Effect of depletion of IGF2BP3 on gene expression in LUAD

IGF2BP3, an oncofetal protein and an m6A reader, is a member of the IGF2BPs family playing an important role in cell migration in early embryogenesis, oncogenesis, metabolism and metastasis. To clarify the effect of depletion of IGF2BP3 on gene expression in LUAD, we conducted IGF2BP3 knocked out experiments in the NCI-H1299 LUAD cell lines. The knockout efficiency was validated by western blot analysis, demonstrating successful silencing of IGF2BP3 at the protein levels in NCI-H1299 cells. To explore the underlying mechanisms of IGF2BP3 in LUAD development, we conducted RNA-sequencing analysis using IGF2BP3 knockout and control NCI-H1299 cells, with each group consisting of three biological replicates. The analysis revealed that IGF2BP3 knockout resulted in differential expression of 1700 genes including 621 genes upregulated and 1079 downregulated. Overall design: NCI-H1299 was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were grown and maintained at 37°C in a humid atmosphere with and 5% CO2 and were cultured in Gibco's RPMI 1640 Medium (Thermo Fisher, Schwerte, Germany) supplemented with 10% FBS and 1% PAA. CHOPCHOP (https://chopchop.rc.fas.harvard.edu/) and CRISPR design (http://crispr.mit.edu/) were used to design two single guide RNAs (sgRNA, Supplementary Table 3) targeted to the BsmBI of IGF2BP3 (NCBI GeneID 10643). Annealed oligonucleotides were ligated into LentiCrisprv2 (GENECARE, GC010100605). The plasmids were sequenced to confirm successful ligation. For transfection, 4*10^5 293T cells were plated overnight in antibiotic-free medium and then transfected with 4µg of LentiCrisprv2 using Lipofectamine 3000. After high speed centrifugation, supernatant was collected and frozen at -80 ° C. NCI-H1299 cells were then infected with the supernatant, and selected under puromycin (2.0 µg/ml) for 48 hours and subsequently plated onto 96 well plates (10 cells/ml). Single cell colonies were expanded for RNA extraction, protein extraction and cryopreservation. The knockout efficiency was validated by western blot analysis, demonstrating successful knockout of IGF2BP3 at the protein levels in NCI-H1299 . Total RNA was isolated from IGF2BP3 knockout and control NCI-H1299 cells using TRIzol Reagent. RNA sequencing (RNA-seq) analysis was performed at Lian Chuan Biotech Inc. (Hangzhou, China).