Construction of Stable MCT1-Knockout RKO Cell Line Based on CRISPR/Cas9 Technology and Its Biological Function Detection / 华中科技大学学报(医学版)

ABSTRACT

@#Objective To precisely knock out MCT1in humancolorectalcancercellline RKO by using CRISPR/Cas9 gene editingtechnique,and to detect its biological function. Methods CRISPRv2-MCT1-KO # 1 and CRISPRv2-MCT1-KO # 2 plasmids weretransfected into RKO cells. The monoclonal cells were selected by the medium containing puromycin after 48 h. The expression of MCT1 was detected by Western blotting and DNA sequencing. The change of expression and distribution of MCT1 protein were detected by immunofluorescence.Intracellular lactic acid level was detected by lactic acid detection kits. Colonyformation assayand CCK-8 assay were performed to detect cell proliferation ability. The potential signaling pathways of MCT1 in colorectal cancer were explored by GSEA software. Results CRISPRv2-MCT1-KO plasmid was well constructed. Western blottingand DNAsequencingresults showedthat MCT1 was successfully knocked outinthe humancolorectal cancer RKOcells. Compared withthecontrol group, MCT1 protein was notobserved onthecell membraneof MCT1-nockoutcells,andtheintracellularlactatelevel was significantlyreduced(P <0.05). The proliferation ability of RKO cells was significantly decreased after MCT1 knockout(P <0.05). GSEA analysis showedthat MCT1 may promotethe occurrence and development ofcolorectal cancerthrough oxidative phosphorylation and MYC signaling pathway. Conclusion The stable MCT1-knockout human colorectal cancer RKO cell line was successfully constructed by applying CRISPR/Cas9 technology, which might become an ffectivetoolforstudyingtherole of MCT1 inthe occurrence and development ofcolorectal cancer.