Nedd4 E3 ubiquitin ligase promotes cell proliferation and autophagy
Nedd4 E3 ubiquitin ligase promotes cell proliferation and autophagy
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Nedd4 E3泛素连接酶促进细胞增殖和自噬
DOI：
10.1111/cpr.12184
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发表时间：
2015-06-01
影响因子：
8.5
通讯作者：
Diao, Aipo
中科院分区：
文献类型：
--
作者：
Li, Yuyin;Zhang, Li;Diao, Aipo
目的Nedd 4（neural precursor cell expressed developmentally downregulated protein 4）是HECT E3泛素连接酶的成员之一，在前列腺癌、膀胱癌和结直肠癌中表达升高，促进结肠癌细胞群的扩增。到目前为止，Nedd 4功能的分子机制还没有得到很好的理解。材料和方法在这项研究中，shRNA被用来减少Nedd 4在人前列腺癌细胞系DU 145中的表达。为了分析Nedd 4对细胞增殖的影响，进行MTT和集落形成测定。DAPI染色和FACS分析用于研究Nedd 4活性对细胞凋亡的结果。结果Nedd 4表达对溶酶体膜透化和自噬的进一步调查，使用吖啶橙子（AO）染色，免疫荧光和westernblot analysis.ResultsWe发现，在HeLa细胞中，Nedd 4的表达促进细胞增殖，而其敲低抑制集落形成和诱导DU 145细胞凋亡。此外，在DU 145细胞中下调Nedd 4促进溶酶体膜透化。我们还发现，下调Nedd 4抑制DU 145和A549细胞中的自噬。有关机制的调查显示，敲低内源性Nedd 4表达显着增加激活的mTOR（p-mTOR）水平，这表明，mTOR信号参与Nedd 4介导的自噬。ConclusionsOur结果表明，Nedd 4的表达促进细胞增殖和集落形成，但防止凋亡。此外，Nedd 4促进自噬并与mTOR信号通路相关。
ObjectivesNedd4 (neural precursor cell expressed developmentally down-regulated protein 4) is a member of the HECT E3 ubiquitin ligases, and is elevated in prostate, bladder and colorectal cancers, and promotes colonic cell population expansion. Up to now, molecular mechanisms of how Nedd4 functions, have not been well understood.Materials and methodsIn this study, shRNA was used to reduce expression of Nedd4 in the human prostate carcinoma cell line DU145. To analyse effects of Nedd4 on cell proliferation, MTT and colony formation assays were performed. DAPI staining and FACS analysis were used to investigate outcomes of Nedd4 activity, on apoptosis. Results of Nedd4 expression on lysosomal membrane permeabilization and autophagy were further investigated using acridine orange (AO) staining, immunofluorescence and western blot analysis.ResultsWe found that in HeLa cells, expression of Nedd4 promoted cell proliferation, whereas its knockdown inhibited colony formation and induced apoptosis in DU145 cells. Furthermore, down-regulation of Nedd4 in DU145 cells promoted lysosomal membrane permeabilization. We also found that down-regulation of Nedd4 inhibited autophagy in both DU145 and A549 cells. Investigation into mechanisms involved revealed that knockdown of endogenous Nedd4 expression notably increased activated mTOR (p-mTOR) levels, which suggests that mTOR signalling was involved in the Nedd4-mediated autophagy.ConclusionsOur results indicate that expression of Nedd4 promoted cell proliferation and colony formation but prevented apoptosis. Moreover, Nedd4 promoted autophagy and was associated with the mTOR signalling pathway.