ESTRO meets Asia 2024 - Abstract Book

S255

Interdisciplinary – Radiobiology

ESTRO meets Asia 2024

Radiotherapy plays a crucial role in the treatment of cervical carcinoma which is one of the most common cancers diagnosed in women worldwide. Unfortunately, nearly 50 % of all patients with cervical carcinoma do not respond to standard treatment due to tumor radioresistance. Acquired radioresistance during irradiation has been considered as one of the most important reasons for local tumor recurrence or treatment failure. tRNA-derived fragments (tRFs) represent a new class of small non-coding RNAs, which are present in a broad range of species and have been reported to involve in various biological processes. The aim of the present study was to investigate the possible biological role of tRFs in the acquisition of radioresistance and reveal the potential targets for improving radiosensitivity in cervical squamous cell carcinoma.

Material/Methods:

Human parental cervical squamous cell carcinoma cell line SiHa was irradiated 5 fractions with a single dose of 6 Gy (dose rate of 1.0 Gy/min) and a total dosage of 30 Gy with X-rays to establish radioresistant cell line SiHa-R. Radioresistance of SiHa-R was validated and identified by cell clone formation and immunofluorescence assay. Total RNA was isolated from SiHa and SiHa-R, and processed for RNA sequencing and tRFs expression analysis. Differential expression of proteins between SiHa and SiHa-R was detected through label free whole proteomics quantitative sequencing. The mRNA and protein expression levels of ferroptosis related genes in SiHa and SiHa-R were detected by quantitative real-time PCR and western blot methods.

Results:

Compared with the parental SiHa cells, SiHa-R showed an increase in colony formation, survival fraction, and proliferation ability after being irradiated with different doses of radiation. The D0 and Dq values confirmed the difference in radiosensitivity between SiHa and SiHa-R. After irradiation with 6 Gy, the number of γH2AX foci in SiHa was more than that of SiHa-R, and the number of 53BP1 foci was less than that of SiHa-R. The results demonstrated that the degree of DNA damage of SiHa-R was weaker than that of SiHa, and the ability of damage repair was stronger than that of the parental cell line, which confirmed that the radioresistant model was successfully established. The RNA sequencing results identified that significant upregulation of 40 tRFs and downregulation of 35 tRFs in SiHa-R were observed compared with SiHa among the detected candidate 2207 tRFs genes. It suggested that tRFs may play a role in the transformation of cellular radioresistance. In addition, GO enrichment analysis indicated that the functions of differentially expressed proteins in the two cell lines were closely related to biological processes such as redox activity and metal ion regulation. And protein interaction network showed that FTH1 and FTL were the key position genes participated in the regulation of iron metabolism after X-ray irradiation. Quantitative real-time PCR and western blot further verified that, consistent with the sequencing results, ferroptosis-related genes including FTH1, NRF2, SQSTM1 and NQO1 were significantly up regulated in radioresistant cell line.

Conclusion:

The experimental findings in the present study revealed noteworthy functional profile of differentially expressed tRFs in radioresistant cervical squamous cell carcinoma cells, indicating possible involvement of these dysregulated tRFs in the regulation of the process of radioresistance mediated by ferroptosis.

Keywords: radioresistance, tRFs, ferroptosis