下调FoxM1表达对人喉癌Hep-2细胞顺铂敏感性的影响

FoxM1 down-regulation promotes sensitivity of laryngeal carcinoma Hep-2 cells to cisplatin

目的探讨下调Fox M1表达对人喉癌Hep-2细胞顺铂敏感性的影响及其可能机制。方法分别选用Fox M1 siRNA以及Fox M1特异性抑制剂(硫链丝菌肽)下调Hep-2细胞Fox M1表达。实时定量PCR、Western blot检测siRNA或硫链丝菌肽处理细胞后Fox M1 mRNA、蛋白表达量;MTT法检测下调Fox M1表达后Hep-2细胞的顺铂敏感性;流式细胞术检测下调Fox M1表达后顺铂诱导的凋亡率变化;实时定量PCR、Western blot检测顺铂作用于Fox M1下调组及对照组细胞,其Fox M1 mRNA、蛋白及凋亡相关蛋白Bcl-2、Bax表达变化。结果 siRNA及硫链丝菌肽均能下调Fox M1表达(P<0.05);两种方式均能降低顺铂作用下细胞存活率以及IC50[NC组顺铂IC50=(2.609±0.102)μg/m L,干扰组IC50=(0.771±0.058)μg/m L,P<0.05;对照组顺铂IC50=(2.142±0.198)μg/m L,抑制剂组IC50=(0.773±0.063)μg/m L,P<0.05],siRNA法处理后NC组、干扰组、NC+顺铂组、干扰+顺铂组凋亡率依次为(4.197±0.273)%、(12.553±0.183)%、(37.465±4.305)%、(82.373±7.214)%,干扰+顺铂组凋亡率显著升高(P<0.05);抑制剂处理后对照组、抑制剂组、顺铂组、抑制剂+顺铂组凋亡率依次为(2.343±0.194)%、(10.127±0.479)%、(35.075±1.995)%、(62.843±1.824)%,抑制剂+顺铂组凋亡率显著升高(P<0.05);下调Fox M1表达,凋亡抑制蛋白Bcl-2下调,促凋亡蛋白Bax表达上调(P<0.05)。结论下调Fox M1表达可提高Hep-2细胞对顺铂的敏感性,其机制可能与凋亡相关蛋白Bcl-2下调、Bax上调相关。

Objective To determine the effect of down-regulation of forkhead box protein M1 （FoxM1） on the cisplatin sensitivity of human laryngeal carcinoma Hep-2 cells and investigate the potential mechanisms. Methods Small interfering RNA （siRNA） and FoxM1 inhibitor thiostrepton were used respectively to suppress FoxM1 expression in Hep-2 cells. The expression of FoxM1 at mRNA and protein levels was detected by qRT-PCR and Western blotting respectively. Cell viability and apoptosis of Hep-2 cells were detected by MTT assay and flow cytometry. Furthermore, qRT-PCR and Western blotting were used to investigate the changes of FoxM1 in Hep-2 cells with FoxM1 suppression and cisplatin treatment. The changes of apoptosis-related proteins were detected by Western blotting. Results Both of siRNA and thiostrepton suppressed FoxM1 expression, reduced the cell survival after cisplatin treatment and IC50 （cisplatin treated normal cells IC50 = 2.609 ±0. 102 g/mL, siRNA infected cells IC50 = 0.771 ±0.058 g/mL, P 〈 0. 05 ; cisplatin treated cells IC50=2. 142±0. 198 g/mL, thiostrepton treated cells IC50 =0.773 ±0. 063 g/mL, P 〈0. 05）. The apoptotic rate was （4. 197 ±0. 273）%, （12. 553 ±0. 183）%, （37. 465 ±4. 305）% and （82. 373 ±7. 214） % respectively in the normal Hep-2 cells and the cells with siRNA infection, cisplatin treatment and infection plus cisplatin treatment, with that of the latter group of cells the highest （P 〈 0.05 ）. The rate was （2. 343 3 ±0. 193 6）%, （10. 127 ±0.479）%, （35. 075 ± 1. 995）% and （62. 843 ±1. 824）% separately in the normal Hep-2 cells and the cells after thiostrepton treatment, cisplatin treatment and thiostrepton plus cisplatin treatment, with that of the latter group of cells the highest （ P 〈 0. 05 ）. Down-regulation of FoxM1 inhibited the expression of Bcl-2 and up-regulated that of Bax. Conclusion FoxM1 promotes the sensitivity of Hep-2 cells to cisplatin, which potentially is due to its down-regulation of Bcl-2 and up-regulation of Bax.