Si adam9

Negative control (NC), miR-526b-3p mimics, miR-526b-3p inhibitor, sh-NC, sh-LINC00689, si-ADAM9, pcDNA3.1 plasmid and pcDNA3.1-LINC00689 were all obtained from Genepharma (Shanghai, China). The cell suspension was prepared by 0.25% pancreatin digested AGS or MGC-803 cells and complete medium. Then, the cells were incubated in six-well plates (1×10⁶ cells/well) for 18–24 h. Three hours before transfection, GC cells at 80–90% confluence were incubated with fresh medium without serum or antibiotics. Transfection was performed using Lipofectamine 2000 (~ 0.6 μg Lipofectamine reagent/1 μg DNA, Life Technologies).

Three EC cell lines, including HEC-1-A (HTB-112), HEC-1-B (HTB-113), and Ishikawa (HTB-113), and human embryonic stem cells (ESC) (SCRC-2002) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in 5% CO₂ at 37°C. The cell culture medium used for each cell line follows the recommended formulation and required additives on the ATCC website (https://www.atcc.org/products/all/HTB-113.aspx).
Cell transfections were performed when cultures reached 80% confluence using Lipofectamine 2000 Transfection Reagent (Invitrogen, Carlsbad, CA, USA). Specific small interfering RNA (siRNA) oligonucleotides targeting HOXB-AS3 (si-HOXB-AS3; 5ʹ-UGCUUGUCUGGAGAUGGAGCCACUA-3ʹ) or negative control siRNA (si-NC) were transfected into EC cells. The si-HOXB-AS3, si-ADAM9 (5ʹ-CUCCUUGGAGAUUAACUAGUU-3ʹ), anti-miR-498-5p (5ʹ-UUUCAAGCCAGGGGGCGUUUUUC-3ʹ), miR-498-5p mimic (5ʹ-TTTCAAGCCAGGGGGCGTTTTTC-3ʹ), and negative control (5ʹ-TGTAGTTGTACTCCAGCTTGUGCTT-3ʹ) were synthesized and purchased from GenePharma (Shanghai, China). In our study, miR-498-5p was used in all experiments. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) assays were used to confirm HOXB-AS3 knockdown 48 hours after transfection. The sequence of si-HOXB-AS3 was designed as UGCUUGUCUGGAGAUGGAGCCACUA.