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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 effect of proteasome inhibitor on the expression of activated transcription factor 4 (ATF-4) in human thyroid carcinoma cell line FRO and the role of ATF-4 in proteasome inhibitor-induced FRO apoptosis. Methods: Human thyroid undifferentiated carcinoma cell line FRO was selected and treated with blank control group and MG132 proteasome inhibitor respectively. The expression of ATF-4 was detected by microRNA interference technique. Real-time quantitative PCR and Western blot were used to detect the expression of ATF- The mRNA and protein expression of ATF-4 and oxygen regulatory protein 150 (ORP150) in group cells were detected by flow cytometry. Apoptosis was detected by flow cytometry (FCM). Results: Compared with the blank control group, the proteasome inhibitor MG132 significantly increased the ATF-4 gene expression in the FRO cell line (P <0.01). Compared with the blank control group and the random sequence siRNA group, ATF-4, ORP150 mRNA and protein expression levels were significantly lower (P <0.01), the apoptosis rate was significantly increased, P <0.01. CONCLUSION: Proteasome inhibitor can up-regulate the expression of ATF-4 gene in FRO cell line of thyroid undifferentiated carcinoma. SiATF-4 can reduce the expression of ATF-4 and OPR150 gene and increase the expression of ATF-4 in undifferentiated thyroid carcinoma FRO cell apoptosis.