Opti mem plus x treme gene sirna transfection reagent

Oligonucleotides for human MMP-9, CB₁R, CB₂R siRNA kit were purchased from GenePharma (Shanghai, China). The kit contains three predesigned duplexes targeting a specific MMP-9 gene. Cells were transfected with MMP-9, CB₁R, CB₂R siRNA or NC using the opti-MEM plus X-treme GENE siRNA transfection reagent (Roche, Mannheim, Germany) according to the instruction. Stably infected cells were selected and processed for further analysis by western blotting. After 48 h of post-transfection, western blot analyses were further performed. For gene overexpression, the recombinant lentiviruses carrying MMP-9 or control were obtained as gifts from colleague and used according to the manufacturer's protocol. Briefly, HEC-1B cells were seeded at 2×10⁵ cells/well in a 6-well plate. After adherent cells reached ~40% confluence, they were infected with an LV-MMP-9 or a control supplemented with 8 µg/ml polybrene (Sigma-Aldrich; Merck KGaA). Treated cells were selected with puromycin to generate puromycin-resistant clones, which were assayed by qPCR and western blotting.

Oligonucleotides for human ITGB1 siRNA kit was purchased from Guangzhou RiboBio Co., Ltd. The kit contains three predesigned duplexes targeting a specific ITGB1 gene. Cells were transfected with ITGB1 siRNA or NC at the concentration of 50 nmol/l using the opti-MEM plus X-treme GENE siRNA transfection reagent (Roche Diagnostics) according to the protocol of the manufacturer. The ITGB1 siRNA sequence was as follows: Forward, 5′-CCAUUCUGAUGAAUCUGAU-3′ and reverse, 5′-AUCAGAUUCAUCAGAAUGG-3′. After 48 h of post-transfection, western blot analyses were further performed.