摘要
目的肾母细胞瘤是一种高发的儿童实体瘤,发病率占儿童恶性肿瘤8%~10%,多发生于<5岁儿童,有研究表明某些信号通路可调控其增殖过程。本研究探讨TGF-β和IGF-1信号通路是否共同参与调控肾母细胞瘤细胞的增殖过程。方法采用siRNA转染实验将TGFBR1-siRNA和IGF1R-siRNA转至肾母细胞瘤细胞株G401,以转染不同siRNA分为TGFBR1-siRNA组和IGF1R-siRNA组,siRNA无关序列(non-siRNA)转染组作为对照组,每组设6个复孔。MTS方法检测TGFBR1-siRNA和IGF1R-siRNA对细胞增殖率的影响,蛋白质印迹法检测TGF-β信号通路TGFBR1及下游蛋白p-ERK、SMAD2/3和p-AKT蛋白表达情况;蛋白质印迹法检测IGF-1信号通路IGF1R及下游蛋白p-AKT蛋白表达情况。ELISA法检测细胞上清中FGF9蛋白表达。结果抑制TGFBR1和IGF1R72和96h后,细胞的增殖率受到抑制,对照组增殖率设为100%。与其相比,转染TGFBR1-siRNA 72h后的实验组和对照组的增殖率分别为(80±14)%和(100±7)%,实验组低于对照组,P=0.034;转染IGF1R-siRNA 72h后实验组和对照组的增值率分别为(85±21)%和(100±7)%,实验组低于对照组,P=0.028。转染TGFBR1-siRNA 96h后的实验组和对照组的增殖率分别为(81±13)%和(100±11)%,实验组低于对照组,P=0.021;转染IGF1R-siRNA 96h后实验组和对照组的增殖率分别为(82±17)%和(100±11)%,实验组低于对照组,P=0.038。抑制TGFBR1后,与non-siRNA组相比,下游蛋白FGF-9、p-ERK、SMAD2/3和p-AKT明显受到抑制。TGFBR1-siRNA转染72h后,实验组细胞上清中FGF9蛋白表达量显著下降,实验组为(533.85±54.64)ρg/mL,对照组为(587.34±46.83)ρg/mL,P=0.041;96h后实验组FGF9蛋白表达量仍显著低于对照组,实验组为(495.03±59.26)ρg/mL,对照组为(605.34±49.28)ρg/mL,P=0.018。TGFBR1-siRNA转染48h后,p-ERK、SMAD2/3和p-AKT蛋白表达量出现降低;72h后,p-ERK和SMAD2/3蛋白表达量仍降低。与non-siRNA转染对照组相比,IGF1R-siRNA转染72和96h后,p-AKT蛋白表达量出现降低。结论TGF-β和IGF-1信号通路共同参与肾母细胞瘤细胞的增殖,两条信号通路可能通过共同的下游蛋白AKT参与对肾母细胞瘤增殖的调控,TGFBR1和IGF1R可以作为肾母细胞瘤治疗的靶点深入研究。
OBJECTIVE Nephroblastoma is one of the most common solid tumours in children,with an annual incidence of approximately 8%-10% of all neoplasms in that group.The peak incidence occurs between 1to 5years of age.The objective of this study was to explore whether TGF-βand IGF-1signaling pathways interact to regulate the proliferation of the Wilms’ tumor.METHODS siRNA transfection experiment was used for TGFBR1-siRNA,IGF1R-siRNA being transferred to the G401 cell line.Cells were grouped as TGFBR1-siRNA,IGF1R-siRNA and non-siRNA（control group）.Each test was repeated in six wells.MTS assay was used for cell proliferation assay.Western-blot was performed to detect the expression of p-ERK,SMAD2/3and p-AKT,the concentration of FGF9 in the supernatant was determined by ELISA.RESULTS The proliferation of G401 cells was significantly decreased at the time point of 72,96-hour of TGFBR1-siRNA,IGF1R-siRNA transferred to the G401.Proliferation of non-siRNA cells were considered as 100%,and the relative proliferation rate of TGFBR1-siRNA group and control group at 72-hour were（80±14）% and（100±7）%,P=0.034.IGF1R-siRNA group and control group at 72-hour were（85±21）% and（100±7）%,P=0.028.For96-hour of TGFBR1-siRNA group and control group were（81±13）% and（100±11）%,P=0.021;IGF1R-siRNA group and control group were（82±17）% vs（100±11）%,P=0.038.The expression of p-ERK,SMAD2/3and p-AKT were decreased significantly at the 48 hfor TGFBR1-siRNA group compared with non-siRNA group detected by Western-blot.p-ERK,SMAD2/3were still decreased significantly at the 72 hcompared with non-siRNA group.The concentrations of FGF9 at 72hwere（533.85±54.64）ρg/mL in TGFBR1-siRNA group compared with non-siRNA group（587.34±46.83）ρg/mL（P=0.041）,and TGFBR1-siRNA group was（495.03±59.26）ρg/mL compared with non-siRNA group（605.34±49.28）ρg/mL for 72 htime point（P=0.018）.p-AKT was decreased significantly at the 72,96 hfor IGF1RsiRNA group compared with non-siRNA group.CONCLUSIONS The TGF-βand IGF-1signaling pathway may regulate the prolifetation of Wilms’ tumor cells.The two pathways regulate the prolifetation process of Wilms’ tumor through downstreame protein AKT that maybe belongs to the two signaling pathway.TGFBR1 and IGF1Ras a therapeutic target is our future research direction.
出处
《中华肿瘤防治杂志》
CAS
北大核心
2017年第12期813-816,共4页
Chinese Journal of Cancer Prevention and Treatment
基金
北京市自然科学基金(7042017)
