ABSTRACT
【Objective】 To investigate the effects of chlorogenic acid on the proliferation, migration and invasion of renal carcinoma A498 and 769-P cells and the possible molecular mechanism. 【Methods】 Human renal carcinoma A498 and 769-P cells were divided into control group and chlorogenic acid group (2 μL,1 μmol/L) and cultured for 72 h. The cell proliferation, invasion and migration were detected with MTT assay, Transwell assay and scratch test, respectively. The expressions of IL-1β, EPAS-1 and AKT/P65 signaling pathway related proteins were detected with ELISA, qRT-PCR and Western blot, respectively. 【Results】 Chlorogenic acid inhibited the proliferation, invasion and migration of renal carcinoma A498 and 769-P cells, and reduced the IL-1β level in the cell supernatant. Anti-IL-1β reduced the protein and mRNA expressions of EPAS-1, p-AKT and p-P65. Compared with the control group, the chlorogenic acid group had reduced mRNA and protein expressions of EPAS-1, p-AKT and p-P65 (P<0.05). 【Conclusion】 Chlorogenic acid can inhibit the invasion and metastasis of renal carcinoma cells, and its mechanism may be related to inhibiting the secretion of IL-1β, thereby inhibiting the AKT/P65/EPAS-1 pathway.
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.
ABSTRACT
Objective:To investigate the inhibitory effect of RNA interference (RNAi) on the expression of multidrug resistance (MDR1) gene and analyze the altered sensitivities of human renal carcinoma cell line to cisplatin.Methods:Three small interfering RNA (siRNA) sequences targeted MDR1 gene were synthesized and transfected into renal carcinoma A498 cells. The expression level of MDRl mRNA was measured by RT-PCR to identify the most effective siRNA sequence. The recombinant plasmid was packed by lentivirus and transfected into A498 cells. RT-PCR was used to screen the A498 cells with the optimal silencing efficacy. The MDR1 protein expression level in the cloned cells was verified by Western blotting. The inhibitory effect of cisplatin on the proliferation of A498 cells was assessed by MTT assay and the IC_(50) value was calculated. Results:The 3 siRNA sequences suppressed MDR1 gene expression at different degrees. The siRNA 1 sequence silenced MDR1 gene more effectively with a significant reduction of 67%. The MDR1 protein expression greatly decreased in screened A498 cells compared with non-transfected cells (P<0.01), and the IC_(50) value of cisplatin on screened A498 cells was significantly decreased by 83.37% (P<0.01). Conclusion: The RNAi could effectively inhibit the expression of MDR1 gene and increase the sensibility to cisplatin in human renal carcinoma A498 cell line, which make it possible to reverse the resistance of renal carcinoma to chemotherapy.