MiR-26b-5p mediates radioresistance and immunosuppression via targeting PRKCD in non-small cell lung cancer.

Purpose: Overcoming miRNA-mediated radioresistance and enhancing its synergy with immunotherapy remained significant challenges.

Methods: A total of 23 patients with locally advanced non-small cell lung cancer (NSCLC) undergoing thoracic radiotherapy from a single center were enrolled. Pre-radiotherapy blood samples were collected and analyzed using real time qPCR array to detect miRNA expression profiles, identifying differential miRNAs between responders and non-responders. In vitro experiments further assessed the impact of radiotherapy on significant differential miRNAs. Targeted immune genes of miRNAs were predicted through bioinformatics websites and validated by cellular experiments. Using TCGA and GEO datasets, the association between immune gene of interest and survival outcomes and immune infiltration were investigated. In vivo experiment was further performed to investigate the relationship between dendritic cell (DC) expression and miR-26b-5p following radiotherapy.

Results: Using pre-radiotherapy blood samples from 23 NSCLC patients, 22 differentially expressed miRNAs were identified between responders and non-responders. Among them, miR-26b-5p exhibited significant differential expression, suggesting its role as a potential radioresistant molecule. The dual-luciferase assay confirmed miR-26b-5p targeted PRKCD, an immune-related gene. After continuous three days of 2-Gy irradiation, the expression of miR-26b-5p decreased significantly, while the expression of PRKCD increased. The effect of radiotherapy on PRCKD expression were further validated in clinical samples, which demonstrated elevated PRCKD expression after thoracic radiotherapy. Bioinformatic analysis using TCGA and GEO datasets revealed that a higher PRKCD expression was correlated with better survival outcomes, increased immune cell infiltration, and better outcomes. Further in vivo experiments showed that, after radiotherapy, the inhibition of miR-26b-5p showed a significantly higher proportion of DCs than the controls, along with increased expression of CD80, CD86, and TNFSF4.

Conclusion: miR-26b-5p and PRKCD modulates dual resistance of both radiotherapy and immunotherapy in NSCLC. These insights demonstrate that downregulating miR-26b-5p could offer a promising therapeutic strategy to enhance radiosensitivity and immune responses.