GTP-binding protein DIRAS2 inhibits ferroptosis in oral leukoplakia via regulating iron metabolism

Oral leukoplakia (OLK) is the most prevalent and extensively studied oral potential malignant disorder (OPMD), with a high risk of progression to malignancy. Ferroptosis has been implicated as a key process in the progression of OLK, and iron metabolism significantly influences the ferroptosis process. DIRAS2, a member of the GTPase family within RAS proteins, is associated with ferroptosis, however, how DIRAS2 affects iron metabolism remains unknown.

Immunohistochemical staining was used to assess the expression of SLC7A11 and LCN2 in human normal oral mucosa and OLK tissues. Erastin-induced Leuk-1 cells were employed to investigate ferroptosis through CCK8 assays, MDA and Fe²⁺ measurements, and alterations in mitochondrial membrane potential and morphology. qRT-PCR and western blotting were performed to detect the expression of iron metabolism-related molecules, additionally, LCN2 inhibitor and small interfering RNAs were employed in this study. Dual-luciferase assays, co-ip, CHX chase assay and Duolink assay were conducted to elucidate the interactions among DIRAS2, HIF1α, and LCN2. The findings were validated in 4-Nitroquinoline-1-oxide (4NQO) -induced mouse OLK models too.

SLC7A11 and LCN2 expression was upregulated in OLK specimens compared to normal oral mucosa tissues. DIRAS2 knockdown in Erastin-induced Leuk-1 cells exacerbated mitochondrial morphology alteration, increased MDA and Fe²⁺ levels, and further upregulated LCN2 and NCOA4 expression while decreasing FTH1 protein expression. Knockdown of LCN2 expression by inhibitor or small interfering RNAs decreased Fe²⁺ levels and related protein expression in Leuk-1 cells. Compared with normal tongue tissues, the expression of ACSL4, SLC7A11, and the LCN2-NCOA4/FTH1 pathway was elevated in 4NQO-induced mouse tongue tissues. Conditional knockout of DIRAS2 resulted in decreased expression of SLC7A11 and FTH1, while increased expression of ACSL4, LCN2, and NCOA4. Furthermore, DIRAS2 regulated the expression of LCN2 via HIF1α mediation.

DIRAS2 may regulate iron metabolism in ferroptosis during the progression of OLK.