5 aza

Pre-miR-5701 expression vectors and control vector were chemically synthesized in the pcDNA6.2-GW/EmGFP vector (Invitrogen, Carlsbad, CA, USA). The overexpressed plasmid of FGFR2, miR-5701 inhibitor, and all siRNA (i.e., siDNMT3A, siHDAC3, and siMBD1) were purchased from the GenePharma Corporation (SGC, Shanghai, China). The FGFR2 wild-type and mutated mRNA were cloned in between the SacI and XhoI sites of the pmirGLO expression vector (Invitrogen, Carlsbad, CA, USA). All related sequences used are listed in the Supplementary Table 2. The SGC-7901 cells were treated with 5-Aza (2.5, 5, 7.5, 10, and 15 μM; MedChemExpress, Shanghai, China) for 3 days and TSA (100, 300, 500, 700, and 900 nM; MedChemExpress, Shanghai, China) for 24 h. Then, the total RNA was extracted using the TRIzol protocol.

The human HCC cell line HepG2 was obtained from the American type culture collection (Manassas, VA, USA) and the two variants of this cell lines which undergo resistance to sorafenib (HepG2S1 and HepG2S3) were generated by the laboratory of hepatology of the University Hospitals Leuven [19 (link)]. Cells were grown in Dulbecco’s modified eagle’s medium (DMEM)-high glucose, supplemented with 10% fetal bovine serum and penicillin/streptomycin (100 U/mL), and they were cultured under a humidified 5% CO₂ atmosphere at 37 °C. Cell culture reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Resistant cell lines were continuously cultured in the presence of 6 μM sorafenib (Santa Cruz Biotechnology, Dallas, TX, USA) to preserve drug resistance. CoCl₂ (Panreac AppliChem, Barcelona, Spain) was added at 100 μM to mimic hypoxia. We used 300 μM CHX and 30 μM MG132 (Tocris Bioscience, Bristol, UK) as protein synthesis and proteasome inhibitors, respectively.
Besides, we inhibited histone deacetylation using 10, 50, and 100 nM of the HDACs inhibitor TSA (AdooQ^® Bioscience, Irvine, CA, USA) and methylation with 10 and 100 μM of the DNMT inhibitor 5-Aza (MedChemExpress, Sollentuna, Sweden).

The BMSCs were divided into five groups: 1) transfected with Tbx18 overexpression vector; 2) transfected with overexpression Tbx18 vector and treatment with 10 μM 5-Aza (Sigma-Aldrich; Merck KGaA); 3) transfected with Tbx18 overexpression vector and treatment with 1 μM BIX01294 (MedChemExpress, Inc.); 4) transfected with Tbx18 overexpression vector and treated with 10 μM 5-Aza and 1 μM BIX01294; 5) no treatment as blank control. All groups of were treated with BIX01294 and/or 5-Aza for one day and then replaced with normal medium. The cells were cultured until day 10, and after digestion and centrifugation, each group of cells was collected for subsequent treatment.