RNA干扰瞬时受体电位阳离子通道1表达抑制缺氧诱导的胶质瘤血管内皮生长因子上调的实验研究

Experimental study on down-regulation of TRPC1 inhibits expression of VEGF induced by hypoxia in glioma cells

目的探讨瞬时受体电位阳离子通道1(TRPC1)是否参与缺氧诱导的胶质瘤细胞血管内皮生长因子(VEGF)表达。方法通过RT-PCR和钙图检测技术观察U87细胞上表达的TRPC亚型;采用RNAi技术、real-time RT-PCR、免疫印迹和ELISA方法,在基因和外分泌蛋白水平观察TRPC1对缺氧U87细胞VEGF表达的影响。结果 U87胶质瘤细胞表达TRPC1,TRPC3,TRPC4和TRPC5亚型。TRPC通道激动剂OAG增加了胞内Ca2+浓度,而TRPC通道阻断剂2-APB可抑制激动剂诱导的胞内Ca2+浓度增加。化学合成的siRNA-1,siRNA-2和siRNA-3分别减少TRPC1 mRNA到64%,39%和36%(P<0.01);分别减少TRPC1蛋白到48%,29%和33%(P<0.01)。siRNA-2和siRNA-3显著抑制了缺氧诱导的VEGF基因上调(P<0.01)。同时缺氧U87细胞VEGF蛋白的分泌亦受到抑制(P<0.01)。结论 U87胶质瘤细胞表达功能性的TRPC通道,TRPC1参与缺氧诱导的胶质瘤VEGF表达。

Objective To investigate whether TRPC1 was involved in VEGF expression induced by hypoxia in U87 MG cells. Methods RT-PCR and calcium image technique were used to observe the TRPC subtypes existed in U87 cells. RNAi, real-time RT-PCR, western blot and ELISA technique were used to observe effects of TRPC1 on hypoxia- induced VEGF gene expression and secretion. Results TRPC1, TRPC3, TRPC4 and TRPC5 were found expressed in U87 cell. Calcium concentration of U-87 cells was increased by OAG （agonist of TRPC）, OAG-induced calcium con- centration rise was attenuated by 2-APB （antagonist of TRPC）. TRPC1 mRNA were reduced to 64%, 39% and 36% respectively by the chemical synthesis of siRNA-1, siRNA-2, siRNA-3 （P 〈 0.01）; TRPC1 protein were reduced to 48%, 29%, 33% respectively by the chemical synthesis of siRNA-1, siRNA-2, siRNA-3 （P 〈 0.01）. Hypoxia en- hanced VEGF gene up-regulation was restrained by siRNA-2 and siRNA-3 （P 〈 0.01）; VEGF gene expression in Hypoxia U87 cell was restrained too （P 〈 0,01）. Conclusion U87 glioma cells express the functional TRPC channels; TRPC1 participates the VEGF expression induced by hypoxia.