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目的探讨瞬时受体电位C1(TRPC1)对人肾癌细胞A498细胞增殖和迁移的影响。方法利用荧光定量PCR检测人肾癌细胞A498、786-O和GRC-1中TRPC1的表达水平。设计并合成靶向TRPC1的特异性sh RNA,通过脂质体转染法转染TRPC1表达最高的人肾癌细胞A498以构建稳定低表达TRPC1细胞株,通过免疫印迹法和荧光定量PCR来验证sh RNA的干扰效率;通过MTT比色法检测干扰后细胞的增殖能力;通过Transwell小室测细胞迁移情况;免疫印迹法检测PI3K和AKT的表达水平。结果在3株肾癌细胞中A498的TRPC1表达量最高;特异性靶向TRPC1的sh RNA能够有效下调A498细胞中TRPC1的表达;特异性sh RNA干扰组细胞较其他两组细胞增殖显著减慢(F=42.31,P<0.01);A498/sh RNA组的迁移细胞数显著低于A498组和A498/Con组(F=24.52,P<0.01);A498/sh RNA组细胞的PI3K和AKT蛋白的表达较A498组和A498/Con组显著下调。结论采用RNA干扰技术能够有效沉默A498细胞的TRPC1基因,并致使其细胞增殖能力下降以及细胞迁移能力减弱,其中可能的机制为通过调节细胞因子PI3K来调控人肾癌细胞的增殖和迁移。提示TRPC1在肾癌的发生发展中起着较为重要作用,TRPC1可能成为治疗肾癌的新靶点。
Objective To investigate the effect of transient receptor potential C1 (TRPC1) on the proliferation and migration of human renal cell carcinoma A498 cells. Methods The expression of TRPC1 in human renal cell carcinoma A498, 786-O and GRC-1 cells was detected by real-time fluorescence quantitative PCR. The specific sh RNA targeting TRPC1 was designed and synthesized, and transfected into human renal carcinoma cell line A498 with the highest expression of TRPC1 by lipofection method to construct stable and low expression TRPC1 cell lines. The expression of sh was confirmed by immunoblotting and real-time PCR RNA interference efficiency; MTT colorimetric detection of cell proliferation after interference; Transwell chamber cell migration assay; Western blot analysis of PI3K and AKT expression levels. Results The expression of TRPC1 in A498 cells was the highest in three human renal carcinoma cell lines. ShRNA targeting TRPC1 could down-regulate the expression of TRPC1 in A498 cells. The expression of TRPC1 in specific shRNA group was significantly slower than that in the other two groups The number of migrated cells in A498 / sh RNA group was significantly lower than that in A498 group and A498 / Con group (F = 24.52, P <0.01); The levels of PI3K and AKT protein in A498 / sh RNA group Compared with A498 group and A498 / Con group, the expression was significantly down-regulated. Conclusion RNA interference can effectively silence the TRPC1 gene in A498 cells and lead to the decrease of cell proliferation and cell migration. The possible mechanism is to regulate the proliferation and migration of human renal cell carcinoma cells by regulating the expression of PI3K. Tip TRPC1 in the occurrence and development of renal cell carcinoma plays a more important role, TRPC1 may be a new target for the treatment of renal cell carcinoma.