Cdc37调控细胞自噬参与骨髓瘤细胞对硼替佐米耐药的研究

Cdc37 Contributes to bortezomib resistance in multiple myeloma via autophagy

目的:探索细胞分裂周期蛋白37(Cdc37)通过调控细胞自噬参与多发性骨髓瘤(MM)对硼替佐米(BTZ)耐药的机制,为MM的治疗提供新的思路。方法:应用实时荧光定量PCR(qRT-PCR)和Western blot(WB)技术测定BTZ耐药细胞株ANBL-6.BR中Cdc37及自噬相关基因LC3b的表达水平。采用慢病毒转染技术上调ANBL-6.BR细胞中Cdc37的表达水平,WB技术检测LC3b的表达,流式细胞术检测BTZ诱导的细胞凋亡。采用shRNA干扰技术下调MM细胞株NCI-H929中Cdc37的表达水平,CCK-8法进一步验证Cdc37低表达与BTZ耐药相关,WB技术检测AKT/mTOR信号通路及自噬相关蛋白的表达变化;通过流式细胞术检测自噬抑制剂氯喹(CQ)是否可以增加耐药MM细胞对BTZ的敏感性。结果:Cdc37在BTZ耐药细胞株ANBL-6.BR中低表达,而自噬相关基因LC3b在耐药株中高表达。上调ANBL-6.BR细胞中Cdc37的表达水平可抑制LC3b的表达,且增加了MM细胞对BTZ的敏感性。在MM细胞系NCI-H929中抑制Cdc37的表达导致BTZ耐药,同时引起自噬水平升高。而自噬抑制剂CQ可逆转Cdc37下调所引起的BTZ耐药。结论:Cdc37可能通过调控细胞自噬参与MM细胞对BTZ的耐药过程。

Objective To explore the role of cell division cycle protein 37(Cdc37)mediating bortezomib(BTZ)resistance in multiple myeloma(MM)via the regulation of autophagy activity to provide a novel strategy for MM therapy.Methods The expressions of Cdc37 and LC3b were investigated in BTZ-resistant MM cell line ANBL-6.BR using quantitative real-time PCR(qRT-PCR)and western blot(WB)analysis.Cdc37 was upregulated in ANBL-6.BR cells owing to lentivirus transfection.The LC3b expression was detected with WB,and BTZ-induced apoptosis was explored using flow cytometry.Cdc37 was then down-regulated by shRNA in the MM cell line NCI-H929.Sensitivity of BTZ was evaluated using CCK-8 analysis.WB analysis was performed to check the expression of the AKT/mTOR pathway and autophagy-associated proteins.The sensitivity of NCI-H929 cells to BTZ in the presence of autophagy inhibitor chloroquine(CQ)was analyzed using flow cytometry.Results Cdc37 was down-regulated,while autophagy-associated gene LC3b was upregulated in BTZ-resistant cell line ANBL-6.BR.Up-regulated Cdc37 in ANBL-6.BR cells could inhibit LC3b expression and increase the sensitivity of MM to BTZ.Suppressing Cdc37 expression in MM cell line NCI-H929 induced BTZ resistance and autophagy activation,while CQ could rescue BTZ resistance caused by Cdc37 inhibition.Conclusion Cdc37 may participate in BTZ resistance in MM via the regulation of autophagy activity.