3161 - HnRNPA1 Regulates MARF1 Alternative Splicing by Interacting with PTBP1 to Enhance the Radioresistance of Oral Squamous Cell Carcinoma
Presenter(s)
F. Xia1,2, J. Yang1,2, L. Zhang1,2, L. Zhou1, J. Sui2,3, and Y. Wang1,2; 1Department of Radiation Oncology, Chongqing University Cancer Hospital, Chongqing, China, Chongqing, China, 2College of Medicine, Chongqing University, Chongqing, China, 3Department of Radiation Oncology, Chongqing University Cancer Hospital, Chongqing,, Chongqing, China
Purpose/Objective(s): Radiotherapy is a curative arsenal for oral squamous cell carcinoma (OSCC), but radioresistance seriously compromises its effectiveness. Alternative splicing (AS) is a tightly regulated post-transcriptional cellular process that increases protein diversity in eukaryotes. Statistically, over 95% of human genes undergo AS, and cancers exhibit up to 30% more AS events than normal tissues, contributing to diverse gene variants involved in tumorigenesis. Nonetheless, the role of splicing factors in radioresistance remains unexplored, mainly in OSCC.
Materials/Methods: We used RNA sequencing(RNA-seq) to detect the changes in alternative splicing factors in the control and radiation groups' OSCC tumor tissues (15Gy, single irradiation). We identified heterogeneous nuclear ribonucleoprotein A1 (hnRNAP1) as the target molecule. By integrating public databases and clinical records from our hospital, we examined the correlation between the expression level of hnRNPA1 and the clinicopathological features and prognosis of OSCC. Following this, we evaluated the impact of hnRNPA1 on OSCC radiation resistance through clonogenic and comet assays. We further explored how hnRNPA1 regulates alternative splicing to influence OSCC radiation resistance by combining alternative splicing analysis from RNA-seq data with immunoprecipitation experiments and clonogenic assays.
Results: Compared to the control group, there are 11 splicing factors significantly elevated in the OSCC tumor tissues after radiotherapy. Among those genes,hnRNAP1 is upregulated substantially in OSCC and is positively associated with advanced clinicopathological features and poor prognosis. Gain- and loss-of-function experiments demonstrate that hnRNPA1 markedly reinforces genomic DNA stability to desensitize OSCC cells to irradiation in vitro and in vivo. RNA-seq results combined with immunoprecipitation experiments and gain- and loss-of-function experiments revealed that hnRNPA1 interacts with polypyrimidine tract binding protein 1(PTBP1), a member of the same hnRNP family, to jointly regulate the splicing of pre-mRNA of DNA repair-related protein-Meiosis arrest female protein 1(MARF1) by promoting the retention of the 8th exon in the pre-mRNA. Furthermore, functional gain-of-function and loss-of-function experiments demonstrated that MARF1-L significantly enhances DNA repair, making OSCC cells more resistant to irradiation.
Conclusion: Our research demonstrates that hnRNPA1 interacts with the PTBP1 and regulates exon eight splicing of MARF1 from MARF1-S to MARF1-L. Furthermore, the upregulation of MARF1-L promoted DNA repair in OSCC cells, enhancing radiotherapy resistance in OSCC. The findings highlight the role of splicing factors in inducing aberrant splicing events in response to radiotherapy and suggest that hnRNPA1 and MARF1-L may potentially play a role in reversing OSCC radiotherapy resistance.