CRISPR/Cas9技术敲除WSB2对MDA-MB-231增殖和迁移的影响  

Effect of WSB2 knockout by CRISPR/Cas9 on proliferation and migration of MDA-MB-231

作　　者：李一菲 邓英姿 李若兵 张运峰[2] 白云 胡芬 LI Yifei;DENG Yingzi;LI Ruobing;ZHANG Yunfeng;BAI Yun;HU Fen(School of Life Sciences,North China University of Science and Technology,Tangshan 063210,China)

机构地区：[1]华北理工大学生命科学学院,河北省唐山市063210 [2]唐山师范学院生命科学系

出　　处：《中国煤炭工业医学杂志》2024年第5期454-460,共7页Chinese Journal of Coal Industry Medicine

基　　金：河北省医学科学研究课题项目(编号:20221512);河北省自然科学基金项目(编号:H2021209007,B2022105015)

摘　　要：目的 分析信号传导抑制蛋白WD重复蛋白2(WSB2)的乳腺组织和细胞系表达量,通过CRISPR/Cas9(Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9)技术构建WSB2基因敲除人乳腺癌(MDA-MB-231)单克隆细胞株,并探究WSB2敲除对MDA-MB-231细胞增殖和迁移的影响。方法 采用生物信息学和实时荧光定量PCR(RT-qPCR)分析WSB2的表达水平;根据CRISPR/Cas9技术原理,设计靶向敲除WSB2基因的向导RNA(sgRNA)序列,构建WSB2敲除重组质粒并进行慢病毒包装后转染至MDA-MB-231细胞,嘌呤霉素筛选、基因组测序和蛋白质免疫印记实验(Western blot)验证WSB2基因敲除效果;分为野生型MDAMB-231细胞和WSB2基因敲除(MDA-MB-WSB2-KO)细胞两组,通过细胞计数试剂盒8(CCK-8)、5-乙炔基-2'-脱氧尿苷(EdU)染色实验、克隆形成实验、迁移(Transwell)实验及划痕实验检测两组细胞的增殖及迁移能力。结果 相较于正常组和Luminal组,WSB2在三阴性组中显著高表达(P<0.05);定量PCR结果显示,相较于人乳腺上皮细胞SVCT和MCF-10A细胞,WSB2 mRNA的表达在MDA-MB-231细胞中明显升高(SVCT细胞,t=5.505,P<0.01;MCF-10A细胞,t=4.508,P<0.05)。与野生型MDA-MB-231细胞相比,MDA-MB-231-WSB2-KO细胞活力(24h,t=8.411,P<0.01;48h,t=4.286,P<0.05;72h,t=4.657,P<0.01)、增殖(t=6.163,P<0.01)和克隆形成(t=7.374,P<0.01)能力,伤口愈合(24h,t=14.662,P<0.001;48h,t=14.724,P<0.001;72h,t=9.411,P<0.001)和迁移(t=8.141,P<0.01)能力也明显下降。结论 利用CRISPR/Cas9技术成功构建MDA-MB-231-WSB2-KO细胞株,WSB2被敲除后抑制了MDA-MB-231细胞的增殖和迁移。Objective The expression of WSB2(WD repeat and SOCS box-containing protein 2,WSB2)in breast tissues and cell lines was analyzed,and the monoclonal cell line of WSB2 gene knockout human breast cancer(MDA-MB-231)was constructed by CRISPR/Cas9(Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9)technology,and the effect of WSB2 knockout on the proliferation and migration of MDA-MB-231 cells was investigated.Methods The expression level of WSB2 was analyzed by bioinformatics and real-time fluorescence quantitative PCR(RT-qPCR).According to the principle of CRISPR/Cas9 technology,the guide RNA(sgRNA)sequence targeting WSB2 gene knockout was designed,the WSB2 knockout recombinant plasmid was constructed,and then transfected into MDA-MB-231 cells after lentviral packaging.Purinomycin screening,genome sequencing and Western blot were used to verify the effect of WSB2 gene knockout.They were divided into wild-type MDA-MB-231 cells and WSB2 gene knockout(MDA-MB-WSB2-KO)cells.The effects of cell counting kit 8(CCK-8),5-acetylidene-2'-deoxyuridine(EdU)staining,clonogenesis,Transwell and scratch tests on the proliferation and migration ability of the two groups of cells were detected.Results Compared with normal group and Luminal group,WSB2 was significantly overexpressed in triple negative group(P<0.05).Quantitative PCR results showed that compared with human mammary epithelial cells SVCT and MCF-10A,WSB2 mRNA expression was significantly increased in MDA-MB-231 cells(SVCT cells,t=5.505,P<0.01;MCF-10A cells,t=4.508,P<0.05).The viability of MDA-MB-231-WSB2-KO cells compared with wild type MDA-MB-231 cells(24h,t=8.411,P<0.01;48h,t=4.286,P<0.05;72h,t=4.657,P<0.01),proliferation(t=6.163,P<0.01),clonal formation(t=7.374,P<0.01),wound healing(24h,t=14.662,P<0.001;48h,t=14.724,P<0.001;72h,t=9.411,P<0.001)and migration ability(t=8.141,P<0.01)were also significantly decreased.Conclusion The MDA-MB-231-WSB2-KO cell line was successfully constructed using CRISPR/Cas9 technology,and the proliferation an

关 键 词：CRISPR/Cas9 WSB2 乳腺癌 增殖 迁移 

分 类 号：Q279[生物学—细胞生物学]