Detection of genes responsible for cetuximab sensitization in colorectal cancer cells using CRISPR-Cas9
Abstract Colorectal cancer (CRC) is a common malignant tumor in digestive tract with highly invasive and metastatic capacity. Drug sensitivity remains a significant obstacle to successful chemotherapy in CRC patients. The present study aimed to explore genes related to cetuximab (CTX) sensitivity in CRC by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9. Celigo image cytometer was used to detect suitable cells and optimal dosage of CTX. Inhibition rate of CTX on Caco-2 cells was evaluated by cell counting kit-8 (CCK-8) method before and after transfection. 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) was performed to explore suitable concentration of puromycin and multiplicity of infection (MOI). CRISPR-Cas9, sequencing data quality analysis and cell viability test were used for the selection of genes related to CTX sensitivity in CRC cells. Finally, the selected genes associated with CTX sensitivity in CRC cells were further validated by colony formation and CCK-8 assays. In the present study, Caco-2 cells had a better prolificacy, and CTX 100 μg/ml exhibited a good inhibition trend on the 7th and 14th days of infection. MTT assay indicated that the minimum lethal concentration of puromycin was 2.5 μg/ml. Forty-six candidate genes were preliminarily screened via sequencing data quality analysis. Subsequently, we found that knockout of any of the four genes (MMP15, MRPL48, CALN1 and HADHB) could enhance CTX sensitivity in Caco-2 cells, which was further confirmed by colony formation assay. In summary, MMP15, MRPL48, CALN1 and HADHB genes are related to the mediation of CTX sensitivity in CRC.
