RNA-sequencing assay to Identify Genes Promoting Lung Metastasis of Esophageal Squamous Cell Carcinoma (ESCC) Cells

Purpose: To elucidate the molecular basis governing lung metastasis in ESCC cells, we established two subpopulations of ESCC cells with enhanced pulmonary metastatic potential, namely K30LM3 and K450LM2 respectively. Then, transcriptional profiling of highly metastatic cells (K30LM3 and K450LM2) and their parental cells (K30 and K450) were performed to characterize differentially expressed genes (DEG). Methods: When parental and highly metastatic cells grew to approximately 80% confluence, these cells were washed twice using pre-chilled PBS. Total RNAs were harvested using Trizol reagents (Thermo Fisher Scientific). Poly(A) mRNA was isolated and enriched using NEBNext Poly(A) mRNA Magnetic Isolation Module. The mRNA was then recovered for library generation with NEBNext ® Ultra™ Directional RNA Library Prep Kit for Illumina (NEB, E7420S) following the manufacturer’s instructions. The cDNA libraries were sequenced at WuXiNextCODE (China). FastQC was used to examine the quality of raw reads. Read alignment was conducted using STAR (v2.5.1b), and R package edgeR (v3.8.5) was used to determine relative transcript abundances and differentially expressed genes (DEGs) between sample pairs. Results: Generally, 1502 genes (FDR<0.05, Fold Change>2) between K30 and K30LM3 cells and 650 genes (FDR<0.05, Fold Change>2) between K450 and K450LM2 cells were detected. Among thses DEGs, 52 genes were co-upregulated while 32 genes were co-downregulated in K30LM3/K450LM2 cells. GSEA showed that these commonly-changed genes were significantly enriched in epithelial-mesenchymal transition (EMT), cell adhesion, and anoikis process. Conclusions: In vivo selection approach enriched highly metastatic subpopulations with acquired essential molecular traits.