Yy1 sirna

In this experiment, four cell lines of which THHL‐3 and HL‐7702 belong to human liver epithelial cells and HepG2 and SMMC‐7721 represent HCC cell lines were involved. All four cell lines were purchased from the ATCC Cell Biology Collection (Maryland, USA) and stored at –80°C. The Dulbecco's Modified Eagle Medium with 10% fetal bovine serum and 100 U/mL penicillin‐streptomycin was used to culture the cells in a chilled atmosphere of 5% CO₂ at 37°C.
The CASC11 siRNAs, EIF4A3 siRNA, YY1 siRNA, negative control (NC) siRNAs, E2F1 overexpression plasmids pcDNA‐E2F1, NC plasmids pcDNA‐NC, and luciferase reporter vectors with wild or mutant CASC11 promoter were synthesized by GenePharma (Shanghai, China). The sequences are detailed in Table S1. The lipofectamine 2000 (Invitrogen Life Technologies, USA) was employed to accomplish the cell transfections as previously described.¹⁷

The YY1-siRNA, pIRES2-EGFP-YY1 and respective negative controls were purchased from GenePharma (Shanghai, China). The specific sequences of YY1 siRNA were as follows: (F) 5′-CAUGUCCAUUAUUCGUGATT-3′(R) 5′-UCACGAAUGGCCAUGTG-3′. A non-coding siRNA (Invitrogen, Carlsbad, CA, USA) was used as a negative control. Transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Briefly, after trypsinization, cells were resuspended in an antibiotic-free medium and mixed with Opti-MEM (Gibco, Carlsbad, CA, USA) including 25 nM siRNA and Lipofectamine 2000. After incubation for 20 min at room temperature, cells were diluted with culture medium and seeded into a 60-mm dish. For cell viability assays, siRNA-transfected cells were re-seeded into a 48-well plate at 48 h after transfection. The silencing efficiency of V-ATPase V1A subunit siRNA was measured by the expression of mRNA and protein.