RNA sequencing analyses of gene expression by CRISPR/Cas9 knockout of CLL-1 gene in acute myeloid leukemia cells

Abstract

CLL-1 has been revealed its potential role in acute myeloid leukemia (AML), however, the underlying mechanisms remain unclear. CRISPR/Cas9 strategy was employed to knock out CLL-1 gene in U937 cells and western-blot was used to validate the success of knock out. CCK8 and Transwell assays were used to detect cells viability and migration, respectively. RNA-sequencing was performed to profile mRNA expression in CLL-1 gene knock-out and wide type U937 cells. A cutoff of 1.5-fold change and false discovery rate (FDR) <0.05 was used to screen differentially expressed genes (DEGs), which were presented by volcano plots and hierarchical cluster heatmap. Protein-protein interaction (PPI) network was constructed by String database and Cytoscape software. Furthermore, hub genes were mined by CytoNCA and MCODE, which were subjected to functional enrichment using R package. Finally, the findings were validated using qRT-PCR and western-blot. The protein level of CLL-1 was significantly lowered, and cell viability and migration were suppressed in knock-out cells compared to wide type. Using RNA-sequencing and bioinformatics analysis, 452 DEGs (179 up-regulated and 273 down-regulated) were obtained, and several important hub genes (such as CCR2, FBXO21, UBB and UBE2C) were filtered out, which were enriched in 132 GO terms and 36 KEGG pathways such as chemokine signaling pathway and ubiquitin mediated proteolysis. A total of 8 representative genes mRNA expressions were validated by qRT-PCR, and the protein levels of 6 genes were confirmed by western-blot. CLL-1 gene might exert its role in AML through modulating genes enriched in multiple functions such as chemokine signaling and ubiquitination. Our results may give us new knowledge of CLL-1 in AML and provide a basis for mining novel targets.