Increased m6A methylation level is associated with the progression of human abdominal aortic aneurysm

Abstract

Background: The role of N6-methyladenosine (m6A) modification in abdominal aortic aneurysm (AAA) has not been extensively studied. This study therefore aimed to investigate m6A RNA methylation and the expressions of the corresponding modulators in AAA.

Methods: A comparative study between AAA tissue samples (n=32) and healthy aortas (n=12) was performed using m6A methylation quantification for messenger RNA (mRNA) m6A status, quantitative polymerase chain reaction (qPCR), and western blot for the expressions of m6A modulators and immunohistochemistry (IHC) to detect locations of the modulators in AAA tissues.

Results: The m6A level significantly increased in AAA as compared to healthy aorta tissues. Among AAA patients, the high m6A level represented an even greater risk of AAA rupture as compared to non-ruptured AAA [odds ratio (OR), 1.370; 95% confidence interval (CI), 1.007-1.870]. The major N6-adenosine modulators, including YTHDF1, YTHDF3, FTO, and METTL14, are the main factors involved in aberrant m6A modification and the expression of both was significantly correlated to the proportion of m6A in total mRNA. Clinically, YTHDF3 represented an even greater risk of rupture (OR, 1.036; 95% CI, 1.001-1.072). Regarding the cellular location, METTL14 seemed to be associated with inflammatory infiltrates and neovascularization. Furthermore, a strong correlation was seen between FTO and aneurysmal smooth muscle cells (SMCs), YTHDF3, and macrophage infiltrate.

Conclusions: We were first to observe m6A modification in human AAA tissues. The results also reveal the important roles of m6A modulators, including YTHDF3, FTO, and METTL14, in the pathogenesis of human AAA and provide a new view on m6A modification in AAA. Our findings suggest a potential mechanism of epigenetic alterations in clinical AAA.

Keywords: Abdominal aortic aneurysm (AAA); N6-methyladenosine RNA methyltransferase (m6A RNA methyltransferase); N6-methyladenosine modification (m6A modification); epigenetics.

Figure 1

Flowchart of AAA patient selection and biopsy tissue or blood sample recovered from CMU-aB. AAA, abdominal aortic aneurysm; FFPE, formalin-fixed paraffin-embedded; CMU-aB, China Medical University Aneurysm Biobank.

Figure 2

Expression of m6A RNA methylation status (A) and m6A methylation modulators (B,C,D,E,F,G,H) at the mRNA level in AAA tissue samples compared with the healthy control aortas, analyzed by qRT-PCR and SYBR Master Mix. The non-parametric Mann-Whitney U test was applied to analyze the mRNA methylation status (A); the m6A “writers” family, METTL3 (B) and METTL14 (C); m6A “erasers” family, ALBH5 (D) and FTO (E); and “readers” family, YTHDF 1 (F), YTHDF2 (G), and YTHDF3 (H). Dates are normalized to the expression of GAPDH and analyzed through the non-parametric Mann-Whitney U test. *, P<0.05; **, P<0.01. m6A, N6-methyladenosine; AAA, abdominal aortic aneurysm; qRT-PCR, quantitative real-time polymerase chain reaction; ns, not significant.

Figure 3

Expression analysis of METTL14 (A), FTO (B), YTHDF1 (C), and YTHDF3 (D) in AAA and healthy aortas at the protein level through the non-parametric Mann-Whitney U test. At top are the raw images of blots by Western blot, and at bottom is quantification of the band intensities relative to the expression of GAPDH. ***, P<0.001. C and Ctrl, control healthy aorta (n=12); A and AAA, specimens of abdominal aortic aneurysm (n=32).

Figure 4

Analysis of METTL14 (A), FTO (B), and YTHDF3 (C) in AAA using IHC for cellular localization. Overview image (left panel) of the whole AAA tissue sample with areas selected for cellular localization of m6A modulator expressions (HE staining). The magnified images depict consecutive staining of individual cell types, as revealed by staining for indicated markers within the AAA wall and indicated corresponding substrates. Scale bar, 50 µm. AAA, abdominal aortic aneurysm; IHC, immunohistochemistry; m6A, N6-methyladenosine; HE, hematoxylin-eosin; SMC, smooth muscle cell; EC, endothelial cell.

Figure 5

Analysis of acetylated METTL14 (A), FTO (C), and YTHDF3 (E) in AAA using IHC, compared with their expressions in healthy control aortic tissues (B,D,F). Scale bar, 50 µm. AAA, abdominal aortic aneurysm; IHC, immunohistochemistry.

Figure S1

AAA is a frequent vascular disorder with high mortality and accumulation of several cell types, including leukocytes, lymphocytes, macrophages, endothelial, and smooth muscle cells. Alternations in m6A modification were clearly observed in AAA compared to healthy aortas. Furthermore, m6A modification in AAA was also correlated with clinical data and associated with a higher risk of AAA rupture. m6A, N6-methyladenosine; AAA, abdominal aortic aneurysm; VSMC, vascular smooth muscle cell.