抑制β-catenin诱导的多发性骨髓瘤细胞自噬与凋亡关系的研究

The study of the relationship between autophagy and apoptosis induced by the silence of β-catenin in multiple myeloma

目的:通过慢病毒介导的siRNA转染沉默β-catenin的表达以证实抑制β-catenin所诱发的细胞自噬与凋亡之间的可能相互关系。方法:培养多发性骨髓瘤细胞系RPMI8226细胞,应用慢病毒转染的方法沉默β-catenin表达,分别联合自噬抑制剂3-MA及凋亡抑制剂Z-VAD,Westernblot检测细胞加药前后β-catenin、自噬相关蛋白LC3及凋亡相关蛋白Caspase-3(active)的表达变化;CCK-8法检测细胞增殖情况;流式细胞术检测细胞凋亡情况。结果:沉默β-catenin后,联合自噬抑制剂3-MA,细胞加药前后β-catenin表达无明显变化,LC3Ⅱ表达降低,Caspase-3(active)表达增高,细胞增殖能力下降,细胞死亡增加;联合凋亡抑制剂Z-VAD,细胞加药前后β-catenin无明显变化,LC3Ⅱ表达增高,Caspase-3(active)表达降低,细胞增殖能力下降,细胞死亡增加。结论:沉默β-catenin所诱发的自噬与凋亡共同促进细胞死亡,自噬和凋亡任一途径受抑,均可使细胞转向另一死亡途径。

Objective:To confirme the relationship between autophagy and apoptosis in multiple myeloma(MM) through the way of silencing β-catenin expression by lentivirus mediated siRNA transfection. Methods:To culture the MM cell line RPMI 8226.Lentivirus transfection method was applied to silence β-catenin expression.After silencing of β-catenin,autophagy inhibitor 3-MA and apoptosis inhibitor Z-VAD was combined separately,using CCK-8 method to detect the MM cell proliferation,flow cytometry to detect cell death,Western blot to detect expression changes of β-catenin,LC3 and Caspase-3(active). Results:After silencing of β-catenin,combining with autophagy inhibitor 3-MA,we found cell death increased by FCM,and cell proliferation decreased by CCK-8,β-catenin didn't change,LC3Ⅱ decreased and Caspase-3(active) increased by Western blot.After silencing of β-catenin,combining with the apoptosis inhibitor Z-VAD,we found cell death increased by FCM,and cell proliferation decreased by CCK-8,β-catenin did not change,LC3Ⅱ increased,Caspase-3(active) decreased by Western blot. Conclusion:Autophagy and apoptosis induced by the silencing of β-catenin both can promote cell death,one of which is inhibited,can promote the other way to death.