Objective To investigate the molecular mechanisms of early mitochondrial stress in keratinocytes induced by ionizing radiation (IR), focusing on the key role of ROMO1 in radiation-induced skin injury (RISI).

Methods HaCaT keratinocytes were used to assess cellular status, mitochondrial dysfunction and oxidative stress at various time points after different irradiation doses. Mitochondrial proteomics at 24 hours post-20 Gy irradiation identified ROMO1 as the protein with the greatest reduction in expression. ROMO1-overexpressing HaCaT cells and skin-specific Romo1 knockout (Romo1-sKO) mice were established to investigate its functional mechanisms. ROMO1 expression and localization were analyzed by Western blot and immunofluorescence; flow cytometry measured ROS, mtROS, and mitochondrial membrane potential in ROMO1-overexpressing cells, while CCK-8, LDH release, and colony formation assays evaluated viability and proliferation. The role of Romo1 in the progression and repair of radiation-induced skin injury was further examined in Romo1-sKO mice.

Results At 24 hours post-20 Gy irradiation, HaCaT cells exhibited inhibited proliferation (P < 0.05) and mitochondrial dysfunction (P < 0.0001). Mitochondrial proteomics identified 43 differentially expressed proteins (26 upregulated, 17 downregulated), with enrichment analysis indicating their involvement in oxidative phosphorylation, mitochondrial assembly and stress response. Among these, reactive oxygen species modulator 1 (ROMO1) was identified as the most significantly downregulated key protein in the early radiation response (P < 0.01, FDR = 0.03). In vitro, ROMO1 overexpression increased mtROS levels and mitochondrial membrane potential (P < 0.0001) but suppressed cell viability and promoted reproductive death (P < 0.05) after radiation. In vivo, Romo1-sKO mice showed accelerated wound healing, with a reduction in skin injury score by approximately 1 point, along with enhanced tissue regeneration at 25 days post-irradiation compared to control mice.

Conclusion The mitochondrial protein ROMO1 is a key regulator of the early keratinocyte stress response to radiation. It impedes the repair of radiation-induced skin injury by suppressing cell proliferation via modulation of mitochondrial function. Targeted inhibition of ROMO1 may represent a novel therapeutic strategy to promote healing and prevent chronic progression of radiation-induced skin injury.