Establishment of TSPAN4 Gene Knockout of Non-Small Cell Lung Cancer Cell Line by CRISPR/Cas9

The CRISPR/Cas9 gene editing system was used to knockdown the TSPAN4 gene and construction of TSPAN4 stable knockdown non-small cell lung cancer A549 cell line. The role of TSPAN4 was detected in invasion and migration of A549 cell line by means of the biological research methods. Results of this study may provide an experimental basis in exploring the role of migrasome in the metastatic process of non-small cell lung cancer. The plasmid pSpCas9(BB)-2A-Puro(px459) was used as the vector, and the knockdown vector px459- sgRNA-TSPAN4 was obtained by ligating it with the designed primers. Px459-sgRNA-TSPAN4 were transfected into A549 cell lines through jetPRIME®. Monoclonal cells were obtained by puromycin pressure screening, and the efficiency of TSPAN4 knockout was identified by Western blot in the A549 cell lines. The cell scratch and Transwell experiment were used to analysis the effect of the invasion and migration in knockout TSPAN4 cell lines. Here, the TSPAN4 knockout plasmid pX459-sgRNA-TSPAN4 was successfully constructed. Western blot results showed that the expression of TSPAN4 protein in A549 cell line was significantly decreased after TSPAN4 gene knockout. Compared with the wild cell lines, there is no statistical difference on the ability of migration and invasion in knockout cell line of TSPAN4, but the TSPAN4 knockout reduces the expression of N-cadherin and has little impact on the expression of E-cadherin. The above study demonstrated that A549 cell lines with knockdown of TSPAN4 gene were successfully constructed using CRISPR/Cas9 gene editing technology, and knockdown of TSPAN4 gene alone did not affect the migration ability of A549 cells.

CSTR: 32200.14.cjcb.2023.02.0003