Development of Regorafenib-Resistant HCC Cells

The sh-circDCAF8 vector was constructed by designing and synthesizing shRNA that targets human circDCAF8. The Lv-circDCAF8 vector was created by constructing lentiviral vectors that also include human circDCAF8. The lentiviral vectors mentioned above were designed by GenePharma (Shanghai, China). Target cells were transfected using lentiviral vectors, and the stable transfected cells were chosen using puromycin and confirmed with qRT-PCR. The shRNA target sequences are listed in Table S1, and the full sequence of circDCAF8 is listed in Table S2. The mimic and inhibitor of miR-217 and their negative controls were obtained from GenePharma (Shanghai, China). The cells were cultured in 6-well plates and transfected plasmid or inhibitor using Lipofectamine 2000 (Invitrogen, USA). Hep-G2 and Hep-3B cells were chosen to be induced regorafenib resistant HCC cells. Regorafenib was purchased from MCE (MedChemExpress, NJ, USA). Regorafenib resistant HCC cells were established by long-term exposure to regorafenib. Specifically, HCC cells were first treated with a modest dosage of regorafenib (0.625 µM) for 2 weeks and then the medium containing regorafenib was exchanged with fresh complete medium for an additional 2 weeks. Afterwards, the regorafenib dose was progressively raised while the culture pattern was maintained. This process continued until the regorafenib dose reached 10 µM, the maximum clinically tolerated dose, and the remaining cells were regorafenib-resistant HCC cells.