus (470) Google Scholar, 22Glass C.K. Witztum J.L. Atherosclerosis. the road ahead.Cell. 2001; 104: 503-516Abstract Full Text Full Text PDF PubMed Scopus (2629) Google Scholar). oxLDL is known to initiate atherogenesis by stimulating endothelial cells to overexpress cell surface adhesion molecules [e.g., intercellular adhesion molecule (ICAM)-1] (22Glass C.K. Witztum J.L. Atherosclerosis. the road ahead.Cell. 2001; 104: 503-516Abstract Full Text Full Text PDF PubMed Scopus (2629) Google Scholar, 23DeGraba T.J. Expression of inflammatory mediators and adhesion molecules in human atherosclerotic plaque.Neurology. 1997; 49: S15-S19Crossref PubMed Google Scholar, 24Kitagawa K. Matsumoto M. Sasaki T. Hashimoto H. Kuwabara K. Ohtsuki T. Hori M. Involvement of ICAM-1 in the progression of atherosclerosis in APOE-knockout mice.Atherosclerosis. 2002; 160: 305-310Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) and inflammatory cytokines, such as E-selectin (25Takei A. Huang Y. Lopes-Virella M.F. Expression of adhesion molecules by human endothelial cells exposed to oxidized low density lipoprotein. Influences of degree of oxidation and location of oxidized LDL.Atherosclerosis. 2001; 154: 79-86Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 26Di Santo S. Diehm N. Ortmann J. Volzmann J. Yang Z. Keo H.H. Baumgartner I. Kalka C. Oxidized low density lipoprotein impairs endothelial progenitor cell function by downregulation of E-selectin and integrin alpha(v)beta5.Biochem. Biophys. Res. Commun. 2008; 373: 528-532Crossref PubMed Scopus (30) Google Scholar), and to induce endothelial cell apoptosis by activating caspase-3 and caspase-9 (27Chen J. Mehta J.L. Haider N. Zhang X. Narula J. Li D. Role of caspases in Ox-LDL-induced apoptotic cascade in human coronary artery endothelial cells.Circ. Res. 2004; 94: 370-376Crossref PubMed Scopus (241) Google Scholar). Lectin-like oxLDLR-1 (LOX-1) overexpression in the APOE−/− mouse model has been shown to promote atherogenesis (28White S.J. Sala-Newby G.B. Newby A.C. Overexpression of scavenger receptor LOX-1 in endothelial cells promotes atherogenesis in the ApoE(-/-) mouse model.Cardiovasc. Pathol. 2011; 20: 369-373Crossref PubMed Scopus (35) Google Scholar). LOX-1 upregulation has also been observed in endothelial cells in human atherosclerotic lesions, especially in the early stage of atherogenesis (29Kataoka H. Kume N. Miyamoto S. Minami M. Moriwaki H. Murase T. Sawamura T. Masaki T. Hashimoto N. Kita T. Expression of lectinlike oxidized low-density lipoprotein receptor-1 in human atherosclerotic lesions.Circulation. 1999; 99: 3110-3117Crossref PubMed Scopus (400) Google Scholar). The most frequently used in vivo mouse models are APOE−/− and LDLR−/− mice (14Loyer X. Potteaux S. Vion A.C. Guerin C.L. Boulkroun S. Rautou P.E. Ramkhelawon B. Esposito B. Dalloz M. Paul J.L. et al.Inhibition of microRNA-92a prevents endothelial dysfunction and atherosclerosis in mice.Circ. Res. 2014; 114: 434-443Crossref PubMed Scopus (289) Google Scholar, 19Soh J. Iqbal J. Queiroz J. Fernandez-Hernando C. Hussain M.M. MicroRNA-30c reduces hyperlipidemia and atherosclerosis in mice by decreasing lipid synthesis and lipoprotein secretion.Nat. Med. 2013; 19: 892-900Crossref PubMed Scopus (217) Google Scholar, 24Kitagawa K. Matsumoto M. Sasaki T. Hashimoto H. Kuwabara K. Ohtsuki T. Hori M. Involvement of ICAM-1 in the progression of atherosclerosis in APOE-knockout mice.Atherosclerosis. 2002; 160: 305-310Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 30Zhang T. Tian F. Wang J. Jing J. Zhou S.S. Chen Y.D. Atherosclerosis-associated endothelial cell apoptosis by miR-429-mediated down regulation of Bcl-2.Cell. Physiol. Biochem. 2015; 37: 1421-1430Crossref PubMed Scopus (44) Google Scholar). These models differ in how plasma lipoprotein clearance is dysregulated, although both show enhanced atherosclerosis. For example, APOE KO in mice results in a significant decrease of HDL and leads to reduced cholesterol efflux capacity (31Hayek T. Oiknine J. Brook J.G. Aviram M. Role of HDL apolipoprotein E in cellular cholesterol efflux: studies in apo E knockout transgenic mice.Biochem. Biophys. Res. Commun. 