Knockdown of DEPTOR induces apoptosis, increases chemosensitivity to doxorubicin and suppresses autophagy in RPMI-8226 human multiple myeloma cells in vitro.
Knockdown of DEPTOR induces apoptosis, increases chemosensitivity to doxorubicin and suppresses autophagy in RPMI-8226 human multiple myeloma cells in vitro.
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DOI：
10.3892/ijmm.2013.1299
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发表时间：
2013-05
影响因子：
5.4
通讯作者：
Haoran Zhang;Junmin Chen;Z. Zeng;Wenzhong Que;Linying Zhou
中科院分区：
文献类型：
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作者：
Haoran Zhang;Junmin Chen;Z. Zeng;Wenzhong Que;Linying Zhou
含有 DEP 结构域的哺乳动物雷帕霉素靶蛋白 (mTOR) 相互作用蛋白 (DEPTOR) 是一种 mTOR 结合蛋白，在 RPMI-8226 人多发性骨髓瘤细胞中过表达，在维持细胞存活中发挥重要作用。然而，关于 DEPTOR 敲低对 RPMI-8226 人多发性骨髓瘤细胞生物学功能影响的了解有限。本研究旨在确定 DEPTOR 在这些细胞的增殖、凋亡和自噬中的作用，并阐明 DEPTOR 促进骨髓瘤细胞化疗敏感性的机制。 RNA干扰用于减少DEPTOR的表达。通过MTT测定评估细胞毒性。通过流式细胞术检查细胞凋亡。通过 RT-PCR、定量 PCR 和蛋白质印迹分析评估经 DEPTOR 特异性短发夹 RNA (shRNA) 处理的 RPMI-8226 细胞中的 DEPTOR mRNA 和蛋白质表达。通过蛋白质印迹分析检测凋亡相关蛋白、自噬相关蛋白的表达以及磷酸肌醇3激酶（PI3K）/Akt信号通路的激活。还通过透射电子显微镜和单丹酰尸胺 (MDC) 测量自噬。在本研究中，RPMI-8226细胞被DEPTOR特异性shRNA转染，导致DEPTOR的转录和表达显着抑制。 DEPTOR 的下调抑制增殖，增强阿霉素诱导的对 RPMI-8226 细胞的生长抑制作用，并增加裂解的 caspase-3 和裂解的聚（ADP-核糖）聚合酶（PARP）的表达。此外，DEPTOR 的下调可抑制 RPMI-8226 细胞中的自噬并抑制 PI3K/Akt 信号传导的激活。总之，我们的数据表明，在 RPMI-8226 人多发性骨髓瘤细胞中，DEPTOR 的下调会诱导细胞凋亡，增加对阿霉素的化疗敏感性，并抑制自噬和 PI3K/Akt 信号通路的激活。
DEP domain containing mammalian target of rapamycin (mTOR)-interacting protein (DEPTOR) is an mTOR binding protein that is overexpressed in RPMI-8226 human multiple myeloma cells, and plays an important role in maintaining cell survival. However, knowledge on the effects of DEPTOR knockdown on the biological functions of RPMI‑8226 human multiple myeloma cells, is limited. This study aimed to determine the role of DEPTOR in the proliferation, apoptosis and autophagy in these cells and to elucidate the mechanisms by which DEPTOR contributes to the chemosensitivity of myeloma cells. RNA interference was used to reduce the expression of DEPTOR. Cytotoxicity was evaluated by MTT assay. Apoptosis was examined by flow cytometry. DEPTOR mRNA and protein expression in RPMI‑8226 cells treated with DEPTOR-specific short hairpin RNA (shRNA) was evaluated by RT-PCR, quantitative PCR and western blot analysis. The expression of apoptosis‑associated proteins, autophagy‑associated proteins, and the activation of the phosphoinositide 3‑kinase (PI3K)/Akt signaling pathway were detected by western blot analysis. Autophagy was also measured by transmission electron microscopy and monodansylcadaverine (MDC). In this study, RPMI-8226 cells were transfected with the DEPTOR-specific shRNA, which resulted in the significant inhibition of the transcription and expression of DEPTOR. The downregulation of DEPTOR inhibited proliferation, enhanced the doxorubicin‑induced growth inhibitory effects on RPMI-8226 cells, and increased the expression of cleaved caspase‑3 and cleaved poly(ADP-ribose) polymerase (PARP). Moreover, the downregulation of DEPTOR suppressed autophagy and inhibited the activation of the PI3K/Akt signaling in RPMI‑8226 cells. In conclusion, our data demonstrated that the downregulation of DEPTOR induces apoptosis, increases chemosensitivity to doxorubicin, and suppresses autophagy and the activation of the PI3K/Akt signaling pathway in RPMI‑8226 human multiple myeloma cells.