氟尿嘧啶调控VHL/HIF信号通路抑制肾癌细胞增殖、侵袭与转移的研究

Fluorouracil regulates VHL/HIF signaling pathway and inhibits proliferation, invasion and metastasis of renal carcinoma cells

目的:探讨氟尿嘧啶通过调控VHL/HIF信号通路影响肾癌细胞增殖、侵袭与转移。方法:体外培养人肾癌786-0细胞,分别用终浓度为6、60、600μg/mL的氟尿嘧啶处理肾癌细胞,噻唑蓝(MTT)检测氟尿嘧啶对肾癌细胞增殖的影响,划痕实验检测细胞转移能力的影响,Transwell实验检测细胞体外侵袭能力的影响。Western blot检测细胞中VHL/HIF信号通路相关蛋白HIF-1α、VEGF及活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved Caspase-3)、基质金属蛋白酶-9(MMP-9)的表达。通过细胞转染法转染pcDNA-HIF-1α后Western blot检测转染效率,同样的方法检测对肾癌细胞增殖、侵袭和转移的影响。结果:氟尿嘧啶处理后的肾癌细胞增殖明显受到抑制,侵袭和转移能力明显降低,且呈现出时间和浓度依赖性(P<0.05),相关蛋白HIF-1α、VEGF、MMP-9表达下调,Cleaved Caspase-3表达上调,转染pcDNA-HIF-1α后,HIF-1α的表达上调,肾癌细胞增殖活性降低,侵袭和转移能力减弱,与对照组相比,差异具有统计学意义(P<0.05)。结论:氟尿嘧啶能抑制肾癌细胞增殖、侵袭与转移,其作用机制与通过阻断VHL/HIF信号通路,调节Cleaved Caspase-3和MMP-9的表达有关。

AIM:To investigate the effect of fluorouracil on the proliferation,invasion and metastasis of renal cell carcinoma by regulating VHL/HIF signaling pathway.METHODS:Human kidney cancer 786-0 cells were cultured in vitro.Renal cancer cells were treated with fluorouracil with final concentrations of 6,60,and 600μg/Ml,respectively.Effects of fluorouracil against kidney cancer cells proliferation was detected by MTT assay.Scratch test was applied to detect the effect of fluorouracil on the metastasis capacity of renal cell carcinoma,and Transwell assay to detect the effect of uracil on the invasion ability of renal cell carcinoma in vitro.Western blot analyzed the VHL/HIF signaling pathway-associated protein HIF-1α,VEGF,activated cysteine-containing aspartate proteinase 3(Cleaved Caspase-3),matrix metalloproteinases in renal cell carcinoma cells treated with fluorouracil and the effect of 9(MMP-9)expression.After transfection of pcDNA-HIF-1αby the cell transfection method,the transfection efficiency was detected by Western blot,and the same method was used to examine the effect on the proliferation,invasion and metastasis of renal cell carcinoma.RESULTS:The proliferation of renal cell carcinoma cells treated with fluorouracil was significantly inhibited,and the invasion and metastasis ability was significantly decreased,and it was time-and concentration-dependent(P<0.05).The expression of related proteins HIF-1α,VEGF,and MMP-9 was down-regulated,and Cleaved Caspase-3 the expression was up-regulated.After transfected with pcDNA-HIF-1α,the expression of HIF-1αis upregulated.The proliferation activity and invasion and metastasis ability of renal cell carcinoma were weakened.Compared with the control group,the difference was statistically significant(P<0.05).CONCLUSION:Fluorouracil can inhibit the proliferation,invasion and metastasis of renal cancer cells,and its mechanism is related to blocking the VHL/HIF signaling pathway and regulating the expression of Cleaved Caspase-3 and MMP-9.