1994; 205: 1072-1078Crossref PubMed Scopus (55) Google Scholar). Comparatively, LDLR KO mice have a high percentage of their cholesterol carried in IDL/LDL particles, which closely resembles the condition of dyslipidemic humans (32Ishibashi S. Brown M.S. Goldstein J.L. Gerard R.D. Hammer R.E. Herz J. Hypercholesterolemia in low density lipoprotein receptor knockout mice and its reversal by adenovirus-mediated gene delivery.J. Clin. Invest. 1993; 92: 883-893Crossref PubMed Scopus (1271) Google Scholar). However, more than 85% of hypercholesterolemia's genetic variants have been identified in the LDLR gene. Of these, approximately 46% of the variants are single missense mutations of LDLR (33Chora J.R. Medeiros A.M. Alves A.C. Bourbon M. Analysis of publicly available LDLR, APOB, and PCSK9 variants associated with familial hypercholesterolemia: application of ACMG guidelines and implications for familial hypercholesterolemia diagnosis.Genet. Med. 2018; 20: 591-598Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), suggesting that a LDLR-null or missense mutation model may be more applicable, mimicking the majority of human hypercholesterolemia cases. In this study, we developed a novel mouse model of atherosclerosis that resembled human familial hypercholesterolemia (FH) by knocking in a single missense mutation of W483STOP in the LDLR gene using the CRISPR/Cas9 technology, as the W483X mutation in LDLR is considered the most prevalent missense mutation in Chinese FH individuals (34Jiang L. Sun L.Y. Pan X.D. Chen P.P. Tang L. Wang W. Zhao L.M. Yang S.W. Wang L.Y. Characterization of the unique Chinese W483X mutation in the low-density lipoprotein-receptor gene in young patients with homozygous familial hypercholesterolemia.J. Clin. Lipidol. 2016; 10: 538-546.e5Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). The microarray technology was then used to profile whole-genomic miRNAs and mRNAs; subsequent bioinformatics analysis was undertaken to screen candidates of atherosclerosis-related miRNA/mRNA pairs in the new atherosclerosis mouse model as well as an in vitro human cell model. In both models, a novel miRNA, miR-23a-3p, was identified that acted as a key regulator of atherosclerosis. By targeting TNFα-induced protein 3 (TNFAIP3), miR-23a-3p promoted inflammation and endothelial apoptosis through the NF-κB and p38/MAPK pathways. Taken together, these results indicated that the newly identified atherosclerosis-related miRNA plays an important role in inflammatory pathways. All experimental procedures performed in both Biomodel Organism (Shanghai, China) and Wuxi Apptech, Inc. (Shanghai, China) were in accordance with standard Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health, and abided by the institution-approved animal protocol and the AAALAC accreditation. C57BL/6-LDLRW483STOP knock-in (KI) mice were generated at the Shanghai Biomodel Organism. The single mutation W483STOP in the human LDLR gene was generated through the CRISPR/Cas9 technology. Oligo donor DNA was synthesized by Sango Biotech (Shanghai, China). The Cas9 mRNA and guide RNA (gRNA) were transcribed in vitro using MEGAscript® T7 transcription kit (Thermo Fisher, AM1334) and purified with MEGAclear™ transcription clean-up kit (Thermo Fisher, AM1908). The Cas9 mRNA and gRNA were injected into C57BL/6 mouse zygotes using a microinjection system under standard conditions. The injected zygotes were then cultured with embryos and transferred into the oviducts of recipient mice. After birth, genomic DNA was extracted from tail biopsies of all mice by the standard ethanol precipitation method (35Kanai N. Fujii T. Saito K. Tokoyama T. Rapid and simple method for preparation of genomic DNA from easily obtainable clotted blood.J. Clin. Pathol. 