木蝴蝶苷A通过调控MEK/ERK信号通路对肝癌细胞增殖侵袭和上皮间质转化的机制研究

Mechanisms of Luteolin A on Proliferation Invasion and Epithelial Mesenchymal Transition of Hepatocellular Carcinoma Cells through Regulation of MEK/ERK Signaling Pathway

目的:探究木蝴蝶苷A通过调控MEK/ERK信号通路对肝癌细胞增殖、侵袭和上皮间质转化的机制。方法:将肝癌细胞HepG2分为Control组、OAL组、OAM组、OAH组、PD98059组和ML-099组。MTT法检测细胞增殖能力;Transwell小室法检测细胞侵袭能力;流式细胞仪检测细胞凋亡能力;蛋白质印迹法检测细胞中MEK.p-MEK、ERK.p-ERK蛋白及EMT相关蛋白表达。结果:OAL组、OAM组和OAH组细胞增殖和侵袭能力(82.34±2.43)、(61.04±1.61)、(32.44±1.04)均明显低于Control(110.42±3.86)组,细胞凋亡率(6.81±0.62)、(11.74±0.94)、(18.36±1.05)明显高于Control组(5.01±0.53)(F侵袭=1068,F凋亡=323.1,P<0.0001);细胞中上皮间质转化蛋白E-cadherin蛋白(0.42±0.04)、(0.58±0.05)、(0.97±0.09)高于Control组(0.23±0.03)(F=181.1,P<0.0001);上皮间质转化蛋白N-cadherin(0.63±0.06)、(0.51±0.05)、(0.38±0.03)、Vimentin蛋白(1.23±0.11)、(0.98±0.09)、(0.51±0.05)及p-MEK/MEK(0.72±0.07)、(0.51±0.05)、(0.32±0.03)和p-ERK/ERK(0.92±0.09)、(0.68±0.06)、(0.41±0.04)比值低于Control组(F_(N-cadherin)=39.04,F_(Vimentin)=111.0,F_(p-MEK/MEK)=107.8,F_(p-ERK/ERK)=82.68,P<0.0001)。PD98059组细胞增殖和侵袭能力(30.86±1.01)均明显低于Control组(t=48.84,P<0.0001),细胞凋亡能力(17.95±0.98)明显高于Control组(t=28.45,P<0.0001),细胞中上皮间质转化蛋白E-cadherin蛋白(0.92±0.09)明显高于Control组(t=17.82,P<0.0001),上皮间质转化蛋白N-cadherin(0.37±0.04)(t=9.585)、Vimentin蛋白(0.41±0.04)(t=19.99)及p-MEK/MEK(0.30±0.03)(t=16.78)、p-ERK/ERK(0.39±0.04)(t=14.65)比值均明显低于Control组(P<0.0001)。ML-099组细胞增殖和侵袭能力(100.86±2.68)均明显高于OAH组(t=12.54,P<0.0001),细胞凋亡能力(5.15±0.86)明显低于OAH组(t=23.84,P<0.0001);和OAH组相比,ML-099组细胞中上皮间质转化蛋白E-cadherin蛋白(0.25±0.03)明显减少(t=18.59,P<0.0001),上皮间质转化蛋白N-cadherin(0.69±0.07)(t=0.971,P<0.0001)、Vimentin蛋白(1.50±0.12)(t=18.65,P<0.0001)及p-MEK/MEK(0.94±0.09)(t=16.01,P<0.0001)、p-ERK/ERK(1.05±0.10)(t=2.367,P=0.0395)比值均明显升高。结论:木蝴蝶苷A可抑制肝癌细胞增殖、侵袭和上皮间质转化,诱导肝癌细胞凋亡,其机制和抑制MEK/ERK信号通路有关。

Objective:To investigate the mechanism of luteolin A on proliferation,invasion and epithelial mesenchymal transformation of hepatocellular carcinoma cells by regulating MEK/ERK signaling pathway.Methods:HepG2 cells were divided into control group,OAL group,OAM group,OAH group,PD98059 group and ML-099 group.MTT assay was used to detect cell proliferation;Transwell chamber method was used to detect the invasive ability of cells;Apoptosis was detected by flow cytometry;the expression of MEK.p-MEK,ERK.p-ERK and EMT related proteins were detected by Western blot.Results:The cell proliferation and invasion ability of OAL group,OAM group and OAH group(82.34±2.43),(61.04±1.61)and(32.44±1.04)were significantly lower than those of the control group(110.42±3.86),and the cell apoptosis rate in those group(6.81±0.62),(11.74±0.94)and(18.36±1.05)were significantly higher than those of the control group(5.01±0.53)(F invasion=1068,F apoptosis=323.1,P<0.001);the expression of E-cadherin protein(0.42±0.04),(0.58±0.05)and(0.97±0.09)in the cells was higher than that in the control group(0.23±0.03)(F=181.1,P<0.001);N-cadherin(0.63±0.06),(0.51±0.05),(0.38±0.03),Vimentin(1.23±0.11),(0.98±0.09),(0.51±0.05)and p-MEK/MEK(0.72±0.07),(0.51±0.05),(0.32±0.03)and p-ERK/ERK(0.92±0.09),(0.68±0.06).The ratio(0.41±0.04)was lower than that of the control group(FF_(N-cadherin)=39.04,FF_(Vimentin)=11.0,FF_(p-MEK/ME)=107.8,FF_(p-MEK/MEK)=82.68,P<0.001).The cell proliferation and invasion ability(30.86±1.01)of PD98059 group was significantly lower than that of the control group(t=48.84,P<0.001),the cell apoptosis ability(17.95±0.98)was significantly higher than that of the control group(t=28.45,P<0.001),the epithelial mesenchymal transformation protein E-cadherin protein(0.92±0.09)was significantly higher than that of the control group(t=17.82,P<0.001),the epithelial mesenchymal transformation protein N-cadherin(0.37±0.04)(t=9.585),vimentin protein(0.41±0.04)(t=19.99)and p-MEK/MEK(0.30±0.03)(t=16.78),p-ERK/ERK(0.39±0.04)(t=14.65)were significantly lower than those in the control group(P<0.001).The cell proliferation and invasion ability(100.86±2.68)of ML-099 group was significantly higher than that of OAH group(t=12.54,P<0.001),and the cell apoptosis ability(5.15±0.86)was significantly lower than that of OAH group(t=23.84,P<0.001);compared with OAH group,E-cadherin protein(0.25±0.03)was significantly decreased in ML-099 group(t=18.59,P<0.001),and N-cadherin protein(0.69±0.07)(t=0.971,P<0.001),Vimentin protein(1.50±0.12)(t=18.65,P<0.0001),p-MEK/MEK(0.94±0.09)(t=16.01,P<0.001),p-ERK/ERK(1.05±0.10)(t=2.367,P=0.0395)were significantly increased in ML-099 group.Conclusion:Luteolin A can inhibit the proliferation,invasion and epithelial mesenchymal transformation of liver cancer cells,and induce apoptosis of liver cancer cells.Its mechanism is related to inhibition of MEK/ERK signaling pathway.