Mettl3 sirna

For construction of the METTL3 overexpression plasmid, please refer to Supporting Doc 1. The nucleotide sequences of the siRNAs (GenePharma, Shanghai, China) were as follows: METTL3 siRNA: sense 5′‐GCAAGUAUGUUCACUAUGATT‐3′; IGF2BP1 siRNA: sense 5′‐UGGAUGCUACGAGUAUAAATT‐3′; TFAP2C siRNA: sense 5′‐UCAGCUCUACGUCUAAAUATT‐3′; Negative control siRNA: sense 5′‐UUCUCCGAACGUGUCACGUTT‐3′. Cells showing logarithmic growth were seeded into six‐well plates, at 5 × 10⁴ cells per well, cultured for 24 hours and then transfected with 5 μL Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) for siRNA 100 pmol or 2.5 μg plasmid according to the manufacturer's instructions.

METTL3 down-regulation was achieved using sequence-specific small interfering RNA (siRNA). METTL3 siRNA and negative control was from GenePharma (Shanghai, China). COV362 cells grown in 6-well plates were transfected with 1 ug/ml sequence-specific siRNA or vector siRNA prepared in a mix with lipo3000 reagent and DMEM containing 10% serum. The cells were then cultured for 6 hours before use.

Human gastric cancer cell TMK1, pancreatic cancer cell PANC-1, liver cancer cell Huh-7 were grown in DMEM media (Gibco) with 10% FBS (Gibco) and 1% 100X Pen/Strep (Gibco). Human colorectal cancer cell line HCT116, and esophageal cancer cell TE-1 were grown in IMEM and RPMI1640 media, respectively, with 10% FBS and 1% 100X Pen/Strep. Control siRNA and METTL3 siRNA were purchased from GenePharma (Shanghai, China) with reported sequences12 (link). Transfection was achieved by using Lipofectamine Lipofectamine 3000 (Invitrogen) with 20 nM of siRNAs. At 48 h post-transfection, the mRNA levels of METTL3, AKT1, mTOR, PIK3CA, and PTEN were checked by Q-PCR.