Alternative End-Joining Activation by miR-21-5p Confers Radiation Resistance in Oral Squamous Cell Carcinoma [RNA-seq of SCC7 mice tumors]

Marked heterogeneity in radiosensitivity is a major challenge in the treatment of oral squamous cell carcinoma (OSCC), often resulting in poor outcomes for radioresistant tumors. The underlying molecular drivers of this resistance remain elusive. Here, we identify a novel mechanism in which miR-21-5p promotes radiation resistance by selectively activating the error-prone DNA double-strand break (DSB) repair pathway alternative end-joining (Alt-EJ). Integrative miRNA profiling, multi-omic analyses, and functional assays demonstrate that miR-21-5p upregulation coupled with suppression of its target gene signature increases Alt-EJ dependence for DSB repair. This Alt-EJ reliance enhances genomic instability, increases tumor mutational burden and microhomology-mediated indels, and confers pronounced radiation resistance in OSCC models, while correlating with poor radiotherapy response in patients. Importantly, pharmacological or genetic inhibition of Alt-EJ components, including PARP1 and POLQ, restores radiosensitivity in vitro and in vivo, with PARP1 inhibition specifically reversing miR-21-5p-mediated resistance in a syngeneic mouse model. Further RNA-seq analyses of these mice tumors confirmed that PARP inhibitor induced selective radiosensitization effects for miR-21-5p-overexpressing tumors were accompanied by a pronounced reduction in alt-EJ expression. Collectively, these findings establish the miR-21-5p/Alt-EJ axis as a key driver of radiation resistance in OSCC and support Alt-EJ targeting as a promising approach for precision radiosensitization.