柚皮苷调节PERK/eIF2α/ATF4/CHOP轴对甲状腺癌细胞增殖凋亡迁移侵袭的影响

Effect of Naringenin Regulation of PERK/eIF2α/ATF4/CHOP Axis on Proliferation Apoptosis Migration and Invasion of Thyroid Cancer Cells

目的:分析柚皮苷通过调节蛋白激酶R样内质网激酶/真核生物起始因子2α/活化转录因子4/CCAAT/增强子结合蛋白同源蛋白(PERK/e IF2α/ATF4/CHOP)轴对甲状腺癌细胞B-CPAP增殖、凋亡、迁移、侵袭的影响。方法:采用1、5、10、20μmoL/L柚皮苷处理B-CPAP细胞,采用5μmoL/L PERK抑制剂GSK2606414+20μmoL/L柚皮苷处理B-CPAP细胞作为抑制剂组,另以不加柚皮苷的BCPAP细胞作为对照组,MTT法检测各组B-CPAP细胞的增殖;流式细胞术检测各组B-CPAP细胞的凋亡;细胞划痕实验检测各组B-CPAP细胞的迁移情况;Transwell实验检测各组B-CPAP细胞的侵袭;Western bolt法检测各组B-CPAP细胞PERK/e IF2α/ATF4/CHOP轴相关蛋白、细胞波形蛋白(Vimentin)、E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)、Bcl-2相关X蛋白(Bax)、B细胞淋巴瘤-2(Bcl-2)的表达水平。结果:与对照组相比,经1、5、10、20μmoL/L柚皮苷处理48h后,B-CPAP细胞增殖率依次下降[(100.00±0.00)%比(90.73±5.25)%、(76.52±3.19)%、(62.84±3.25)%、(48.39±2.14)%],呈浓度依赖性(P<0.05);20μmoL/L柚皮苷处理12h、24h、48h,B-CPAP细胞增殖率依次下降[(82.37±3.06)%比(71.45±2.73)%比(48.39±2.14)%],呈时间依赖性(P<0.05)。与对照组相比,随柚皮苷浓度的递增(1、5、10、20μmoL/L),B-CPAP细胞凋亡率增加[(6.37±0.44)%比(9.46±0.83)%、(18.59±2.16)%、(33.41±4.29)%、(50.76±5.17)%],B-CPAP细胞划痕愈合率[(62.74±2.86)%比(55.26±2.49)%、(46.38±2.17)%、(39.15±1.84)%、(33.42±1.39)%]、穿膜细胞数[(70.54±6.12)个比(57.78±5.62)个、(42.59±4.86)个、(29.27±3.91)个、(65.62±5.83)个]减少,B-CPAP细胞p-PERK/PERK、peIF2α/eIF2α、ATF4、CHOP、E-cadherin、Bax蛋白水平逐渐升高,Vimentin、N-cadherin、Bcl-2蛋白水平逐渐降低,呈浓度依赖性(P<0.05)。抑制剂组B-CPAP细胞增殖率[(94.16±5.38)%比(48.39±2.14)%]、划痕愈合率[(58.65±2.73)%比(33.42±1.39)%]、穿膜细胞数[(65.62±5.83)个比(16.49±3.04)个]高于20μmo L/L组(P<0.05),细胞凋亡率[(7.82±0.65)%比(50.76±5.17)%]及p-PERK/PERK、p-eIF2α/e IF2α、ATF4、CHOP、E-cadherin、Bax蛋白水平均低于20μmoL/L组,Vimentin、N-cadherin、Bcl-2蛋白水平均高于20μmoL/L组(P<0.05)。结论:柚皮苷能通过激活PERK/eIF2α/ATF4/CHOP轴抑制B-CPAP细胞增殖、促进其凋亡,降低B-CPAP细胞迁移、侵袭能力。

Objective:To analyze the effects of naringin on the proliferation,apoptosis,migration and invasion of thyroid cancer cells B-CPAP by regulating protein kinase R-like ER kinase/eukaryotic translation initiation factor alpha 2/activating transcription factor 4/CCAAT/enhancer-binding protein homologous protein(PERK/eIF2α/ATF4/CHOP)axis.Methods:B-CPAP cells were treated with 1,5,10,20μmol/L naringin,B-CPAP cells treated with 5μmol/L PERK inhibitor GSK2606414+20μmol/L naringin were used as the inhibitor group,and B-CPAP cells without naringin as control group.MTT method was used to detect the proliferation of B-CPAP cells in each group;flow cytometry was used to detect apoptosis of B-CPAP cells in each group;cell scratch test was used to detect the migration of B-CPAP cells in each group;Transwell experiment was used to detect the invasion of B-CPAP cells in each group;and Western blot method was used to detect the expression levels of PERK/eIF2α/ATF4/CHOP axis-related proteins,Vimentin,E-cadherin,N-cadherin,Bcl-2 related X protein(Bax),B-cell lymphoma-2(Bcl-2)in B-CPAP cells of each group.Results:Compared with the control group,the proliferation rate of B-CPAP cells[(100.00±0.00)%vs.(90.73±5.25)%,(76.52±3.19)%,(62.84±3.25)%,(48.39±2.14)%]decreased sequentially after treatment with 1,5,10,and 20μmol/L naringin for 48,in a concentration-dependent manner(P<0.05);after treatment with 20μmol/L naringin for 12 h,24 h,and 48 h,the proliferation rate of B-CPAP cells decreased sequentially[(82.37±3.06)%vs.(71.45±2.73)%vs.(48.39±2.14)%],in a time-dependent manner(P<0.05).Compared with the control group,the apoptosis rate of B-CPAP cells[(6.37±0.44)%vs.(9.46±0.83)%,(18.59±2.16)%,(33.41±4.29)%,(50.76±5.17)%]increased with naringin concentration increasing(1,5,10,20μmol/L),the wound healing rate[(62.74±2.86)%vs.(55.26±2.49)%,(46.38±2.17)%,(39.15±1.84)%,(33.42±1.39)%]and the number of transmembrane cells of B-CPAP cells[(70.54±6.12)vs.(57.78±5.62),(42.59±4.86),(29.27±3.91),(65.62±5.83)]decreased,the levels of p-PERK/PERK,p-eIF2α/eIF2α,ATF4,CHOP,E-cadherin and Bax proteins in B-CPAP cells gradually increased,the levels of Vimentin,N-cadherin and Bcl-2 proteins in B-CPAP cells gradually reduced,in a concentration-dependent manner(P<0.05).The B-CPAP cell proliferation rate[(94.16±5.38)%vs.(48.39±2.14)%],scratch healing rate[(58.65±2.73)%vs.(33.42±1.39)%],and number of penetrating cells[(65.62±5.83)%vs.(16.49±3.04)%]in the inhibitor group were higher than those in the20μmol/L group(P<0.05),the apoptosis rate[(7.82±0.65)%vs.(50.76±5.17)%]and levels of p-PERK/PERK,p-e IF2α/eIF2α,ATF4,CHOP,E-cadherin and Bax proteins were lower than those in the20μmol/L group,the levels of Vimentin,N-cadherin and Bcl-2 proteins were higher than those in the 20μmol/L group(P<0.05).Conclusion:Naringin can inhibit the proliferation of B-CPAP cells,promote their apoptosis,and reduce the migration and invasion abilities of B-CPAP cells by activating the PERK/eIF2α/ATF4/CHOP axis.