METTL3 Mediates Epithelial–Mesenchymal Transition by Modulating FOXO1 mRNA N6‐Methyladenosine‐Dependent YTHDF2 Binding: A Novel Mechanism of Radiation‐Induced Lung Injury

Abstract The biological roles of epithelial–mesenchymal transition (EMT) in the pathogenesis of radiation‐induced lung injury (RILI) have been widely demonstrated, but the mechanisms involved have been incompletely elucidated. N6‐methyladenosine (m6A) modification, the most abundant reversible methylation modification in eukaryotic mRNAs, plays vital roles in multiple biological processes. Whether and how m6A modification participates in ionizing radiation (IR)‐induced EMT and RILI remain unclear. Here, significantly increased m6A levels upon IR‐induced EMT are detected both in vivo and in vitro. Furthermore, upregulated methyltransferase‐like 3 (METTL3) expression and downregulated α‐ketoglutarate‐dependent dioxygenase AlkB homolog 5 (ALKBH5) expression are detected. In addition, blocking METTL3‐mediated m6A modification suppresses IR‐induced EMT both in vivo and in vitro. Mechanistically, forkhead box O1 (FOXO1) is identified as a key target of METTL3 by a methylated RNA immunoprecipitation (MeRIP) assay. FOXO1 expression is downregulated by METTL3‐mediated mRNA m6A modification in a YTH‐domain family 2 (YTHDF2)‐dependent manner, which subsequently activates the AKT and ERK signaling pathways. Overall, the present study shows that IR‐responsive METTL3 is involved in IR‐induced EMT, probably by activating the AKT and ERK signaling pathways via YTHDF2‐dependent FOXO1 m6A modification, which may be a novel mechanism involved in the occurrence and development of RILI.


Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
Total RNA was extracted from cells or tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA was reverse transcribed into cDNA using a PrimeScript RT kit (Takara, Shiga, Japan) according to the manufacturer's instructions. qRT-PCR was performed using a SYBR Green Master Mix Kit (Takara, Shiga, Japan) on an ABI ViiA 7 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). The sequences of the specific primer pairs are listed in Supplementary Table 1. Relative mRNA expression levels were calculated by the 2 -ΔΔ CT method with normalization to GAPDH or β-actin.

Immunofluorescence Assay
Cells were fixed with 4% formaldehyde and blocked with 3% bovine serum albumin (BSA) for 1 h at room temperature. The cells were incubated with antibodies against E-cadherin overnight at 4°C and then with a Cy3-conjugated secondary antibody (goat anti-mouse or goat anti-rabbit, 1:1000; Beyotime, Nantong, China) for 1 h at room temperature. 4',6-diamidino-2-phenylindole (DAPI) was used for nuclear staining, and images were acquired under an FV1200 confocal microscope (Olympus, Tokyo, Japan).
Luciferase activity was detected using a Dual-Luciferase Reporter Assay System.
Promoter activities were expressed as the ratio of Firefly luciferase activity to Renilla luciferase activity as previously described. [1] The following sequences were used:

shRNA Adeno-associated Virus (AAV) Production and Transfection
Four different mouse shRNAs for MELLT3 silencing were designed by Vigene Biosciences (Jinan, China). Two hundred microliters (5.0×10 12 vp/mL) of AAV9 containing four METTL3 shRNAs or scrambled shRNAs were injected via the tail vein. Two weeks later, the mice were exposed to radiation as described above. The shRNA sequences are shown in Supplementary Table 2.

Hematoxylin and Eosin (H&E) Staining
Lung tissues were fixed in 10% neutral-buffered formalin and embedded in paraffin.
Three-micrometer paraffin sections were deparaffinized and heat-treated with citrate buffer (pH = 6.0) for seven min following an epitope retrieval protocol. Lung sections were stained with H&E. [1]

Pulmonary Coefficient Calculation
The body weight and lung wet weight were measured at 1, 4, 8 and 16 weeks after irradiation. The ratio of the lung wet weight (g) to the body weight (kg) was used as the pulmonary coefficient.

Vascular Permeability Assessment
To analyze vascular leakage, Evans blue dye was injected into the tail vein. [2] The mice were euthanized 3 hours later and transcranially perfused with saline to remove the blood.
Evans blue dye was extracted from the pulmonary interstitium after incubation in formamide at 65°C for 24 h. The concentration of Evans blue dye was measured by absorbance at 620 nm and normalized to the lung tissue weight.

Immunohistochemistry (IHC)
Lung tissues were fixed in 10% neutral-buffered formalin and embedded in paraffin.
Three-micrometer paraffin sections were deparaffinized and heat treated with citrate buffer (pH = 6.0) for 7 min following an epitope retrieval protocol. Three-micrometer paraffin sections were incubated with antibodies against E-cadherin (1:150), N-cadherin Counterstaining was performed with hematoxylin.

Enzyme-Linked Immunosorbent Assay (ELISA)
After the body weight of the mice was measured, 1 mL of blood was obtained via cardiac puncture. The serum was collected and stored at -80°C for later analysis by ELISA. The serum levels of IL-1β, IL-6, TGF-β and TNF-α were determined using ELISA kits according to the manufacturer's instructions. The optical absorbance of the samples was measured at 450 nm.
Supplementary Figure S1. The m 6 A RNA modification mediates IR-induced EMT during RILI in MLE-12 cells.