TGF-β和IGF-1信号通路调控肾母细胞瘤细胞增殖机制探讨

发布时间：2018-03-07 09:51

本文选题：肾母细胞瘤　切入点：信号通路　出处：《中华肿瘤防治杂志》2017年12期 　论文类型：期刊论文

【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。
[Abstract]:Objective nephroblastoma is a kind of high incidence solid tumor in children. The incidence of nephroblastoma is 8% and 10% of malignant tumors in children, most of which occur in children aged 5 years. Some studies have shown that some signaling pathways can regulate the proliferation process. This study is to investigate whether TGF- 尾 and IGF-1 signaling pathways are involved in the regulation of the proliferation of nephroblastoma cells. Methods TGFBR1-siRNA and IGF1R-siRNA were transferred to the nephroblastoma cells by siRNA transfection experiment. The tumor cell line G401 was divided into two groups: TGFBR1-siRNA group and IGF1R-siRNA group. The effects of TGFBR1-siRNA and IGF1R-siRNA on cell proliferation were detected by six multiple holes. The expression of TGFBR1 and p-ERKN SMAD2 / 3 and p-AKT proteins in TGF- 尾 signaling pathway and downstream proteins were detected by Western blot. IGF-1 signaling pathway IGF1R and downstream protein p-AKT protein expression were detected by Western blot. Elisa method was used to detect the expression of FGF9 protein in the supernatant. Results after inhibiting TGFBR1 and IGF1R72 for 96 h, the cell proliferation rate was inhibited, and the control group proliferation rate was set to 100th. The proliferation rates of the experimental group and the control group were 80 卤14% and 100 卤7, respectively, which were lower than those of the control group after 72 hours of transfection of IGF1R-siRNA, and the proliferation rates of the experimental group and the control group were 85 卤21% and 100 卤7% respectively after 72 hours of IGF1R-siRNA transfection, and were lower than that of the control group after 96 h of TGFBR1-siRNA transfection. The proliferative rates of the test group and the control group were 81 卤13% and 100 卤11%, respectively. The proliferative rate of the experimental group was lower than that of the control group (0.021), the proliferation rate of the experimental group and the control group was 82 卤17% and 100 卤11% respectively after transfection of IGF1R-siRNA for 96 h, and the proliferation rate of the experimental group was lower than that of the control group (0.038%). Compared with the non-siRNA group, the expression of FGF9 protein in the supernatant of the experimental group decreased significantly 72 hours after transfection of the downstream protein FGF-9, p-ERKT, SMAD2 / 3 and p-AKT. The expression of FGF9 protein in the supernatant of the experimental group was significantly lower than that in the control group (533.85 卤54.64) 蟻 g / mLL, and in the control group (587.34 卤46.83) 蟻 / mLPU 0.041 and 96h after transfection, the expression of FGF9 protein in the experimental group was still significantly lower than that in the control group. The expression of p-ERK and p-AKT protein were decreased in the experimental group (495.03 卤59.26) 蟻 g / mLand the control group (605.34 卤49.28) 蟻 g / mLPt0.018.18 TGFBR1-siRNA transfection 48 h after transfection. Conclusion the expression of p-ERK and p-AKT protein is still decreased after 72 and 96 hours compared with the control group transfected with non-siRNA. Conclusion the expression of p-ERK and p-AKT protein is decreased after 72 and 96 hours compared with the control group transfected with non-siRNA. And IGF-1 signaling pathway involved in the proliferation of nephroblastoma cells. Two signaling pathways may be involved in the regulation of the proliferation of nephroblastoma by common downstream protein AKT. TGFBR1 and IGF1R may be used as targets for the treatment of nephroblastoma.
【作者单位】：首都儿科研究所生物化学研究室;首都儿科研究所分子免疫研究室;
【基金】：北京市自然科学基金(7042017)
【分类号】：R737.11

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