利用CRISPR/Cas9技术构建人MCT1基因敲除结肠癌RKO稳定细胞系及其生物学功能检测

Construction of Stable MCT1-Knockout RKO Cell Line Based on CRISPR/Cas9 Technology and Its Biological Function Detection

目的利用CRISPR/Cas9基因编辑技术在人结肠癌细胞系RKO中稳定敲除MCT1基因,并检测其生物学功能。方法将CRISPRv2-MCT1-KO#1、CRISPRv2-MCT1-KO#2质粒转染至RKO细胞中,48 h后用含嘌呤霉素的培养液进行筛选,挑取单克隆细胞;利用Western blot和DNA测序技术检测MCT1基因表达情况;利用免疫荧光技术检测MCT1蛋白表达分布变化;通过乳酸试剂盒检测细胞内乳酸水平;通过细胞克隆形成实验以及CCK-8实验检测细胞增殖能力;通过GSEA软件分析MCT1基因在结肠癌中可能参与的信号通路。结果 CRISPRv2-MCT1-KO质粒成功构建;Western blot和DNA测序结果显示稳定敲除MCT1基因的人结肠癌RKO细胞构建成功;与对照组相比,MCT1基因敲除细胞的细胞膜上不能观察到MCT1蛋白,且细胞内乳酸水平明显降低(P<0.01);MCT1基因敲除后RKO细胞增殖能力明显减弱(P<0.01);GSEA分析显示,MCT1可能通过氧化磷酸化、MYC信号通路促进结肠癌发生发展。结论通过CRISPR/Cas9技术成功构建MCT1基因稳定敲除的结肠癌RKO细胞系,可能成为研究MCT1基因在结肠癌发生发展中作用的有效工具。

Objective To precisely knock out MCT1 in human colorectal cancer cell line RKO by using CRISPR/Cas9 gene editing technique, and to detect its biological function.Methods CRISPRv2-MCT1-KO #1 and CRISPRv2-MCT1-KO #2 plasmids were transfected 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.Colony formation assay and 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 blotting and DNA sequencing results showed that MCT1 was successfully knocked out in the human colorectal cancer RKO cells.Compared with the control group, MCT1 protein was not observed on the cell membrane of MCT1-knockout cells, and the intracellular lactate level was significantly reduced(P<0.05).The proliferation ability of RKO cells was significantly decreased after MCT1 knockout(P<0.05).GSEA analysis showed that MCT1 may promote the occurrence and development of colorectal cancer through 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 effective tool for studying the role of MCT1 in the occurrence and development of colorectal cancer.