Objective  To investigate the changes in the expression of N6-methyladenosine (m6A) demethylase fat mass and obesity-associated protein (FTO) and its downstream target gene, transforming growth factor beta receptor 1 (TGFBR1), during the process of radiation-induced damage in lung fibroblasts, as well as their regulatory mechanisms, in order to provide a theoretical basis for the development of targeted drugs for radiation-induced pulmonary fibrosis (RIPF).

Methods  Human pulmonary fibroblasts (HFL-1) were exposed to a single 6 Gy X-ray irradiation (dose rate: 2 Gy/min), and the total m⁶A methylation level was quantitatively detected using a colorimetric method. Mice were subjected to whole-thoracic irradiation at a dose of 20 Gy (with a dose rate of 2.0 Gy/min) to establish an RIPF (radiation-induced pulmonary fibrosis) mouse model, and primary lung fibroblasts were extracted from the mice. HFL-1 cells with stable knockdown of FTO were established using lentiviral infection. Downstream target genes of FTO were predicted through the m⁶A2 Target website. The expression changes of messenger RNA (mRNA) of FTO, TGFBR1, and fibrosis markers collagen type Ⅰ alpha 1 chain (COL1A1) and fibronectin 1 (FN1) in irradiated HFL-1 cells and primary lung fibroblasts were detected using real-time quantitative PCR (qRT-PCR). The expression changes of FTO, transforming growth factor-beta receptor 1 (TGFBR1), and fibrosis marker proteins Fibronectin and Collagen1 in irradiated HFL-1 cells were detected using Western Blots. The m⁶A enrichment of TGFBR1 mRNA in irradiated HFL-1 cells was detected using MeRIP-qPCR.

Results  Compared to the control group, the m⁶A level of total RNA in the irradiation group was reduced (t=20.970, P<0.01). In comparison with the control group, the expression of FTO in the irradiation group was elevated (t=14.370, 9.247, 8.744, 18.840, 12.640, 10.010, all P<0.01), and the expression of fibrosis markers (Collagen1, Fibronectin) was also increased (t=3.370, 3.075, both P<0.05). Additionally, compared to the control group, the expression of TGFBR1 in the irradiation group was upregulated (t=2.995, P<0.05). The mRNA expression levels of FTO, COL1A1, and FN1 in primary lung fibroblasts of RIPF mice at 8 weeks and 16 weeks post-irradiation were elevated in the irradiation group compared to the control group (t=13.310, 4.095, 4.188, 5.951, 3.335, 8.694, all P<0.05). Furthermore, the m6A enrichment of TGFBR1 mRNA in the irradiation group was significantly decreased compared to the control group (t=3.536, P<0.05). Knockdown of FTO could inhibit the irradiation-induced upregulation of TGFBR1 and fibrosis-related molecules.

Conclusions  Radiation caninduce up-regulation of demethylase FTO expression in lung fibroblasts that enhances TGFBR1 expression by inhibiting m⁶A modification, which in turn promotes RIPF progression.