Exosomal hnRNPA1-delivered miR-27a-3p inhibits the radiation-induced bystander effect in cervical cancer.

Abstract

Purpose: The radiation-induced bystander effect (RIBE) refers to biological responses in non-irradiated cells exposed to signals from irradiated cells and may contribute to tumor control during radiotherapy. Periodic dose heterogeneity caused by the thread effect of helical tomotherapy (TOMO) may favor RIBE, but the underlying intercellular mechanisms remain unclear. This study investigated exosome-mediated bystander responses and their molecular and clinical relevance in cervical cancer.

Methods: Thread effect and uniform TOMO irradiation plans were established, and their effects on cervical cancer cell viability, DNA damage, apoptosis, and cell-cycle progression were compared. Cell-mixture, conditioned-medium transfer, exosome-depletion, and xenograft models were used to characterize RIBE. Proteomic analysis and exosomal miRNA sequencing identified candidate regulators. Gain- and loss-of-function experiments evaluated the effects of hnRNPA1 on exosomal miRNA levels and bystander tumor suppression. Dual-luciferase and rescue assays validated the miR-27a-3p/HIPK2 relationship. Survival associations were assessed in 218 TOMO-treated patients and an independent TCGA-CESC cohort.

Results: The thread plan produced growth-inhibitory effects consistent with a contribution of RIBE. Co‑culture with irradiated tumor cells significantly increased DNA damage and inhibited cell proliferation in non‑irradiated bystander cells. Exosome-depleted conditioned medium largely lost its growth-inhibitory activity, supporting exosomes as major mediators of this bystander response. Irradiation did not markedly alter exosome yield, size, morphology, or cellular uptake but reduced exosomal hnRNPA1 levels. hnRNPA1 overexpression attenuated exosome-mediated growth inhibition. Conversely, hnRNPA1 depletion decreased exosomal miR-27a-3p levels and enhanced tumor suppression. Target prediction and pathway enrichment prioritized miR-27a-3p for functional validation. miR-27a-3p directly targeted the HIPK2 3' untranslated region and promoted cervical cancer cell proliferation, while HIPK2 overexpression counteracted this effect. These findings support an hnRNPA1/miR-27a-3p/HIPK2 regulatory model in which radiation-induced loss of exosomal hnRNPA1 reduces exosomal miR-27a-3p, relieves HIPK2 repression, and enhances tumor-inhibitory bystander responses. High hnRNPA1 expression was associated with shorter OS and PFS in the TOMO-treated cohort and with adverse survival outcomes in the TCGA-CESC cohort. High HIPK2 expression was associated with longer OS and PFS in the TOMO-treated cohort.

Conclusion: Tumor cell-derived exosomes are major mediators of cervical cancer RIBE. Radiation-induced reduction of exosomal hnRNPA1 may enhance tumor-inhibitory bystander responses through the miR-27a-3p/HIPK2 axis. This pathway represents a candidate target for modulating RIBE, while the prognostic utility of hnRNPA1 warrants validation in independent radiotherapy cohorts.