Establishment of two renal carcinoma A498 cell lines stably expressing CXCR4 / 第二军医大学学报

ABSTRACT

Objective To transfect CXCR4 and enhanced green fluorescent protein (EGFP)-CXCR4 plasmids into renal carcinoma cell line A498 cells to preparecell lines stably expressing CXCR4. Methods Two specific plasmids containing CXCR4 or EGFP-CXCR4 were transfected into renal cell carcinoma cell line A498. Then the cells stably expressing CXCR4 were screened by using G418. Confocal microscopy was used to observe the changes of EGFP-CXCR4 fusion protein in A498 cells before and after stimulation with SDF-1. Western blotting analysis was used to determine CXCR4 expression after transfection. Proliferation of A498 cells was detected by MTT and the invasion ability of cells was detected by transwell assay. Results The sequencing result of two plasmids was consistent with CXCR4 DNA sequence, and two cell lines were screened out by G418 screening after the plasmids were transfected into A498 cells. EGFP-CXCR4 fusion protein was found in the cell membrane and cytoplasm of EGFP-CXCR4 transfection group under confocal microscopy. EGFP-CXCR4 migrated into cells after SDF-1 stimulation. Western blotting analysis revealed higher CXCR4 expression in A498 cells stably transfected with CXCR4 plasmids compared with normal A498 cells. The proliferation of cells in pCNDA-CXCR4 and pEGFP-CXCR4 groups were significantly higher than that in normal A498 cell group (P<0. 01). Transwell assay showed that the cell invasion ability of cells with stable CXCR4 expression was significantly increased compared with that in the normal A498 cell group (P<0. 01). Conclusion We have successfully established A498 cell lines stably expressing CXCR4, which have enhanced proliferation levels and higher invasive ability.