敲除ESR1基因对人乳腺癌细胞侵袭能力的影响

Effect of Human ESR1 Gene Knock-Out on the Invasive Ability of Breast Cancer Cells

目的:建立CRISPR/Cas9系统用于敲除人源雌激素受体α(ERα)基因(ESR1),并利用此细胞模型初步检测ESR1基因对乳腺癌细胞侵袭能力的影响。方法:设计一个靶向人源ESR1基因第2外显子的单向导RNA(sg RNA),分别克隆表达载体后,通过慢病毒转入人乳腺癌细胞株MCF-7,Western印迹检测MCF-7中ESR1基因的敲除效果,通过Transwell、反向侵袭试验观察ESR1基因敲除后对细胞侵袭能力的影响。结果:测序结果显示靶向ESR1基因CRISPR/Cas9重组质粒构建成功;Western印迹显示Cas9-ERα组的MCF-7细胞内ERα表达水平较对照组显著降低;Tanswell、反向侵袭试验证实ESR1基因敲除能够促进乳腺癌细胞的侵袭能力。结论:通过CRISPR/Cas9系统获得了靶向ESR1基因的重组质粒,构建的重组质粒能有效敲除ESR1基因;ERα能够抑制乳腺癌细胞的侵袭能力。

Objective:To knock out human estrogen receptorα1(ERα)gene(ESR1)by CRISPR/Cas9system.Methods:A sgRNA targeting the exon2of human ESR1gene was designed and cloned into vector,then it was transfected into human MCF-7cells by lentivirus.The effect of recombination plasmid on the expression of ESR1gene was identified by Western blot assays.The invasive ability of cells was examined by the transwell assay.Results:Western blotting showed that ERαsignificantly decreased in Cas9-ERαgroup.Inverse invasion assay and transwell assay showed that ESR1gene knock out promoted the invasion of breast cancer cells.Conclusion:The CRISPR/Cas9palsmid targeting human ESR1gene was successfully constructed and could knock out the expression of ESR1gene effectively.ERαcan inhibit the invasion of breast cancer cells.