摘要
目的:通过CRISPR/Cas9基因编辑技术在乳腺癌细胞MCF7中稳定敲除FOXQ1,并初探其在缺氧环境中的功能。方法:Cas9慢病毒感染MCF7构建工具细胞,在工具细胞中分别转染靶向3个不同靶位点的FOXQ1-crRNA+tracrRNA,用T7E1酶筛选出编辑效率高的细胞挑取单克隆细胞株;利用T7E1酶、Sanger测序和Western印迹检测FOXQ1基因的编辑和表达情况;Western印迹检测常氧/缺氧条件下细胞FOXQ1表达情况;在常氧/缺氧条件下通过划痕实验和transwell实验检测细胞迁移能力;通过METABRIC数据库分析乳腺癌中FOXQ1与HIF1A及其编码蛋白靶基因LOX、LDHA和GLUT3表达的相关性。结果:筛选出转染靶向CCCGTGCGCATCCAGGACAT序列的FOXQ1-crRNA的细胞具有最高编辑效率;T7E1酶切、Sanger测序和Western印迹结果显示稳定敲除FOXQ1的乳腺癌MCF7细胞构建成功;缺氧促进MCF7细胞FOXQ1的表达;缺氧促进MCF7的迁移能力(t=3.78,P<0.01;t=11.94,P<0.001),敲除FOXQ1后缺氧对MCF7迁移能力的促进作用被抑制(t=0.63,P=0.55;t=2.54,P=0.064)、(t=2.46,P<0.05;t=4.95,P<0.05),乳腺癌中FOXQ1与HIF1A、LOX、LDHA和GLUT3的表达均显著正相关(P<0.0001)。结论:通过CRISPR/Cas9系统成功构建了FOXQ1基因稳定敲除的乳腺癌MCF7细胞株,并发现FOXQ1介导了缺氧对MCF7细胞迁移能力的促进作用,为进一步探究乳腺癌的缺氧微环境中FOXQ1的功能及其机制奠定了基础。
Objective:To stably knock out FOXQ1 using CRISPR/Cas9 gene editing technology in MCF7 breast cancer cells and to explore its function in hypoxic conditions.Methods:Cas9 lentivirus was infected with MCF7 cells to create tool cells,and FOXQ1-crRNA+tracrRNA targeting three different target sites were transfected into the tool cells.T7E1 enzyme was used to screen for cells with high editing efficiency,and single clone cell lines were selected.T7E1 enzyme,Sanger sequencing,and Westernblotting were used to detect the editing and expression of the FOXQ1 gene.Western blotting was used to detect FOXQ1 expression in cells under normoxic/hypoxic conditions.The wound healing and transwell assay were used to detect cell migration ability under normoxic/hypoxic conditions.The METABRIC database was used to analyze the correlation between FOXQ1 and HIF1A,as well as its encoded protein target genes LOX,LDHA and GLUT3 in breast cancer.Results:Cells transfected with FOXQ1-crRNA targeting the CCCGTGCGCATCCAGGACAT sequence showed the highest editing efficiency.T7E1 enzyme cleavage,Sanger sequencing,and Western blotting results confirmed the successful construction of stable FOXQ1-knockout MCF7 breast cancer cells.Hypoxia promoted the expression of FOXQ1 in MCF7 cells and also promoted their migration ability(t=3.78,P<0.01;t=11.94,P<0.001).Knocking out FOXQ1 inhibited the promoting effect of hypoxia on MCF7 migration(t=0.63,P=0.55;t=2.54,P=0.064),(t=2.46,P<0.05;t=4.95,P<0.05).FOXQ1 expression in breast cancer was significantly positively correlated with HIF1A,LOX,LDHA,and GLUT3(P<0.0001).Conclusion:Astable FOXQ1-knockout MCF7 breast cancercell line has beensuccessfully constructed using the CRISPR/Cas9 system.It is found that FOXQ1 mediatesthe promoting effect of hypoxia on MCF7 cells migrationability,laying the foundation for further exploration of the function and mechanism of FOXQ1 in the hypoxic microenvironment of breast cancer.
作者
杨欢
冯玉梅
YANG Huan;FENG Yu-mei(Department of Biochemistry and Molecular Biology,Cancer Institute and Hospital,Tianjin Medical University,National Clinical Research Center for Cancer,Key Laboratory of Cancer Prevention and Therapy,Tianjin,Tianjin′s Clinical Research Center for Cancer,Key Laboratory of Breast Cancer Prevention and Therapy,Tianjin Medical University,Ministry of Education,Tianjin 300060,China)
出处
《天津医科大学学报》
2023年第5期500-506,共7页
Journal of Tianjin Medical University
基金
国家自然科学基金资助项目(82173349)。
