热休克蛋白27通过降低内质网应激抑制硼替佐米诱导的骨髓瘤细胞凋亡

目的 探讨多发性骨髓瘤(MM)细胞热休克蛋白27(HSP27)表达对硼替佐米诱导凋亡的影响及机制。 方法 人MM细胞株U266细胞暴露于硼替佐米,实时荧光定量PCR检测HSP27 mRNA,蛋白质印迹法检测HSP27、磷酸化-HSP27(p-HSP27)、葡萄糖调节蛋白78(GRP78)、磷酸化蛋白激酶R样内质网激酶(p-PERK)、活化的天冬氨酸特异性半胱氨酸蛋白酶3/9(cl-Caspase 3/9)表达。收集12例初治和10例复发MM患者骨髓瘤细胞,荧光原位杂交法检测染色体异常,比较HSP27 mRNA和蛋白表达差异。将U266细胞分为观察组和对照组,分别转染HSP27-siRNA和NC-siRNA,流式细胞术检测硼替佐米诱导的细胞凋亡率。慢病毒介导HSP27基因在U266细胞过表达,蛋白质印迹法检测硼替佐米诱导的GRP78和cl-Caspase 3表达。 结果 硼替佐米暴露显著上调U266细胞HSP27 mRNA和蛋白的表达(P<0.01),上调GRP78和p-PERK表达(P<0.05),活化Caspase-3和Caspase-9(P<0.05); 4-苯基丁酸预处理显著抑制硼替佐米诱导的Caspase-3和Caspase-9活化(P<0.01)。复发MM患者HSP27 mRNA和蛋白的表达显著高于初治MM患者(P<0.01)。沉默HSP27表达显著增加硼替佐米诱导的U266细胞凋亡(P<0.01)。慢病毒介导HSP27过表达显著抑制硼替佐米诱导的GRP78表达和Caspase-3活化(P<0.01)。 结论 HSP27负反馈抑制内质网应激,减少硼替佐米诱导的MM细胞凋亡。

Heat Shock Protein 27 Inhibits Bortezomib Induced Apoptosis of Myeloma Cells by Reducing Endoplasmic Reticulum Stress

Objective To investigate the effect of heat shock protein 27(HSP27)on bortezomib induced apoptosis of multiple myeloma cells and its mechanism. Methods The human multiple myeloma U266 cells were exposed to bortezomib, and HSP27 mRNA expression was measured by real-time fluorescent quantitative PCR. The expression of HSP27, p-HSP27, GRP78, p-PERK, cl-Caspase 3, and cl-Caspase 9 were determined by western blot analysis. Myeloma cells from 12 newly treated and 10 relapsed myeloma patients were collected, chromosomal abnormalities were detected by fluorescence in situ hybridization, and expression of HSP27 mRNA and protein were compared. HSP27-siRNA was transfected into U266 cells in observation group and NC-siRNA was transfected in control group, the apoptotic rate induced by bortezomib was detected by flow cytometry. Lentivirus-mediated overexpression of HSP27 gene in U266 cells, bortezomib-induced expression of GRP78, and cl-Caspase-3 were detected by western blotting. Results Bortezomib exposure significantly increased the expression of HSP27 mRNA and protein in U266 cells(P<0.01). Bortezomib exposure significantly increased the expression of GRP78, p-PERK, cl-Caspase-3, and cl-Caspase-9(P<0.05), while pretreatment with 4-phenylbutyric acid attenuated the effect of bortezomib exposure on activation of Caspase-3 and Caspase-9 of U266 cells(P<0.01). The expression of HSP27 mRNA and protein in relapsed multiple myeloma patients were significantly higher than that in newly treated multiple myeloma patients(P<0.01). Silencing the expression of HSP27 significantly increased the apoptosis of U266 cells induced by bortezomib(P<0.01). Lentivirus mediated HSP27 overexpression significantly inhibited bortezomib exposure induced GRP78 and cl-Caspase-3 expression in U266 cells(P<0.01). Conclusion HSP27 negative feedback inhibited endoplasmic reticulum stress and reduced bortezomib induced apoptosis of myeloma cells.