稳定沉默CXCR4表达的肾癌A498细胞株的构建

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。

Establishment of a renal carcinoma cell line A498 with CXCR4 stably silenced

ObjectiveTo establish a renal carcinoma cell line A498 with CXCR4 stably silenced by using RNAi technique. MethodsWe designed three sequence-specific small interfering RNAs (siRNA) targeting CXCR4 gene and transfected siRNA into renal carcinoma cell line A498; the change of CXCR3 gene expression was observed by RT-PCR. The effective siRNA sequence was used to construct the recombinant plasmid, which was used to transfect A498 cells by liposome. Then the stably CXCR4 silenced cell lines were screened by using G418. RT-PCR and Westen blotting analysis were used to determine CXCR4 mRNA and protein expression. Flow cytometry was employed for determination of apoptosis in A498 cells. The invasion ability of cells was detected by transwell assay. ResultsGreen fluorescence was seen in A498 cells transfected with recombinant shRNA plasmid. G418 screening yielded stably CXCR4-silenced A498 cell lines; RT-PCR and Western blotting analysis revealed that CXCR4 expression in CXCR4-shRNA group was significantly lower than that in the control group(P<0.05). The early apoptotic rate, late apoptotic rate, and total apoptotic rate in the CXCR4-shRNA group were significantly higher than those in the control group(P<0.05); transwell assay showed that the cell invasion ability in the CXCR4-shRNA group was significantly decreased compared with that in the control group (P<0.05). ConclusionWe have successfully established an A498 cell line with CXCR4 stably silenced; the cell line has a higher apoptotic rate and lower invasion ability, which paves a way for future research.