蛋白酶体抑制剂对甲状腺未分化癌细胞ATF-4表达影响的研究

Effects of proteasome inhibitors on expression of activating transcription factor-4 of undifferentiated thyroid cancer cells

目的:探讨蛋白酶体抑制剂对甲状腺未分化癌FRO细胞中活化转录因子4(ATF-4)表达的影响,以及ATF-4在蛋白酶体抑制剂诱导FRO细胞凋亡中的作用。方法:选取人甲状腺未分化癌细胞系FRO,分别设空白对照组和蛋白酶体抑制剂MG132处理组;利用微小RNA干扰技术,减少ATF-4的表达,实时定量PCR法、蛋白质印迹法分别检测各组细胞中ATF-4、氧调节蛋白150(ORP150)mRNA和蛋白表达;流式细胞仪(FCM)检测细胞凋亡。结果:与空白对照组相比,蛋白酶体抑制剂MG132显著增加FRO细胞系中ATF-4基因表达水平,P<0.01;与空白对照组和随机序列核酸siRNA组相比,siATF-4处理组中ATF-4、ORP150mRNA及蛋白表达水平显著降低(P<0.01),其凋亡率显著增加,P<0.01。结论:蛋白酶体抑制剂能够上调甲状腺未分化癌FRO细胞系中ATF-4基因的表达水平,siATF-4具有降低ATF-4及OPR150基因表达的作用,同时增加蛋白酶体抑制剂诱导甲状腺未分化癌FRO细胞的凋亡作用。

OBJECTIVE： To investigate the role of activating transcription factor （ATF）-4 in thyroid cancer ceil death induced by proteasome inhibitors. METHODS： The undifferentiated thyroideaneer FRO cells were treated with vehicle, proteasome inhibitor MG132; ATF-4 expression levels were knockdown by RNA interference; ATF-4 and 150 × 10^3 oxygen-regulated protein （ORP150） mRNA and protein levels were analyzed by using real time RT-PCR and Western blot,respectively; the apoptotie rate was analyzed by using flow cytometry（FCM）. RESULTS： MG132 significantly in- creased the mRNA and protein levels of ATF-4 （P〈0.01） in undifferentiated thyroid cancer FRO cells small interfering RNA against ATF-4 （si ATF-4） markedly reduced the ATF-4 and ORP150 mRNA and protein levels when compared to vehicle or scramble siRNA （P〈0. 01） ,in addition siATF-4 dramatically increased MG132 mediated increase in apoptotic cells （P〈0.01）. CONCLUSION： Proteasome inhibitors increase the ATF-4 expression in undifferentiated thyroid cancer FRO cells and the suppression of ATF-4 and ORP150 expression by siATF-4 significantly enhances the apoptosis of FRO cells induced by proteasome inhibitors.