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. 2023 Nov 28;148(22):1819-1822.
doi: 10.1161/CIRCULATIONAHA.123.065455. Epub 2023 Nov 27.

Epitranscriptomic Modification of MicroRNA Increases Atherosclerosis Susceptibility

Ming He #  1 , Jianjie Dong #  1 , Junmao Wen  1 , Yuqing Zhang  1 , So Yun Han  1 , Chen Wang  2 , Brendan Gongol  1 , Tong-You Wade Wei  1 , Jian Kang  1 , Hsi-Yuan Huang  3 , Susan Cheng  4 , John Y-J Shyy  1
Affiliations

Epitranscriptomic Modification of MicroRNA Increases Atherosclerosis Susceptibility

Ming He et al. Circulation. .
No abstract available

Keywords: atherosclerosis; endothelium; microRNAs.

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Conflict of interest statement

Disclosures None.

Figures

None
Epitranscriptomic modification of miR-483 to 8OH-G miR-483 impairs EC function and increases atherosclerosis susceptibility.
(A-E) Human umbilical vein endothelial cells (HUVECs) were treated with H2O2 (100 μM; with the same volume of PBS as controls) for 4 hr or oxidized-LDL (ox-LDL, 100 μg/mL; with the same amount of native-LDL as controls) for 24 hr or exposed to oscillatory shear stress [OS, 0.5±4 dyn/cm2; with pulsatile shear stress (PS), 12±4 dyn/cm2 as controls] for 16 hr. In (A), cells were stained with an 8OH-G antibody (revealed by red staining; Catalog No, 12501, clone 15A3, QED Bioscience; dilution 1:500) and co-stained with DAPI (Catalog No, D1206, Invitrogen) and images were captured and analyzed by Olympus FV1000 confocal microscopy. Scale bars: 50 μm. In (B), total cell lysates were isolated and immunoprecipitated with an 8OH-G antibody (5 μL per 1 mg protein) or isotype mouse IgG control antibody, then total RNA was isolated by Trizol reagent (Invitrogen). Total RNA was analyzed by electrophoresis by using the TapeStation system (Agilent). In (C), miRs were isolated from the 8OH-G antibody-immunoprecipitated RNAs or input total RNAs by using the mirVana miRNA Isolation Kit (Invitrogen). cDNA sequencing libraries were prepared by using TruSeq Small RNA Library Preparation Kits (Illumina) and then sequenced by using the NovaSeq 6000 system (Illumina) as 50 single-end reads. The x-axis of volcano plot represents the binary logarithm modified fold change of 8OH-G modified miRs (ox-LDL v.s. Ctrl), and the y-axis represents the minus of the binary logarithm modified FDR (false discovery rate) value. In (D, E), the thoracic aorta (TA) or aortic arch (AA) was isolated from 30 C57BL/6 mice (15 males). Each dot represents samples pooled from 5 mice. Serum was isolated from C57Bl/6j mice under an 8-week chow diet (n=8, 4 males) or mice with a single-dose tail vein injection of 1.0×1011 vector genome rAAV8-D377Y-mPCSK9 (Addgene #58376) followed by an 8-week high-fat diet (HF, 40 kcal% fat, 1.25% cholesterol, 0.5% cholic acid, Catalog No. D12109, Research Diets. N=8, 4 males). Serum was collected from hypercholesterolemia individuals (LDL-C>130mg/dL, n=13) or sex- and age-matched ones with normal lipid profile (LDL-C <110 mg/dL, n=8). The lipid profiles were detected by automatic chemical analysis (Hitachi LABOSPECT 008AS). All individuals were enrolled at the First Affiliated Hospital of Xi’an Jiaotong University during 2018 to 2019. Purified RNAs were incubated with an anti-8OH-G antibody for immunoprecipitation (IP). The input, miR-483, and 8OH-G miR-483 were analyzed by qPCR. Mouse IgG was an isotype IP control. The level of miR-483 (D) and ratio of 8OH-G miR-483-to-miR-483 (E) were shown. HC, healthy controls; HCL, hypercholesterolemia. (F, G) HUVECs were transfected with miR-483, 8OH-G miR-483, or scramble control (Ctrl) for 48 hr, then transcriptomes were analyzed by RNA-seq. All miRs were customized in Integrated DNA Technologies. (F), mapping of transcriptomes from each sample for a 2-D PCA analysis. In (G), heatmap comparison of log2 fold change (FC) for differential expressed genes in ECs overexpressing miR-483 or 8OH-G miR-483 (FC >1.5, FDR <0.1). Gene Ontology enrichment analyzed by using Metascape for the top 300 miR-483– or 8OH-G miR-483–downregulated genes. (H) Schematic diagram demonstrating the epitranscriptomic modification of miR-483 to 8OH-G miR-483 and the altered targeting from EC dysfunctional genes (e.g., CTGF) to EC homeostatic genes (e.g., KLF4). (I) Bovine aortic ECs transfected with different molar ratios of miR-483 to 8OH-G miR-483 together with Luc-hCTGF-3’-UTR (shown in red) or Luc-hKLF4–3’-UTR (shown in blue) for 48 hr. Luciferase activity was measured by pRL-TK activity as a transfection control. (J) HUVECs were transfected with anti-sense oligonucleotide against 8OH-G-miR-483 (Anti-8OH-G 483, 5’- dTdTØCAAGUAUØCAAGUAUØCAAGUAUØCAAGUAUØdTdT-3’) or anti-control (scramble sequence) at the dose as indicated, followed by H2O2 (100 μM; with the same volume of PBS as controls) treatment for 4 hr. The protein expression levels of KLF4 (abcam, ab272860; 1:1000 dilution) and CTGF (abcam, ab6992; 1:750 dilution) were assessed by western blotting, with β-Actin (abcam, ab8227; 1:5000 dilution) used as an internal control. mRNA levels were determined through qPCR. (K) Six-week-old EC-483-Tg and wild-type (WT) mice (8 males and 6 females in each group) were administered a single dose of rAAV8-D377Y-mPCSK9 (1.0×1011 vector genomes) via tail vein injection and fed a high-fat diet for 8 weeks. En face oil-red O staining of aortae are shown. (L) Eight-week-old male and female C57BL/6 mice were administered AAV8-PCSK9 and fed western diet to induce atherosclerosis. One week later, the left carotid artery was partially ligated and administered Lentivirus overexpressing scramble miRNA (Lenti-Ctrl, 5 male and 8 female mice), miR-483 (Lenti-miR-483, 5 male and 5 female mice), or the 8OH-G miR-483 mimic oligonucleotide (Lenti-8OH-G miR-483, with G-to-U mutation in the seed sequence of mmu-miR-483, 7 male and 8 female mice) through tail vein injection. The right carotid artery was shamed operated. All mice were sacrificed three weeks later and carotid arteries were isolated. The representative images showed arteries isolated from 3 male and 3 female mice in each groups. In (D), (E), (J), (K), and (L), data are expressed as mean ± SEM. Data were initially tested for normality and equal variance to confirm the appropriateness of parametric tests. Data with normal distribution and equal variance were analyzed by 2-tailed Student t test. If the normality or equal variance test failed, nonparametric data were analyzed by 2-tailed Mann-Whitney U test. * P<0.05 was considered statistically significant.
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