Hsa_circ_0002301 inhibits ferroptosis in gastric cancer by encoding the de novo protein HECTD1-463aa.

Background: CircRNAs are closely related to ferroptosis in gastric cancer cells; however, the mechanism by which circRNAs regulate ferroptosis in gastric carcinogenesis remains unknown. CircRNA-encoded novel peptides are functional products translated from the open reading frames (ORFs) within circular RNAs, demonstrating that circRNAs not only serve as non-coding regulators but also have the capacity to encode biologically active peptides. Compared with noncancerous cells, cancer cells have greater iron requirements, and ferroptosis occurs in response to radiotherapy, chemotherapy, and immunotherapy; therefore, ferroptosis activation may be a potential strategy to overcome the shortcomings of conventional cancer therapy.

Methods: A mouse model of ferroptosis in gastric cancer was constructed, and a bioinformatics analysis was performed to analyze and characterize the circRNAs involved in ferroptosis in gastric cancer. The inhibitory effect of hsa_circ_0002301 on ferroptosis in tumors was confirmed both in vitro and in vivo. The presence and expression of HECTD1-463aa were verified using mass spectrometry, protein blotting, and immunofluorescence staining. The molecular mechanism of hsa_circ_0002301 was investigated using mass spectrometry and immunoprecipitation.

Results: We designed and synthesized antibodies specific for the small protein HECTD1-463aa encoded by hsa_circ_0002301 to verify its presence and purified HECTD1-463aa by constructing hsa_circ_0002301 overexpression vectors with FLAG tags and used liquid chromatography-tandem mass spectrometry (LC‒MS/MS) to detect the characterized peptides. In addition, HECTD1 binding to HECTD1-463aa was identified by immunoprecipitation (Co-IP) and mass spectrometry. We found that HECTD1-463aa inhibited HECTD1-mediated GPX4 ubiquitination by binding to HECTD1, an important regulator of cell death in ferroptotic cancer cells.

Conclusions: hsa_circ_0002301 competitively inhibits the degradation of the GPX4 protein by HECTD1 through the encoded proteins HECTD1-463aa and HECTD1 to affect the ferroptosis level in gastric cancer cells.