1994; 47: 1043-1044Crossref PubMed Scopus (94) Google Scholar). Genotyping was conducted by PCR (primer forward: 5′-ctctacctgccctgcttcccatcc-3′; reverse: 5′-accagccccctctttctttcttgt-3′) and sequencing. Homozygous (HO) mutant mice were obtained by interbreeding heterozygous (HE) mice; the genotype was confirmed by sequencing and Western blot. The atherosclerosis mouse model was established at Wuxi Apptech, Inc. (Shanghai, China). C57BL/6-LDLRW483STOP KI mice were fed a HFD (0.2% cholesterol and 21% saturated fat; Harlan Teklad, TD88137) for 12 weeks. Two independent groups of WT mice were included in the study and fed normal diet (ND) and HFD, respectively. APOE KO mice were purchased from the Jackson Laboratory (stock number 002052) and used as controls for atherosclerosis. Mouse body weights were recorded weekly for 12 weeks. For all biochemical analyses, mouse blood was collected by submandibular bleeding or cardiac puncture at the endpoint. Serum was separated by centrifugation at 1,300 g for 10 min at 4°C and stored at −80°C. Serum total cholesterol (TC), TG, LDL-C, and HDL-C amounts were determined every 2–3 weeks on a Hitachi 7180 biochemistry automatic analyzer (Tokyo, Japan). The mouse aorta was removed from the heart, opened longitudinally from the intercostal ostia to the iliac bifurcation, and pinned open. Each aorta was fixed with 4% paraformaldehyde (Affymetrix, 19943) and rinsed with 60% isopropanol (Sigma, I9030). Aorta specimens were then stained with 60% Oil red O (Sigma, O0625) solution in isopropanol and washed with 60% isopropanol. The stained aortic tissues underwent a final wash with deionized water prior to imaging. For immunohistochemical analysis, mouse aorta samples were first immersed in 4% paraformaldehyde overnight. Then, the tissues were dehydrated through a graded ethanol series and embedded in paraffin. Next, 4 μm paraffin sections were obtained for subsequent staining with hematoxylin (Sigma, GHS132) and eosin (Sigma, HT110132). Stained slides of aortic root sections were evaluated by light microscopy on an Aperio ImageScope v12.3.3.7014 (Leica Biosystems, Nussloch, Germany) to assess plaque formation. In each animal, plaque, open lumen, and total vessel areas were averaged from three separate slides. HAECs were purchased from the ATCC (PCS-100-011) and cultured in vascular cell basal medium (ATCC, PCS-100-030) supplemented with the endothelial cell growth kit (ATCC, PCS-100-041) and 0.1% penicillin/streptomycin (ATCC, PCS-999-002) at 37°C in an incubator with 5% CO2. HAECs were transiently transfected with miR-23a-3p mimic (Thermo Fisher, 4464067, #MC10644), negative control miRNA (miRNA NC) (5′-UUCUCCGAACGUGUCACGU-3′, Sangon Biotech), TNFAIP3 siRNA (5′-GGCCAAUCAUUGUCAUUUCTT-3′, Thermo Fisher, AM16708, #2510), or miR-23a-3p mimic and its inhibitor (Thermo Fisher, 4464084, #MH10644). All transfections utilized the Lipofectamine 3000 reagent (Invitrogen, L3000015) according to the manufacturer's instructions. After 24 h of cell transfection, the culture medium was replaced with fresh medium with or without oxLDL (Beijing Xiesheng Bio-Technology); inclusion or exclusion of oxLDL was determined based on the design. Total RNA was separately extracted from aortic endothelial cells and mouse aortic tissue specimens and purified with a mirVana™ miRNA isolation kit without phenol (Ambion, AM1561). Total RNA was amplified and labeled with a Low Input Quick Amp Labeling Kit, one-color (Agilent Technologies, 5190-2305). Individual miRNAs obtained from the extracted total RNA were labeled with Cy3 using a miRNA Complete Labeling and Hyb kit (Agilent Technologies, 5190-0456). Labeled miRNAs were then purified with an RNeasy mini kit (Qiagen, 74106). Each slide was hybridized with the Cy3-labeled miRNAs in a hybridization oven (Agilent Technologies, G2545A) at 55°C and 20 rpm for 20 h. After hybridization, slides were washed in staining dishes with Gene