Radiation Modifies Let-7 miRNA Binding to AGO2 Independent of Changes in Transcription to Influence Tumor Cell Radiosensitivity

Alterations in gene expression induced by ionizing radiation (IR) were commonly explained by transcriptional activation. However, the weak correlation between mRNA and protein levels following IR indicates the significant role for post-transcriptional regulation. microRNAs (miRNAs) bound to AGO2 play a significant role in post-transcriptional regulation; however, their role in radiation response is not clear. miRNA sequencing was performed to analyze the miRNAome of glioma cells. The effect of IR on Let-7 miRNAs and their association with AGO2 was examined using RT-qPCR and RNA immunoprecipitation (RIP) assays. Clonogenic assays were performed to measure radiosensitivity following Let-7a overexpression or knockdown. DNA damage (γH2AX foci) and cell cycle distribution were analyzed by immunofluorescence and flow cytometry. Let-7 miRNA regulatory networks were identified through target prediction and pathway enrichment analysis. AGO2-Let-7 binding decreased post IR, indicating impaired RISC loading. Let-7 overexpression increased radiosensitivity, DNA damage and G2/M cell cycle arrest in glioma and other cells (HeLa and MDA-MB-231). Let-7 miRNAs mainly targeted cell cycle and DNA damage response (DDR) pathways. Our study showed radiation impairs AGO2-miRNA binding, while restoring Let-7-AGO2 interaction enhances radiosensitivity by modulating DNA repair and cell cycle checkpoint activation. Targeting AGO2-miRNA dynamics represents a promising approach to improve radiotherapy outcomes.