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目的应用RNA干扰技术降低肾癌A498细胞株的CXCR4基因表达水平,并建立稳定转染细胞株。方法针对CXCR4基因设计合成siRNA,转染肾癌A498细胞株,采用RT-PCR法检测CXCR4基因表达变化,根据有效干扰片段结果合成重组shRNA质粒,稳定转染至A498细胞系并进行G418抗性筛选细胞株。采用RT-PCR和Westen印迹法检测CXCR4mRNA及蛋白表达水平,应用流式细胞技术检测CXCR4shRNA诱导A498细胞凋亡的效应,Transwell试验检测细胞侵袭能力的改变。结果将CXCR4重组shRNA质粒成功转染A498细胞后,肾癌细胞内可见绿色荧光,通过G418筛选,获得了CXCR4基因沉默的A498细胞系,RT-PCR及Western印迹检测证实该细胞系的CXCR4水平明显低于对照组的表达水平(P<0.05)。CXCR4shRNA组细胞的早期凋亡率、晚期凋亡率及总凋亡率均显著高于对照组(P<0.05),Tran-swell体外侵袭实验显示CXCR4shRNA能明显抑制A498细胞的体外侵袭力(P<0.05)。结论成功构建稳定沉默CXCR4表达的肾癌A498细胞株,转染后的细胞凋亡率升高,体外侵袭能力降低,为后续研究奠定了基础。
Objective To reduce the expression of CXCR4 gene in renal cell carcinoma A498 cell line by RNA interference and to establish a stable transfected cell line. Methods siRNA targeting CXCR4 gene was transfected into A498 cell line and the expression of CXCR4 was detected by RT-PCR. Recombinant shRNA plasmids were synthesized and transfected into A498 cell line with G418 resistance screening Cell lines. The expression of CXCR4 mRNA and protein was detected by RT-PCR and Westen blot. Flow cytometry was used to detect the effect of CXCR4 shRNA on the apoptosis of A498 cells. Transwell assay was used to detect the change of cell invasiveness. Results The CXCR4 recombinant shRNA plasmid was successfully transfected into A498 cells, and green fluorescence was observed in renal cell carcinoma cells. A498 cell line with CXCR4 silencing was obtained by G418 screening. The CXCR4 level was confirmed by RT-PCR and Western blotting Lower than the control group (P <0.05). The early apoptotic rate, the late apoptosis rate and the total apoptosis rate of CXCR4 shRNA group were significantly higher than those of the control group (P <0.05). Tran-swell invasion assay showed that CXCR4 shRNA could significantly inhibit the invasiveness of A498 cells in vitro (P < 0.05). Conclusion A498 cell line with stable silencing of CXCR4 expression was constructed successfully. The apoptosis rate of transfected A549 cells was increased and the invasion ability in vitro was reduced, which laid the foundation for further study.