敲低膜联蛋白A2对食管鳞癌细胞恶性表型的抑制作用

Inhibitory effects of ANXA2 knockdown on malignant phenotype of esophageal squamous carcinoma cells

目的:利用CRISPR/Cas9系统构建稳定敲低膜联蛋白A2(ANXA2)基因的食管鳞癌细胞,研究该基因对食管鳞癌细胞表型的影响。方法:在公共数据库中查找ANXA2基因及对照的向导RNA(gRNA),选取6个ANXA2 gRNA及1个对照gRNA,使用CRISPR/Cas9载体构建表达ANXA2 gRNA的重组质粒,转入HEK293FT细胞制备gRNA/Cas9慢病毒,将慢病毒感染食管鳞癌KYSE30和KYSE150细胞后,使用嘌呤霉素筛选阳性细胞并扩大培养。采用Western blot检测ANXA2蛋白及其下游MYC蛋白的表达情况,逆转录实时荧光定量PCR(RT-qPCR)检测ANXA2 mRNA表达情况,Transwell和集落形成实验分别检测细胞迁移侵袭和集落形成能力的变化。结果:成功构建了CRISPR/Cas9质粒;Sanger测序结果表明,ANXA2基因敲低质粒及对照质粒均构建成功。与对照组比较,病毒感染细胞后,Western blot结果显示,其中两个gRNA靶点病毒感染的KYSE30细胞中ANXA2蛋白及其下游MYC蛋白水平均显著降低(P<0.05);同时RT-qPCR结果证实,上述两个靶点显著下调ANXA2的mRNA表达水平(P<0.05)。Transwell和集落形成实验结果显示,敲低ANXA2后,细胞迁移侵袭能力和集落形成能力均显著减弱(P<0.05)。结论:ANXA2基因敲低显著抑制了食管鳞癌细胞的恶性表型,为进一步研究ANXA2促进食管鳞癌细胞恶性表型的作用机制提供了细胞模型和实验基础。

OBJECTIVE:To construct esophageal squamous cell carcinoma(ESCC)cell lines with ANXA2 gene knockdown using CRISPR/Cas9 system,and to study the effect of ANXA2 on cell phenotype and downstream molecules.METHODS:The guide RNAs(gRNAs)of ANXA2 and control were searched in the public database,and the recombinant plasmids expressing the gRNAs were constructed using CRISPR/Cas9 vector,which was transferred into HEK293FT cells to prepare CRISPR/Cas9 lentivirus.After ESCC cells KYSE30 and KYSE150 were infected with the lentivirus,positive cells were screened with purinomycin and expanded in culture.The Western blot assay was used to detect expressions of the ANXA2 protein and its downstream molecule MYC,RT-qPCR assay for the expression of ANXA2 mRNA,and the colony formation and The transwell assays for the changes of colonies as well as migration and invasion abilities,respectively.RESULTS:CRISPR/Cas9 plasmids were successfully constructed.Based on the Sanger sequencing results,the ANXA2 gene knockdown plasmids and the control plasmid were successfully constructed.After virus infection,Western blotting showed that the levels of ANXA2 protein and its downstream MYC protein were significantly decreased in KYSE30 infected with two gRNA target viruses(P<0.05).The RT-qPCR results confirmed that the above two targets significantly down-regulated the mRNA expression level of ANXA2(P<0.05).Cell phenotype experiments showed that invasion and migration as well as cell colony formation abilities were significantly weakened after knockdown of ANXA2(P<0.05).CONCLUSION:ANXA2 gene knockdown significantly inhibited the malignant phenotype of ESCC cells,which provides a cell model and experimental basis for further study on mechanisms of ANXA2 in promoting the malignant phenotype of ESCC cells.