Expression Wash Buffer kit (Agilent Technologies, 5188-5327), scanned on an Agilent microarray scanner (Agilent Technologies), and analyzed with the Feature Extraction software v10.7 (Agilent Technologies). For HAEC samples, mRNAs and miRNAs were profiled using Agilent SurePrint G3 Human Gene Expression 8×60K microarray and Agilent SurePrint Human miRNA microarray (V21.0), respectively. In this analysis, 26,083 genes were included in the gene expression microarray; meanwhile, 2,549 miRNAs were included in the miRNA microarray (supplemental Table S1). Each probe was labeled as P (present, expressed) or A (absent, unexpressed) based on its fluorescence intensity in sample testing. Only the probes labeled as P in all samples from at least one group (control vs. experimental groups in the cell model or WT vs. HO groups in the mouse model) were retained for further analysis (supplemental Table S3). Gene and miRNA expression profiles in all samples were quantile-normalized after a log2-based normalization. The log2-transformed fold-change ratio for each gene or miRNA was then calculated between the treatment and control groups. Genes with absolute log2-transformed fold change ≥1 and P < 0.05 were considered to be differentially expressed mRNAs (DE mRNAs); miRNAs with P < 0.05 were considered to be differentially expressed miRNAs (DE miRNAs). In tissue samples from mice, mRNAs and miRNAs were profiled with Agilent SurePrint G3 mouse GE V2 8×60K microarray and Agilent Mouse miRNA microarray 8×60K (V21.0), respectively. Totally, 27,122 genes were included in the gene expression microarray; meanwhile, 1,881 miRNAs were included in the miRNA microarray (supplemental Table S1). Subsequent analysis was performed as described above for cell microarray data. Overlapping DE miRNAs between in vitro and in vivo models were retained for target gene prediction using TargetScan (Release 7.2) (36Agarwal V. Bell G.W. Nam J.W. Bartel D.P. Predicting effective microRNA target sites in mammalian mRNAs.eLife. 2015; 4e05005 Crossref PubMed Scopus (4377) Google Scholar). These miRNAs were further filtered based on the reverse regulation paradigm of miRNA-mRNA pairs on gene expression. The miRNAs in these pairs were defined as candidate atherosclerosis-related miRNAs. Candidate miRNAs targeting previously reported atherosclerosis-related genes were prioritized for selection. Any miRNAs already reported to be related to atherosclerosis were excluded. The remaining candidate miRNAs were further validated by quantitative PCR. Total RNA was separately extracted from collected cells and/or tissues samples with RNeasy Plus mini kit (Qiagen, 74134) according to the manufacturer's instructions. In brief, cDNA was obtained from total RNA using FirstStrand synthesis system (Thermo Fisher, 18091050). Then, qPCR analysis was performed in triplicate using SYBR Green PCR Master Mix (Thermo Fisher, 4367659) with the primers shown in supplemental Table S2. Relative mRNA levels were normalized to the housekeeping gene GAPDH. miRNA expression was determined using TaqMan miRNA reverse transcription kit (Thermo Fisher, 4366597) and TaqMan miRNA assays (Thermo Fisher, 4440886). U6 snRNA was used for normalization. The 3′ untranslated regions (UTRs) of WT and mutant TNFAIP3 were amplified by PCR and cloned into PGL3-CMV-Luc vector using XhoI and MluI restriction sites followed by DNA sequencing verification. Then, 293T cells were cotransfected with luciferase reporter plasmids containing WT TNFAIP3, mutant TNFAIP3, or empty vectors, and miR-23a-3p mimic or miRNA NC, respectively, using Lipofectamine 3000. Luciferase activities were evaluated using a dual-luciferase reporter assay system (Promega, E2940) after 48 h. The apoptosis assay was performed with the Annexin V-FITC Apoptosis kit (Sigma, APOAF-50TST). Briefly, after appropriate treatment, cells were collected and washed with Dulbecco's PBS. Then, cells were resuspended at a density of 1 × 106 