Objective To investigate the changes in the expression levels 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 injury 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 lung fibroblasts-1 (HFL-1) were divided into 4 groups according to the irradiation dose, namely the control group (0 Gy) and the irradiation groups with 2 Gy, 4 Gy, and 6 Gy respectively, and the irradiation doses were selected. HFL-1 cells were divided into the control group (0 Gy) and the groups at 24 h, 48 h, and 72 h after irradiation according to the time after irradiation, and the time points after irradiation were selected. The subsequent experiments groups were divided into control group and irradiation group. The subsequent experiments were grouped into control and irradiated groups based on the selected radiation dose and post-irradiation time. The m⁶A methylation content in total RNA was quantitatively detected. Mice were randomly divided into six groups using simple random sampling: 4-week, 8-week, and 16-week post-irradiation groups and their corresponding control groups (5 mice per group). The irradiation groups received 20 Gy whole-thorax irradiation at a dose rate of 2.0 Gy/min to establish the RIPF mouse model. Primary lung fibroblasts were then extracted from the mice. HFL-1 cells with stable FTO knockdown were established through lentiviral infection. The downstream target genes of FTO were predicted through the m⁶A2 Target website. The changes in the expression levels of the messenger RNA (mRNA) of FTO, TGFBR1, and the fibrosis-related molecules collagen type Ⅰ alpha 1 chain (COL1A1) and fibronectin 1 (FN1) in irradiated HFL-1 cells and primary lung fibroblasts were detected by using real-time quantitative polymerase chain reaction (qRT-PCR). The changes in the protein expression levels of FTO, TGFBR1, and the fibrosis-related molecules proteins Fibronectin and Collagen1 in irradiated HFL-1 cells were detected by using Western blot analysis. The m⁶A enrichment of TGFBR1 mRNA in irradiated HFL-1 cells was detected through methylated RNA immunoprecipitation-qRT-PCR (MeRIP-qRT-PCR). The independent samples t-test (with equal variances) was used to compared the measurement data that conform to the normal distribution between groups.

Results Compared with the control group, the m⁶A content in the total RNA of HFL-1 cells in the irradiation group decreased (t=20.970, P<0.01). Compared with the control group, the FTO mRNA and protein expression in HFL-1 cells of the 2, 4, and 6 Gy irradiation groups and the groups at 24, 48, and 72 h after irradiation increased (t=14.370, 9.247, 8.744, 18.840, 12.640, 10.010, 7.510, 10.010, 4.508, 25.080, 11.400; all P<0.05). Compared with the control group, the mRNA and protein expression of TGFBR1, fibrosis-related molecules (COL1A1, FN1) in the irradiation group increased (t=2.995, 3.370, 3.075, 20.560, 9.339, 9.478; all P<0.05). At 8 and 16 weeks post-irradiation, the mRNA expression levels of FTO, COL1A1, and FN1 in the primary lung fibroblasts of RIPF mice in the irradiation group were elevated compared with those in the control group (t=13.310, 4.095, 4.188, 5.951, 3.335, 8.694; all P<0.05). Compared with the control group, the m⁶A enrichment of TGFBR1 mRNA in HFL-1 cells of the irradiation group was significantly decreased (t=3.536, P<0.05). Compared with the irradiated group, knockdown of FTO suppressed the mRNA and protein expression of TGFBR1 and fibrosis-related molecules (COL1A1, FN1) induced by irradiation to be elevated (t=8.424, 10.990, 9.663, 5.580; all P<0.05).

Conclusions Radiation can induce the upregulation of demethylase FTO expression in lung fibroblasts, enhancing TGFBR1 expression by inhibiting m⁶A modification. This inhibition, in turn, promotes RIPF progression.