CLTRN, Regulated by NRF1/RAN/DLD Protein Complex, Enhances Radiation Sensitivity of Hepatocellular Carcinoma Cells Through Ferroptosis Pathway

• DOI: 10.1016/j.ijrobp.2020.12.062
• Publication Date: 2021-01-27T12:54:10Z
• Authors: Yin Yuan; Wen Cao; Hongbing Zhou; Haixin Qian; Honggang Wang
• Keywords: 0301 basic medicine; 03 medical and health sciences; Carcinoma, Hepatocellular; Membrane Glycoproteins; ran GTP-Binding Protein; Nuclear Respiratory Factor 1; Liver Neoplasms; Ferroptosis; Humans; Hep G2 Cells; Radiation Tolerance; 3. Good health

Abstract Background Radiotherapy is a viable treatment option for patients with unresectable hepatocellular carcinoma (HCC). However, radiation resistance is an issue that needs to be addressed. In this context, cumulative evidence supports the functional roles of a variety of RNA or proteins in radioresistance, and suggests that the modulation of their expression may constitute a novel radiosensitization approach. Here, we investigated the ability of collectrin (CLTRN) to enhance the radiosensitivity in HCC patients for the first time. Methods Transcriptome sequencing technology (RNA-seq technology) was used to analyze the transcription-level changes in the genes from the HepG2 cells before and after X-ray irradiation. Combining the results with the HCC tissue RNA-seq data, we determined the ultimate target gene through bioinformatics analysis and cellular verification. A series of cellular and molecular biology techniques were applied in vitro and in vivo to confirm whether CLTRN can enhance radiosensitivity in HCC cells. Subsequently, the downstream action mechanism, the upstream transcription factor, and the interaction proteins of CLTRN were determined. Results First, we confirmed the association between CLTRN and radiosensitivity. We observed that CLTRN overexpression led to a significant reduction in the proliferation, migration, and invasion potential of X-ray-irradiated HCC cells, whereas no observable effect was exerted on cell cycle and apoptosis. The same results were observed in nude mice in vivo. Investigation of the gene regulatory mechanism revealed that the genes analyzed at transcriptome level after CLTRN overexpression were mostly enriched in the glutathione metabolic pathway. As glutathione metabolism forms a vital link in ferroptosis, we surmised that CLTRN is associated with ferroptosis. This was confirmed through the detection of cellular iron, ROS level determination, transmission electron microscopy, and monitoring of ferroptosis-related protein indicators. Lastly, we investigated whether nuclear respiratory factor 1 (NRF1) is the upstream transcription factor of CLTRN, and whether dihydrolipoamide dehydrogenase (DLD) and members of the RAS oncogene family (RAN) are its interacting proteins. Conclusion Our results indicate that CLTRN is a vital regulator of radiation sensitivity and could serve as a novel therapeutic target or prognostic marker in HCC treatment.