摘要
目的探讨DEC2(Differentiated embryonic chondrocyte express gene2,又称BHLHE41或Sharp1)在乳腺癌细胞系MCF-7中对凋亡的调控作用,及其在他莫昔芬(Tamoxifen)药物抵抗中的意义。方法应用siRNA下调内源性DEC2后,通过TUNEL assay和Hoechst33258染色,观察肿瘤细胞的凋亡有无明显改变,并通过Real-time PCR和Western blot检测凋亡相关因子(Fas,caspase-8,ADP核糖聚合酶(PARP)和(Bax)在mRNA和蛋白水平的改变。应用Tamoxifen处理MCF-7,寻找诱导凋亡的合适处理浓度,并观察DEC2的表达变化。联合应用Tamoxifen与DEC2siRNA处理MCF-7,通过Western blot检测凋亡相关因子的表达变化。结果通过siRNA下调内源性DEC2后,凋亡相关因子(Fas和Bax)以及caspase-8和PARP的裂解产物均显著上调,并促进肿瘤细胞的凋亡。Tamoxifen可以上调DEC2的表达,联合应用Tamoxifen与DEC2siRNA处理与单一使用DEC2siRNA处理MCF-7相比,PARP和caspase-8的裂解产物明显上调,同时明显促进凋亡。相反,联合应用Tamoxifen与DEC2过表达质粒处理MCF-7后,PARP和caspase-8的裂解产物明显下调,同时明显抑制凋亡。结论 DEC2具有抑制乳腺癌细胞凋亡的作用,并能抑制由Tamoxifen诱导的乳腺癌细胞MCF-7的凋亡。
Objective To investigate the role and significance of differentiated embryonic chondrocyte express gene 2（DEC2, BHLHE41/Sharpl） in tamoxifen-induced apoptosis of MCF-7 breast cancer cells. Methods TUNEL assay and Hoechst33258 were performed to examine the effect of DEC2 on the apoptosis of MCF-7 ceils, Real-time PCR and Western blot were performed to examine the variation of apoptosis-related factors such as Fas, caspase-8, poly （ADP-ribose） polymerase（PARP） and Bax. MCF-7 ceils were incubated with various concentrations of tamoxifen to find the appropriate condition. PCR and Western blot were performed to find the variation of DEC2 after treatment of tamoxifen. We also performed dual regulation of DEC2 after treatment to find the variation of apoptosis-related gene. Results siRNA- mediated knockdown of DEC2 resulted in marked enhancement of apoptosis compared with control cells transfected with nonspecific siRNA, upregulated the expressions of mRNA or proteins related to apoptosis, such as Fas, Bax, cleaved caspase-8, and cleaved PARP in MCF-7 cells. Moreover, tamoxifen also upregulated the expression of DEC2, combination of tamoxifen and DEC2 siRNA had stronger effect on cleaved caspase-8 and cleaved PARP levels than DEC2 siRNA, but combination of tamoxifen and DEC2 reduced the levels of cleaved PARP and caspase-8. Conclusion DEC2 has an anti- apoptotic effect and inhibits the apoptosis induced by tamoxifen in human breast cancer MCF-7 cells.
出处
《解剖科学进展》
CAS
2013年第2期133-137,141,共6页
Progress of Anatomical Sciences
基金
国际自然科学基金资助项目(No.30870977)
