The effect of N6-methyladenosine (m6A) factors on the development of acute respiratory distress syndrome in the mouse model

Abstract

Acute respiratory distress syndrome (ARDS) can cause loss of alveolar-capillary membrane integrity and life-threatening immune responses. The underlying molecular mechanisms of ARDS remain unclear. N6-methyladenosine (m6A)-RNA modification plays an important part in many biological processes. However, it is not clear whether ARDS alters RNA methylation in lung tissue. We tried to investigate the changes of m6A-RNA methylation in lung tissues of lipopolysaccharide (LPS)-induced ARDS mice. Lung tissue samples were collected to detect the expression of m6A factors through hematoxylin and eosin (HE) staining, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), immunohistochemical analysis and western blot. The overall m6A levels in lung tissue of ARDS in mouse were detected by UPLC-UV-MS. HE staining showed that the degree of the inflammatory response was more severe in the LPS-3 h group. The mRNA expression of YTHDF1, YTHDC1 and IGFBP3 was remarkably up-regulated at, respectively, 6, 6 and 12 h after LPS treatment. The mRNA expression of METTL16, FTO, METTL3, KIAA1429, RBM15, ALKBH5, YTHDF2, YTHDF3, YTHDC2 and IGFBP2 was significantly down-regulated at 24 h after LPS treatment. The protein expression of METTL16 and FTO increased, YTHDC1, IGFBP3 YTHDF1 and YTHDF3 showed a down-regulation trend after LPS induction. Overall m6A-RNA methylation levels were significantly increased at 6 h after LPS induction. In ARDS mice, LPS-induced m6A methylation may be involved in the expression regulation of inflammatory factors and may play important roles in the occurrence and development of lung tissue. It is suggested that m6A modification may be a promising therapeutic target for ARDS.

Keywords: Acute respiratory distress syndrome; RNA methylation; inflammatory response; lung; m6A.

Graphical abstract

Figure 1.

HE staining in lung tissue of ARDS mouse. Left and right side represent the picture with magnification × 100 and magnification × 400, respectively. In NC group, the red arrow represents macrophages; in VC group, red and green arrow represents red blood cells and neutrophils, respectively; in the LPS-3 h group, green, yellow and blue arrow represents lymphocyte, neutrophils and foam cells, respectively; in the LPS-6 h group, green arrow represents neutrophils; in the LPS-12 h group, green, yellow and red represents lymphocyte, neutrophils and macrophages, respectively.

Figure 2.

The mRNA expression of methylated transferases and demethylases in the lung tissue samples of ARDS mouse in NC, VC, LPS-3 h, LPS-6 h, LPS-12 h and LPS-24 h groups. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 3.

The mRNA expression of recognition factors in lung tissue samples of ARDS mouse in NC, VC, LPS-3 h, LPS-6 h, LPS-12 h and LPS-24 h groups. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 4.

Overall m6A levels in lung tissue of ARDS mouse.

Figure 5.

The protein expression of METTL16, WTAP, FTO, YTHDF1, YTHDF3, YTHDC1 and IGFBP3 in the lung tissue samples of ARDS mouse in NC, VC, LPS-3 h, LPS-6 h, LPS-12 h and LPS-24 h groups.

Figure 6.

Immunohistochemical tests of FTO, METTL16, YTHDC1, YTHDF1 and YTHDF3 in right lung tissue of ARDS mouse. Magnification: 400 ×.

Figure 7.

Immunohistochemical quantitative results of FTO, METTL16, YTHDC1, YTHDF1 and YTHDF3 in right lung tissue of ARDS mouse. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.