Mirna mimic

The GAS5 sequence synthesized according to the full length of the GAS5 RNA sequence (NCBI Reference Sequence: NR_002578.3) was cloned into the pHB-EF1-MCS-GFP vector (Hanbio Biotechnology, Shanghai, China) to construct the GAS5-pHB plasmid. The empty pHB vector was used as a negative control. The miR-21 mimic was purchased from RiboBio Co., Ltd (Guangzhou, China). Both plasmid and miRNA mimic were transfected into LSCC cell lines using Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, USA).

Full-length ANAPC11 coding region (ANAPC11 forward, 5′-ATG AAG GTG AAG ATT AAG TGC TGG AAC G-3′ and reverse, 5′-TCA GGA TGC CCC TCC AGC GAG AG-3′) was cloned into pMy vectors (Addgene, Inc.) to overexpress ANAPC11. An empty vector was used as a negative control. miR-1261 mimic (5′-ACU AUG UUG ACA CUU UUA UCC AA-3′), miR-1261 inhibitor (5′-UGA UAC AAC YGA AAA UAG GUU-3′), mimic control (5′-ACA UCU GCG UAA GAU UCG AGU CUA-3′), control inhibitor (5′-UAA CUA AUA CAU CGG AUU-3′), short hairpin (sh)RNA targeting lncRNA BCAR4 (1 mg; shBCAR4-1; 5′-GCU GCG AGG GUA GAC AUC U-3′ and shBCAR4-2; 5′-GUG AUU GCC AAA CGC UCC C-3′), shANAPC11 (5′-UCC CAG GAC AGG CAC AGG C-3′) and scramble control shRNA (1 mg; 5′-UAA GGC UAU GAA GAG AUA C-3′) were purchased from Shanghai GenePharma Co, Ltd. and cloned into a PLKO.1 puro vector (cat. no. 8453; Addgene, Inc.). Huh7 cells were counted and seeded in a 6-well plate at 1×10⁶ cells/well. When the confluence reached 90%, the cells were transfected with 50 nM miRNA mimic, 50 nM miRNA inhibitor or 4.0 µg corresponding plasmids using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Cells were cultured at 37°C in a 5% CO₂ incubator for 48 h and harvested for subsequent experimentation.

miR-195 mimic (5′-UAG CAG CAC AGA AAU GGC-3′), antagomir (5′-GCC AAU AUU UCU GUG CUG CUA-3′) and scrambled negative control (NC) miRNAs (NC mimic, 5′-UUC UCC GAA CGU GUC ACG UTT-3′ and NC antagomir, 5′-CAG UAC UUU UGU GUA GUA CAA-3′) were purchased from GenePharma Co., Ltd. Cells were seeded into 96-well plates and cultured to 70-80% confluence. The cells were then transfected with miRNA mimic (100 nM) or antagomir (200 nM) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. A total of 48 h after transfection, the cells were harvested.