Targeting alternative end-joining overcomes miR-21-5p -mediated radioresistance in oral squamous cell carcinoma

Radiation resistance remains a major barrier to effective radiotherapy in oral squamous cell carcinoma (OSCC), yet its molecular basis is incompletely defined. Here, we uncover activation of the alternative end‑joining (alt‑EJ) DNA repair pathway as a key driver of miR‑21‑5p-mediated radioresistance in OSCC. Comprehensive miRNA profiling of radioresistant OSCC clones revealed distinct expression signatures marked by robust upregulation of miR‑21‑5p and miR‑486‑5p, coupled with reduced miR‑320b and miR‑1248. Functional assays, including clonogenic survival and syngeneic mouse tumor models, demonstrated that enforced expression of miR‑21‑5p markedly enhanced radioresistance, accompanied by sustained DNA damage response signaling and improved double‑strand break (DSB) repair capacity. Transcriptomic and genomic analyses of the TCGA‑HNSC cohort revealed that high miR‑21‑5p expression correlates with suppression of canonical DNA repair targets, poor radiotherapy outcomes, elevated tumor mutational burden (TMB), and an increased frequency of microhomology‑mediated insertions/deletions, consistent with heightened alt‑EJ activity. Cross‑validation in independent OSCC datasets and local tumor panels confirmed that miR‑21‑5p-overexpressing tumors exhibit strong activation of alt‑EJ gene programs, including upregulation of PARP1, POLQ, and associated accessory factors. Analysis of diverse cancer cell line databases further established that miR‑21‑5p activity positively correlates with alt‑EJ gene expression independent of microenvironmental influence. Mechanistically, miR‑21‑5p overexpression shifted DSB repair dependence from homologous recombination and classical non‑homologous end‑joining toward alt‑EJ. Pharmacologic or genetic suppression of alt‑EJ components (PARP1 or POLQ) disrupted DSB resolution and effectively restored radiosensitivity in miR‑21‑5p-high OSCC cells. Notably, radiosensitization by PARP inhibition was abrogated in POLQ‑deficient cells, confirming a POLQ‑dependent mechanism. In vivo, miR‑21‑5p-driven OSCC tumors exhibited enhanced expression of alt‑EJ signatures, increased radioresistance, and high TMB. Combination treatment with irradiation and PARP blockade suppressed tumor growth, diminished alt‑EJ gene expression, and prolonged survival in a syngeneic mouse model. Collectively, these findings establish a novel miR‑21‑5p/alt‑EJ signaling axis as a central determinant of radioresistance in OSCC. Targeting alt‑EJ represents a promising therapeutic strategy to overcome miR‑21‑5p-mediated treatment failure and improve radiotherapy efficacy in head and neck cancers.