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Lina Zhao, MD, PhD
Xijing Hospital, Air Force Medical University
Xian, ShaanXi
Mechanistic of RBM3 regulating Rad51 alternative splicing mediated DNA damage repair promoting radioresistance of nasopharyngeal carcinoma
Purpose/Objective(s): Radioresistance remains the major obstacle to the failure of radiotherapy for nasopharyngeal carcinoma (NPC). RNA alternative splicing (AS) plays an important role in tumor radioresistance. Our previous study reported that AS factors RNA binding motif protein 3 (RBM3) promoted radioresistance of NPC. However, the molecular mechanism is not clear. The aim of this study was to elucidate the mechanism of DNA damage repair by RAD51 variable splicing regulate mediated.
Materials/
Methods: Splicing events regulated by RBM3 either in its wild type or knockdown status were evaluated by the multivariate analysis of transcript splicing after performing RNA-seq detection. KEGG analyze RBM3 regulation of AS-related signal pathways. RT-PCR was performed the change of these validate genes. 𝛾-H2AX foci in cell nuclei was detected by confocal microscopy. Furthermore, to explore the RBM3 upstream genes, RBM3 binding molecules were screened via RIP-seq. Subsequently, Colony formation was assessed to evaluate the radio-sensitization effect of the ATM inhibitor and Rad51-AS. Subcutaneous xenograft models were established to investigate the inhibitory effect of ATM inhibitor and Rad51-AS under X-ray radiation on tumor growth in vivo.
Results: Differential alternative splicing frequency showed RBM3 knockdown would change the alternative splicing of some genes in CNE1 cells in response to radiation and most of the genes present exon skipping (SE). KEGG analysis showed that SE were enriched in DNA damage repair signaling transduction. We further validated RBM3 knockdown causes a switch of the Rad51 splicing isoform from Rad51WT to Rad51-¦¤ex9 during the response to radiation. Subsequently, RIP-seq data revealed that the interaction of CTBP1 and RBM3 causes a switch of Rad51 alternative splicing isoforms from Rad51WT to Rad51-¦¤ex9. Moreover, we found that irradiation could extremely increase the expression of ATM, while ATM inhibitor induced CTBP1 phosphorylation and attenuates its association with RBM3. The experiments in vivo and vitro uncovered that ATM inhibitor and Rad51-¦¤ex9 enhanced the sensitivity to irradiation in NPC.
Conclusion: Irradiation-induced ATM phosphorylates CTBP1 and attenuates its association with RBM3, thus inhibiting Rad51 variable splicing and DNA damage repair to promote radioresistance of NPC.
Funding: This investigation was supported by the Foundation of Shaanxi Educational Committee of China (Grant No. 2021JM-245).