Unraveling Sorafenib Resistance in Hepatocellular Carcinoma: The Role of circ_0001944 and miR-1292-5p in Ferroptosis Regulation

Introduction

Hepatocellular carcinoma (HCC) is the fourth leading cause of death globally. Many patients are diagnosed in advanced stages due to subtle symptoms. Sorafenib is a first-line treatment for advanced HCC but has limited efficacy; only 35–43% of patients respond, and many relapse within six months. The average survival benefit after treatment for those resistant to sorafenib is only 2.8 months. Understanding the mechanism behind sorafenib resistance is critical for improving treatments.

Ferroptosis is a form of regulated cell death characterized by iron accumulation, leading to increased reactive oxygen species (ROS) and lipid peroxidation. Changes in ferritin heavy chain 1 (FTH1), Divalent metal-ion transporter-1 (DMT1), and Transferrin Receptor 1 (TFR1) contribute to iron overload and ROS production. Conversely, Quinone oxidoreductase 1 (NQO1) helps eliminate lipid peroxides and ROS, providing resistance to ferroptosis. Research indicates that ferroptosis resistance is linked to chemotherapy resistance in various cancers.

Non-coding RNAs (ncRNAs), particularly circular RNAs (circRNAs) and microRNAs (miRNAs), play roles in cancer progression. circRNAs possess a unique circular structure, making them resistant to degradation. They can act as miRNA “sponges,” reducing miRNA activity and increasing target mRNA expression. The roles of circRNAs in cancer have been well-documented, with examples including Circ-E-Cad in glioblastoma and Circ-Foxo3 in breast cancer. While recent studies have improved understanding of circRNAs in HCC, the specific mechanisms by which they influence sorafenib resistance through ferroptosis remain unclear.

In this study, 70 upregulated and 45 downregulated circRNAs were found in sorafenib-resistant SMMC7721/SOR cells, while 20 upregulated and 13 downregulated circRNAs were identified in Huh7/SOR cells. Additionally, 91 upregulated and 90 downregulated miRNAs in SMMC7721/SOR cells and 50 upregulated and 38 downregulated miRNAs in Huh7/SOR cells were determined. Notably, circ_0001944 and circ_0078607 were upregulated, while circ_0002874 and circ_0069981 were downregulated in sorafenib-resistant HCC cells.

Circ_0001944 specifically targets miR-1292-5p. The “circ_0001944-miR-1292-5p-mRNA” network was mapped to identify target mRNAs regulated by this pair. FBLN2 was confirmed as a target of miR-1292-5p, which negatively regulates ferroptosis by influencing the expression of DMT1 and TFR1.

Materials and Methods

Cell lines SMMC7721 and Huh7 were sourced from the National Collection of Authenticated Cell Cultures. Sorafenib and Ferrostatin-1 were purchased for treatment. Various plasmids were obtained for transfections and gene analyses. RNA integrity was assessed, followed by library preparation for RNA sequencing. Clean data were mapped to a reference genome, with circRNA, miRNA, and mRNA identified using specific software. Differential expression analysis was conducted to identify significant changes.

Results

RNA sequencing revealed distinct patterns of differentially expressed non-coding RNAs in sorafenib-resistant cells compared to their sensitive counterparts. Key circRNAs and miRNAs emerged as central players in promoting sorafenib resistance. Specifically, circ_0001944 demonstrated a direct interaction with miR-1292-5p, leading to downregulation of FBLN2, which is instrumental in ferroptosis regulation.

Discussion

Sorafenib resistance in HCC remains a challenge. The study highlights circ_0001944’s role in targeting the miR-1292-5p/FBLN2 axis, inhibiting ferroptosis, and promoting drug resistance. This offers insights into new therapeutic targets that may enhance treatment efficacy in HCC patients. Further clinical investigations are warranted to validate these findings and their implications for reversing drug resistance.

Conclusion

Circ_0001944 is crucial in HCC resistance to sorafenib by impeding ferroptosis through the miR-1292-5p/FBLN2 pathway. This study lays the groundwork for future research aimed at overcoming drug resistance in hepatocellular carcinoma.