N. Wu1, and S. Jin2; 1Department of Radiation Oncology, China-Japan Union Hospital of Jilin University, Changchun, China, 2School of Public Health, NHC Key Laboratory of Radiobiology, Jilin University, Changchun, China
Purpose/Objective(s): Radiotherapy is one of the most common treatments for cervical cancer, which can reduce local recurrence and prolong survival in patients with cervical cancer. Unfortunately, the resistance of cancer cells to radiotherapy are the main cause of treatment failure in patients with cervical cancer. tRNA-derived fragments (tRFs) are a class of newly defined functional small non-coding RNAs,which may play an important role in radioresistance. Previous studies have discovered that an increase in copper content is directly associated with the proliferation and metastasis of cancer cells. This research aimed to investigate the possible biological functions of tRFs associated with cuproptosis in the acquisition of radioresistance and reveal the potential targets for improving radiosensitivity in cervical cancer. Materials/
Methods: Human cervical squamous cell carcinoma (SiHa) radioresistant cell line (SiHa-RR) was established by X-ray irradiation. Total RNA was isolated and processed for RNA sequencing and tRFs expression analysis. Downstream target genes of differentially expressed tRFs were verified by bioinformatics analysis and label free whole proteomics quantitative sequencing. tRFs overexpression and knockdown models were established with synthesize mimics and inhibitors for transient transfection. The regulatory relationship between tRFs and target genes was explored by detecting ROS, copper metabolism indicators, and cuproptosis markers. The mechanism of tRFs in radioresistance of cervical cancer was elucidated by application of rescue experiment, RIP and Dual-Luc assays. Results: The parental SiHa cells were cultivated and exposed to a radiation dose of 6 Gy in 2-week cycles, for a total dosage of 30 Gy. Subsequently, the successful establishment of radioresistant strains SiHa-RR was confirmed usingcolony formation assays, cell proliferation and apoptosis analyses. qPCR verified the tRF-29-Q99P9P9NH525 with high differential expression between SiHa and SiHa-RR cells. The intersection of downstream target genes predicted by tRF-29-Q99P9P9NH525 and cuproptosis-related genes was focused on SLC31A1. RIP and Dual-Luc assays verified the regulatory relationship between tRF-29-Q99P9P9NH525 and SLC31A1. The high expression of SLC31A1 might be related to the low survival rate of patients with cervical cancer according to analysis of clinical relevance through GEPIA database. There were significant differences in the level of ROS and intracellular copper, contents of a-ketoglutaric acid and pyruvic acid, and changes of Fe-S cluster protein and mitochondrial oxidative phosphorylation complex between SiHa and SiHa-RR. Conclusion: Our results revealed a possible novel role of tRFs-mediated SLC31A1-dependent cuproptosis on conferring cervical cancer radioresistance. This study indicated that disrupting tRFs-SLC31A1 pathway maybe a promising potential strategy to overcome cervical squamous cell carcinoma radioresistance.
