Department of Gastroenterology and Hepatology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325003, P.R. China.
Department of Colorectal and Anal Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325003, P.R. China.
Biosci Rep. 2020 Oct 30;40(10). doi: 10.1042/BSR20201125.
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.
结直肠癌（CRC）是一种常见的消化道恶性肿瘤，具有高度侵袭性和转移性。药物敏感性仍然是 CRC 患者化疗成功的重大障碍。本研究旨在通过 CRISPR-Cas9 技术探索与西妥昔单抗（CTX）敏感性相关的基因。Celigo 图像细胞仪用于检测合适的细胞和 CTX 的最佳剂量。在转染前后，通过细胞计数试剂盒-8（CCK-8）方法评估 CTX 对 Caco-2 细胞的抑制率。3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）用于探索合适的嘌呤霉素浓度和感染复数（MOI）。CRISPR-Cas9、测序数据质量分析和细胞活力测试用于筛选与 CRC 细胞中 CTX 敏感性相关的基因。最后，通过集落形成和 CCK-8 测定进一步验证与 CRC 细胞中 CTX 敏感性相关的选定基因。在本研究中，Caco-2 细胞具有更好的繁殖能力，CTX 100μg/ml 在感染后第 7 天和第 14 天表现出良好的抑制趋势。MTT 试验表明嘌呤霉素的最小致死浓度为 2.5μg/ml。通过测序数据质量分析初步筛选出 46 个候选基因。随后，我们发现敲除任何四个基因（MMP15、MRPL48、CALN1 和 HADHB）中的任何一个都可以增强 Caco-2 细胞中 CTX 的敏感性，这一结果进一步通过集落形成试验得到证实。综上所述，MMP15、MRPL48、CALN1 和 HADHB 基因与 CRC 中 CTX 敏感性的调节有关。
