RNA-seq Analysis of Differential Gene Expression in Wild-type Versus FOXC2-deficient B16-F1 Melanomas

The FOXC2 transcription factor regulates a variety of developmental and biological processes in both embryonic and adult tissues. Importantly, overexpression or dysregulation of FOXC2 is also associated with oncogenic activity in numerous cancer types, though the function of FOXC2 in the context of melanoma has not been previously investigated. Therefore, the goal of this study was to assess FOXC2's regulation of gene expression in melanoma cells. To this end, we employed CRISPR-Cas9 gene editing technology to disrupt the Foxc2 gene in B16-F1 melanoma, and we performed RNA-seq analysis to assess differential gene expression between the wild-type B16-F1 melanoma cell line and our novel FOXC2-deficient B16-F1?FOXC2 gene-edited variant cell line. Overall design: B16-F1 or B16-F1?FOXC2 melanoma cells were cultured for 24 hours before isolating RNA for gene expression analysis using the Arraystar Inc. Illumina Hi-seq RNA-sequencing service. Samples were run in replicates of five per group. So that FOXC2's contribution to the induction or repression of gene expression in B16-F1 melanoma could be most easily assessed, B16-F1?FOXC2 melanoma cells lacking this transcription factor served as the reference sample. Genes expressed at 1.5-fold higher or lower levels in B16-F1 (with a p value = 0.05) were defined as genes exhibiting differential expression between the two tumors with statistical significance.