cells per milliliter and double stained with FITC-Annexin V and propidium iodide. Cells were finally analyzed on an LSRFortessa X-20 (BD) with the FlowJo v10.1 software. For cytokine detection, HAECs at a density of 3 × 105 cells per well were treated with miRNA negative control, miR-23a-3p mimic, or miR-23a-3p inhibitor in 6-well plates. Extracellular levels of ICAM and E-selectin were measured with a customized Luminex human magnetic assay kit (R&D, LXSAHM-04) and ELISA kit (Abcam, ab174445), respectively. For TNFAIP3 protein level detection, cells at a density of 8,000 cells per well in 96-well plates were transfected with miRNAs followed by oxLDL treatment. TNFAIP3 expression was measured with an ELISA kit (Aviva Systems Biology, OKCD08256) following the manufacturer's instructions. Cell adhesion was measured with a leukocyte adhesion assay kit (Cell Biolabs, CBA-210). Briefly, HAECs at a density of 5 × 104 in 96-well plates were transfected with miRNA mimic or its inhibitor for 48 h, followed by oxLDL treatment for 24 h. Then, THP-1 cells at 3 × 105 cells per well with LeukoTracker were added to the endothelial monolayer for an additional 90 min incubation before reading, according to manufacturer's instructions. Cells were lysed with RIPA (Thermo Fisher, 89901) containing 1% protease inhibitors (Sigma, 78444). After centrifugation, the supernatants were collected and total protein amounts were determined with a BCA kit (Thermo Fisher, 23227). Proteins were then separated by SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked and incubated with primary antibodies against TNFAIP3 (CST, 5630), p38 MAPK (CST, 8690), p-p38 MAPK (CST, 4511), IκBα (CST, 9242), p-IκBα (CST, 2859) and GAPDH (R&D Systems, MAB5718) at 4°C overnight. After incubation with appropriate secondary antibodies, the membranes were scanned on a Gel Documentation System. Statistical analysis was performed with GraphPad Prism 7. Multiple comparisons were performed by ANOVA followed by Tukey's test (Figs. 1C–F, 4B–E, 5D and E, 6B–D) or Dunnett's test (Figs. 1H–J, 2B, 5C). Comparison between two groups was carried out by Student's t-test (Figs. 2C and D, 3D and E, 5B). Differences were considered statistically significant at P < 0.05. All data are mean ± standard deviation. Recently, a very common but unique single missense mutation of the LDLR gene (p. W483STOP) has been identified as a pathogenic mutation in Chinese FH individuals (34Jiang L. Sun L.Y. Pan X.D. Chen P.P. Tang L. Wang W. Zhao L.M. Yang S.W. Wang L.Y. Characterization of the unique Chinese W483X mutation in the low-density lipoprotein-receptor gene in young patients with homozygous familial hypercholesterolemia.J. Clin. Lipidol. 2016; 10: 538-546.e5Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). The W483 of LDLR is highly conserved across human, mouse, and rat based on the multiple sequence alignment analysis in the LDLR sequences (37Waterhouse A.M. Procter J.B. Martin D.M. Clamp M. Barton G.J. Jalview version 2–a multiple sequence alignment editor and analysis workbench.Bioinformatics. 2009; 25: 1189-1191Crossref PubMed Scopus (5690) Google Scholar). To develop a human mouse model of atherosclerosis that could mimic the human condition mentioned above, we generated a LDLR mutation KI mouse model by the CRISPR-Cas9 technology with gRNA and oligo donor DNA, as shown in Fig. 1A. The chronological steps involved in the generation of C57BL/6-LDLRW483STOP KI mice are highlighted in supplemental Fig. S1. Sequencing results showed that the gRNA was effective (supplemental Fig. S1). Genotyping of HE and HO mice for the LDLRW483STOP mutation was validated by Western blot (Fig. 1B). A total of 10 LDLR HO mice (seven males and three females) and 10 LDLR HE mice (five males and five females) were obtained. There were eight (four males and four females) and five (three males, two females) of WT and APOE KO mice, respectively. The body weights of LDLR HO mice
Referência(s)