Gene mutation kit

A total of 3 × 10⁴ cells were seeded into each well of the 96-well plates. The 3′-UTR sequences of MIAT or FOXM1 encompassing the miR-149-5p wild-type or mutant binding sites were synthesized. The sequences were inserted into pmirGLO luciferase reporters (Promega, Madison, WI, USA) between Sacl and Sall restriction sites, respectively. The binding sites for miR-149-5p were mutated by Gene Mutation Kit (Takara, JAPAN) to generate the mutant MIAT or FOXM1 3′-UTR. The plasmids, including 3′-UTR of wild-type or mutant sequences from MIAT or FOXM1 and miRNA mimic were co-transfected into NSCLC cells using Lipofectamine 2000 (Invitrogen, Foster city, USA). After incubation for 48 h, a dual-luciferase reporter assay (Promega, Madison, WI, USA) was used to test the firefly and Renilla luciferase activities. Results were presented as relative luciferase activities of Renilla, which were normalized to the activity of firefly luciferase.

The fragment of wild-type USP14 CDS (USP14) containing predicted YTHDF1 target sites was amplified by PCR and cloned into pGL3 vector (Promega, United States), which included Firefly luciferase reporter genes. The wild-type YTHDF1(YTHDF1-WT) was amplified by PCR and cloned into pcDNA3.1 vector. The mutant YTHDF1 (YTHDF1-MUT) was then generated by mutating the YTH domain of YTHDF1 using Gene Mutation Kit (Takara, JAPAN). Next, the YTHDF1-MUT or YTHDF1-WT plasmid were co-transfected with USP14 and pRL-TK vector which included Renilla luciferase reporter genes (Promega, United States) into AGS cells. The pcDNA3.1 vector was used as negative control. After 48 h, the cells were harvested, and the Firefly and Renilla luciferase activities were measured based on a dual-luciferase reporter assay system (Promega, United States). The relative luciferase activity was normalized to Renilla luciferase activity (F-Luc/R-Luc).