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.
Highlights
Colorectal cancer (CRC) is a highly invasive and metastatic malignant tumor of the digestive tract, with high mortality [1]
genome-scale CRISPR-Cas9 knockout (GeCKO) screening and cell viability test results revealed that MMP15, MRPL48, CALN1 and HADHB genes might be involved in drug sensitivity to CTX in CRC cell (Caco-2)
This finding suggested that MMP15, MRPL48, CALN1 and HADHB genes were potential targets for the treatment of CRC, which fills in the gap of genes responsible for CTX sensitivity in CRC
Summary
Colorectal cancer (CRC) is a highly invasive and metastatic malignant tumor of the digestive tract, with high mortality [1]. Most CRC cells are currently assumed to be stem cells or stem cell-like cells. These stem cells are the outcomes of progressive accumulation of genetic and epigenetic changes that activate oncogenes and inactivate tumor-suppressor genes [2]. Due to the low early diagnosis rate, most patients are diagnosed as middle or advanced CRC, resulting in difficult treatment and poor prognosis [3]. Despite therapeutic advancements, the prognosis for metastatic CRC patients remains poor, with a median overall survival of 18–21 months [5]. Drug resistance is the main reason for the failure of chemotherapy in cancers
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