FanJing Jing,1 YunYan Shi,1 Dong Jiang,2 Xiao Li,1 JiaLin Sun,1 Qie Guo1 

1Department of Clinical Pharmacy, The Affiliated Hospital of Qingdao University, Qingdao, Shandong, 266003, People’s Republic of China; 2Navy Qingdao Special Service Rehabilitation Center, Qingdao, Shandong, 266003, People’s Republic of China

Correspondence: Qie Guo, Email guoqie822a@qdu.edu.cn

Background: Sorafenib, an orally active potent tyrosine kinase inhibitor (TKI), represented a primary treatment in patients with advanced hepatocellular carcinoma (HCC). Unfortunately, sorafenib resistance was regarded as a huge obstacle for HCC treatment.
Methods: RNA-sequencing including circRNA Sequencing (circRNA-Seq) for circular RNAs (circRNAs), miRNA Sequencing (miRNA-Seq) for microRNAs (miRNAs), as well as mRNA Sequencing (mRNA-Seq) for mRNAs in sorafenib-resistant HCC cells vs sorafenib-sensitive HCC cells, were performed. Then, interaction correlation analysis between differentially expressed circRNAs and miRNAs and their target genes in Huh7/SOR and SMMC7721/SOR cells was exhibited. The “circRNA-miRNA-mRNA” network was constructed through the Cytoscape software application, Circular RNA Interactome and Targetscan prediction, RNA binding protein immunoprecipitation (RIP), RNA pull-down, and Dual luciferase reporter assay. Furthermore, mRNA-Seq, Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis for the downstream genes involved in the “circRNA-miRNA-mRNA” network was implemented. Iron detection assay, Lipid peroxidation quantification assay, ROS measurement assay, CCK-8 assay, and tumor challenge in vivo were used to determine the mechanisms promoting sorafenib resistance in HCC, where the “circRNA-miRNA-mRNA” network is clearly involved in.
Results: : circ_0001944 and circ_0078607 with upregulation and 2 downregulated expressed circRNAs (circ_0002874 and circ_0069981), as well as 11 upregulated miRNAs including miR-193a-5p, miR-197-3p, miR-27a-5p, miR-551b-5p, miR-335-3p, miR-767-3p, miR-767-5p, miR-92a-1-5p, miR-92a-3p, miR-3940-3p, and miR-664b-3p and 3 downregulated expressed miRNAs (miR-1292-5p, let-7c-5p, and miR-99a-5p) in sorafenib-resistant HCC cells were determined. Among these non-coding RNAs (ncRNAs), circ_0001944 and miR-1292-5p should not be drop out of sight; circ_0001944 has been proved to target miR-1292-5p to inhibit its expression in HCC. Subsequent findings also raise that miR-1292-5p directly targeted the 3’-noncoding region (3’-UTR) of Fibulin 2 (FBLN2) mRNA. Furthermore, circ_0001944 targets the miR-1292-5p/FBLN2 axis to inhibit cell ferroptosis in which the indicated regulators associated with iron overload and lipid peroxidation were “rearranged”. Most importantly, circ_0001944 advanced sorafenib resistance in HCC through mitigating ferroptosis, where the miR-1292-5p/FBLN2 axis cannot be left unrecognized.
Conclusion: : Circ_0001944 is a putative target for reversing sorafenib resistance in HCC. Our findings are expected to provide new targets and new directions for sorafenib sensitization in the treatment of HCC.

Keywords: Circular RNAs, Sorafenib, hepatocellular carcinoma, MicoRNAs