[The application of ANXA2 gene knockout mouse models in lung cancer metastasis].

Abstract

Objective: ANXA2 plays a crucial role in cancer metastasis, but its mechanism is not yet fully understood. Therefore, it is necessary to establish an ANXA2 gene knockout mouse model to provide an effective tool for subsequent studies on ANXA2-related mechanisms. Methods: A gene knockout mouse model was constructed using CRISPR/Cas9 technology. The model was validated through tissue DNA extraction followed by polymerase chain reaction (PCR), sequencing, and western blot to confirm ANXA2 genotype and protein expression. The successfully constructed models were divided into a model group and a wild-type (WT) group for the creation of a mouse tail vein injection Lewis lung carcinoma (LLC) metastasis model. Metastatic foci formation was monitored using in vivo imaging technology, and the survival rates of the two groups were compared. Results: An sgRNA sequence targeting the first exon of ANXA2 was designed, and 16 founder mice were obtained through microinjection. Through consanguineous hybridization, 30 homozygous offspring were ultimately acquired. After establishing the strains of the mouse model, mice were divided into the ANXA2 knockout group and the WT group, with 8 mice in each group. An LLC lung metastasis model was established in both groups. Compared with the WT group, the number of metastatic foci was significantly increased in the ANXA2 knockout group (7 vs. 1), and the fluorescence intensity was stronger in the WT group than in the knockout group (P=0.002). Using the GEPIA2 database to analyze ANXA2 gene expression in tumor tissues and normal tissues of lung cancer patients, it was found that ANXA2 expression levels were significantly higher in lung cancer tumor tissues compared to normal tissues (P＜0.05). The database included data from 478 lung cancer patients, and patients were stratified into high-expression and low-expression groups based on ANXA2 levels. Compared to the low-expression group, patients in the high-expression group exhibited significantly shorter disease-free survival and overall survival (P＜0.05, respectively). The survival time of mice in the ANXA2 knockout group (median survival time, 43 days) was significantly longer compared to the WT group (median survival time, 26 days; P=0.017). Additionally, ANXA2 expression is significantly associated with the prognosis of lung cancer patients (P=6.4e-14). Conclusions: ANXA2 is closely associated with cancer metastasis and holds potential as a new target for metastasis treatment. Further in-depth research will greatly facilitate the transition of ANXA2 from basic research to clinical application.