Abstract: 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.

Key words: CRISPR/Cas9, ANXA2, esophageal squamous cell carcinoma cell line, migration and invasion