华蟾素对TGF-β1诱导人结肠癌细胞上皮-间质化的实验研究

摘要

目的 研究华蟾素在体外对肿瘤坏死因子-β1(TGF-β1)诱导的人结肠癌细胞上皮-间质化(EMT)的作用.方法 将体外培养的人结肠癌细胞株(SW480)分为:(1)正常对照组;(2)TGF-β1 (10 ng/mL)单独处理组;(3)TGF-β1(10 ng/mL)+华蟾素(2.5、5、10、20、40、80 mg/mL)共同处理组,并在体外培养48 h.CCK8检测细胞增殖抑制情况,利用倒置相差显微镜观察各处理组细胞形态学变化,Transwell小室检测细胞侵袭与迁移能力,实时荧光定量PCR(QRT-PCR)和Western blot分别检测E-钙黏蛋白(E-cadherin)、波形蛋白(Vimentin)在mRNA及蛋白水平表达的变化.结果 (1)与TGF-β1(10 ng/mL)单独处理组比较,TGF-β1(10 ng/mL)+华蟾素(10、20、40、80 mg/mL)共同处理组对SW480具有显著的增殖抑制作用(P＜0.05).(2)与正常对照组相比,TGF-β1单独处理组、TGF-β1(10 ng/mL)+华蟾素(2.5 mg/mL)共同处理组呈现典型的间质化表型,TGF-β1 (10 ng/mL)+华蟾素(5 mg/mL)共同处理组则呈现经典的上皮表型.(3)TGF-β1(10 ng/mL)+华蟾素(2.5、5mg/mL)共同处理组与TGF-β1单独处理组相比侵袭和迁移能力均明显减弱(P＜0.05).(4) QRT-PCR和Western blot结果显示TGF-β1单独处理组与正常对照组比较,Vimentin表达水平明显增强,E-cadherin表达显著减弱.TGF-β1 (10 ng/mL)+华蟾素(2.5、5 mg/mL)共同处理组与TGF-β1单独处理组、对照组相比Vimentin表达水平显著降低,E-cadherin表达显著增强.结论 华蟾素可抑制TGF-β1诱导的人结肠癌细胞SW480的增殖,其机制可能与促进E-钙黏蛋白表达增强,同时使波形蛋白表达减弱,从而与抑制TGF-β1诱导的EMT过程有关.%Objective To investigate the effects of cinobufacini on TGF-β1-induced epithelial-to-mesenchymal transition of human colorectal carcinoma cells in vitro.Methods The cultured colorectal carcinoma cell line(SW480) was divided into the control group,TGF-β1 (10 ng/mL) individual treatment group and co-treatment groups with TGF-β1 (10 ng/mL) + cinobufacini (2.5,5,10,20,40,80 mg/mL),which were cultured in vitro for 48 h.The proliferation of the cells were measured by CCK8 assay.The morphological changes were observed by inverted phase contrast microscope.The ability of cell invasion and migration was detected by Transwell assay.The mRNA and protein expressions of E-cadherin and Vimentin were detected with QRT-PCR and Western Blot.Results (1)Compared with the TGF-β1 (10 ng/mL) individual treatment group,TGF-β1 (10 ng/mL) + cinobufacini (10,20,40,80 mg/mL) co-treatment groups significantly had significantly proliferation inhibitory effect on SW480 (P＜0.05).(2) Compared with the normal control group,TGF-β1 individual treatment group and TGF-β1 (10 ng/mL) + cinobufacini(2.5 mg/mL) group exhibited classical mesenchymal phenotype,while the TGF-β1 (10 ng/mL) + cinobufacini (5 mg/mL) co-treatment group showed classical epithelial phenotype.(3) The ability of invasion and migration in the TGF-β1(10 ng/mL)+ cinobufacini(2.5,5 mg/mL) co-treatment group were significantly weakened compared with the TGF-β1 individual treatment group (P＜0.01).(4) QRT-PCR and Western Blot results indicated that compared with the normal control group,the Vimentin expression in the in the TGF-β1 individual treatment group was significantly increased and the E-cadherin expression was significantly decreased.Furthermore,compared with the TGF-β1 individual treatment group and control group,the Vimentin expression level in the TGF-β1 (10 ng/mL) + cinobufacini (2.5,5 mg/mL) groups was significantly decreased and E-cadherin expression was significantly increased.Conclusion Cinobufacini can inhibit TGF-β1-induced cell proliferation in human colorectal carcinoma SW480 cells,and its mechanism may be related with promoting E-cadherin expression increase,meanwhile decreasing the vimentin expression,thus inhibiting the EMT process induced by TGF-β1.