核心蛋白聚糖对膀胱癌细胞增殖、迁移及侵袭的影响

Effects of decorin on proliferation,migration and invasion of bladder cancer cells

目的:探讨核心蛋白聚糖(DCN)对膀胱癌细胞增殖、迁移及其侵袭的影响。方法:以膀胱癌T24细胞株为研究对象,利用MTT法检测24、48、72、96 h不同浓度(0、5、10、20、30、40、50 mg/L)DCN对T24细胞增殖的抑制作用,流式细胞仪分析DCN对T24细胞周期及凋亡的影响,MTT法、Transwell细胞迁移和细胞侵袭实验分别检测DCN对T24细胞的黏附、迁移及侵袭的影响,ELISA及蛋白质印迹法分别检测DCN对TGF-β1和P21蛋白表达的影响。结果:用浓度为0、5、10、20、30、40、50 mg/L DCN处理T24细胞,在24、48、72、96 h时细胞增殖活力差异均具有统计学意义(F=168.64,P<0.001;F=165.81,P<0.001;F=291.02,P<0.001;F=148.93,P<0.001);用0、5、10、20、30、40和50 mg/L浓度的DCN处理T24细胞72 h时,细胞增殖活力分别为(60.71±3.03)%、(40.82±2.09)%、(37.24±1.63)%、(25.65±2.55)%、(23.00±2.67)%、(10.78±1.17)%、(11.04±0.96)%,差异具有统计学意义,40 mg/L浓度时增殖活力达到最低水平,对细胞增殖的抑制作用最强。40、50 mg/L浓度时G 1期细胞达到高峰值,S期达最低,整个处理过程G 2期始终保持不变。0、5、10、20、30、40、50 mg/L浓度DCN作用72 h时的细胞凋亡率分别为(12.18±1.17)%、(21.24±1.05)%、(19.80±1.20)%、(26.52±1.40)%、(30.86±1.40)%、(52.99±1.22)%、(43.04±2.16)%,差异具有统计学意义(F=178.54,P<0.001);5、10、20、30、40、50 mg/L浓度DCN凋亡率与0 mg/L浓度DCN相比,差异均具有统计学意义(均P<0.001)。DCN作用于T24细胞72 h时,0、40 mg/L浓度时的细胞黏附率分别为(37.14±1.35)%、(59.86±1.95)%,差异具有统计学意义(t=25.25,P<0.001);迁移细胞数分别为53.86±3.18、12.86±1.35,差异具有统计学意义(t=31.36,P<0.001)。DCN作用于T24细胞48 h时,0、40 mg/L浓度时的侵袭数分别为235.14±3.44、160.86±3.13,差异具有统计学意义(t=2.27,P<0.001)。当T24细胞被DCN处理72 h后,0、5、10、20、30、40、50 mg/L浓度时的TGF-β1相对表达量分别为85.67±3.35、45.51±1.19、49.93±4.15、47.64±3.53、46.05±3.18、25.54±2.25、33.44±4.05,差异具有统计学意义(F=324.58,P<0.001),与0 mg/L相比,5、10、20、30、40、50 mg/L浓度均对TGF-β1的表达有明显的抑制作用(均P<0.001);与0 mg/L浓度相比,P21蛋白经40 mg/L DCN处理72 h后上调。结论:DCN在体外能够抑制T24细胞增殖,诱导其凋亡,并具有抗T24细胞转移的作用。

Objective To investigate the effects of decorin(DCN)on the proliferation,migration and invasion of bladder cancer cells.Methods Bladder cancer T24 cell line was used as the research object.MTT assay was used to detect the inhibitory effect of DCN at different concentrations(0,5,10,20,30,40,50 mg/L)on T24 cell proliferation at 24,48,72 and 96 h.The effects of DCN on T24 cell cycle and apoptosis were analyzed by flow cytometry.MTT assay,Transwell migration and invasion experiments were used to detect the effects of DCN on the adhesion,migration and invasion ability of T24 cells.The effects of DCN on TGF-β1 and P21 protein expression were detected by ELISA and Western blotting.Results T24 cells were treated with 0,5,10,20,30,40 and 50 mg/L DCN at 24,48,72 and 96 h,and there were statistically significant diffe-rences in cell proliferation activity(F=168.64,P<0.001;F=165.81,P<0.001;F=291.02,P<0.001;F=148.93,P<0.001).T24 cells were treated with 0,5,10,20,30,40 and 50 mg/L DCN for 72 h,and the cell proliferation activities were(60.71±3.03)%,(40.82±2.09)%,(37.24±1.63)%,(25.65±2.55)%,(23.00±2.67)%,(10.78±1.17)%,(11.04±0.96)%,respectively,and there was a statistically significant difference.At the concentration of 40 mg/L,the proliferation activity reached the lowest level,and the inhibitory effect on cell proliferation was the strongest.At concentrations of 40 and 50 mg/L,the cells in G1 phase reached the peak value,while the cells in S phase reached the lowest value,and the cells in G2 phase remained unchanged throughout the treatment process.T24 cells were treated with 0,5,10,20,30,40 and 50 mg/L DCN for 72 h,and the apoptosis rates of cells were(12.18±1.17)%,(21.24±1.05)%,(19.80±1.20)%,(26.52±1.40)%,(30.86±1.40)%,(52.99±1.22)%,(43.04±2.16)%,respectively,and there was a statistically significant difference(F=178.54,P<0.001).The differences between 5,10,20,30,40,50 mg/L DCN and 0 mg/L DCN were all statistically significant(all P<0.001).When T24 cells were treated with 0,40 mg/L DCN for 72 h,the cell adhesion rates were(37.14±1.35)%and(59.86±1.95)%,the numbers of migrated cells were 53.86±3.18 and 12.86±1.35,and there were statistically significant differences(t=25.25,P<0.001;t=31.36,P<0.001).When DCN was applied to T24 cells for 48 h,the numbers of invasion at 0,40 mg/L were 235.14±3.44 and 160.86±3.13,and there was a statistically significant difference(t=2.27,P<0.001).When T24 cells were treated with 0,5,10,20,30,40 and 50 mg/L DCN for 72 h,the relative expression levels of TGF-β1 were 85.67±3.35,45.51±1.19,49.93±4.15,47.64±3.53,46.05±3.18,25.54±2.25,33.44±4.05,and there was a statistically significant difference(F=324.58,P<0.001).Compared with 0 mg/L DCN,5,10,20,30,40 and 50 mg/L DCN could significantly inhibited the expression of TGF-β1(all P<0.001).Compared with 0 mg/L DCN,P21 protein was upregulated 72 h after treatment with 40 mg/L DCN.Conclusion DCN can inhibit proliferation and induce apoptosis of T24 cells in vitro,and has the effect of anti-metastasis of T24 cells.