[Study on mechanism underlying the role of HLA-DRA in promoting radiation-induced sinusitis]

Objective: To verify whether the HLA-DRA gene regulates the radiosensitivity of nasal mucosal epithelial cells and to elucidate its underlying mechanism in the pathogenesis of radiation-induced sinusitis (RNS). Methods: Sinonasal mucosal specimens were collected from five patients with RNS following radiotherapy and five without RNS (control group). HLA-DRA expression levels were assessed by real-time reverse transcription quantitative PCR (RT-qPCR), western blotting (WB), and immunohistochemistry (IHC). HLA-DRA knockout (KO) and overexpression (OE) nasal mucosal epithelial cell models were generated using CRISPR/Cas9 technology. Cells were divided into three groups: control (wild-type), HLA-DRA KO, and HLA-DRA OE (overexpression/rescue). Cells in all groups were subjected to X-ray irradiation at identical doses. Cell proliferation and apoptosis following irradiation were assessed using colony formation assay, CCK-8 assay, and flow cytometry. The expression of inflammatory factors and the activation of the JAK-STAT signaling pathway were analyzed by RT-qPCR, enzyme-linked immunosorbent assay (ELISA), and WB. Statistical analysis was conducted using SPSS 23.0 software. Results: Both RT-qPCR and WB revealed significantly elevated HLA-DRA expression at both mRNA and protein levels in sinonasal mucosal tissues from patients with RNS compared to non-RNS controls. Immunohistochemistry demonstrated diffuse HLA-DRA immunoreactivity throughout the full thickness of the epithelium, accompanied by marked inflammatory cell infiltration. HLA-DRA knockout and overexpression nasal mucosal epithelial cell models were successfully established. Colony formation assay revealed significantly suppressed clonogenic survival in the OE group compared to the KO group (130.46±3.74 vs 393.18±10.13, P<0.01), with the OE group showing the lowest colony counts overall. CCK-8 assay demonstrated significantly enhanced proliferative capacity in HLA-DRA KO cells following irradiation compared to control cells, whereas, OE cells exhibited markedly suppressed proliferation. Flow cytometric analysis revealed a significantly higher apoptotic rate in the OE group compared to controls (80.41% vs 77.82%, P<0.05), whereas, the KO group exhibited a dramatic reduction in the apoptotic rate (0.49% vs 77.82%, P<0.001). RT-qPCR analysis demonstrated significantly decreased mRNA expression of interferon-γ (IFN-γ) and interleukin-6 (IL-6) in the KO group compared to controls (IFN-γ: 0.25±0.06 vs 0.54±0.07; IL-6: 0.13±0.05 vs 0.31±0.06; both P<0.01). Conversely, the OE group exhibited upregulated expression of both cytokines (IFN-γ: 0.48±0.08 vs 0.25±0.06; IL-6: 0.39±0.05 vs 0.13±0.05; both P<0.05). ELISA confirmed significantly reduced secretion of IFN-γ and IL-6 in the culture supernatant of KO cells compared to controls (IFN-γ: 12.53±1.04 vs 20.62±2.09; IL-6: 11.31±2.52 vs 30.12±4.53; both P<0.05). In contrast, OE cells showed increased cytokine secretion. WB further revealed that combined IL-6 and IFN-γ stimulation markedly enhanced signal transducer and activator of transcription (STAT) phosphorylation compared to untreated controls. Conclusions: HLA-DRA regulates the radiosensitivity of nasal mucosal epithelial cells by modulating inflammatory cytokine expression and activating the JAK-STAT signaling pathway, thereby contributing to the pathogenesis of RNS.