| 陈暮霖,穆星宇,李维国,蒋立人,凡 杰.敲除含α-Arrestin结构域蛋白3对肾癌细胞PI3K/AKT信号通路及增殖能力的影响[J].现代生物医学进展英文版,2020,(13):2401-2405. |
| 敲除含α-Arrestin结构域蛋白3对肾癌细胞PI3K/AKT信号通路及增殖能力的影响 |
| Effects of α-Arrestin-Domain-Containing-3 Deficiency on PI3K/AKT Signaling Pathway and Proliferation of Renal Cell Carcinoma Cells |
| Received:March 05, 2020 Revised:March 28, 2020 |
| DOI：10.13241/j.cnki.pmb.2020.13.001 |
| 中文关键词: ARRDC3 AXL 信号通路 增殖 肾细胞癌 |
| 英文关键词: ARRDC3 AXL Signaling pathway Proliferation Renal cell carcinoma |
| 基金项目:国家自然科学基金项目（81372753） |
| Author Name | Affiliation | E-mail | | CHEN Mu-lin | Department of Urology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200080, China | cml0613@sina.com | | MU Xing-yu | Department of Urology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200080, China | | | LI Wei-guo | Department of Urology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200080, China | | | JIANG Li-ren | Department of Pathology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200080, China | | | FAN Jie | Department of Urology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200080, China | |
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| 中文摘要: |
| 摘要 目的：探究敲除含α-Arrestin结构域蛋白3（α-Arrestin-Domain-Containing-3，ARRDC3）对肾透明细胞癌786-O细胞PI3K/AKT信号通路和增殖能力的影响及作用机制。方法：使用Crispr-Cas9技术构建稳定敲除ARRDC3的786-O细胞系；通过免疫印迹检测敲除ARRDC3对AXL蛋白水平的影响；借助免疫沉淀技术研究敲除ARRDC3对AXL泛素化水平的影响；利用免疫印迹、shRNA干扰蛋白表达技术和及构建的敲除ARRDC3细胞检测ARRDC3和AXL表达水平对PI3K/AKT信号通路活性的影响；CCK-8技术检测ARRDC3和AXL表达水平对肾癌细胞增殖能力的影响。结果：与野生型786-O细胞相比，稳定敲除ARRDC3的786-O细胞内AXL的蛋白水平显著升高。进一步检测细胞内AXL的泛素化水平发现，ARRDC3稳定敲除组细胞内AXL蛋白的泛素化水平显著降低；免疫印迹和CCK-8实验结果显示，敲除ARRDC3会显著增加AXL/PI3K/AKT信号通路磷酸化和细胞增殖能力，而在ARRDC3缺陷细胞内降低AXL蛋白的表达，会导致AXL/PI3K/AKT的活性和细胞增殖能力恢复到与野生型相似的水平。结论：在786-O细胞内敲除ARRDC3可通过降低ARRDC3对AXL的泛素化降解，上调AXL蛋白水平和PI3K/AKT信号通路的活性，并促进肾癌细胞的增殖。 |
| 英文摘要: |
| ABSTRACT Objective: To investigate the effects and mechanisms of α-Arrestin-domain-containing-3(ARRDC3) deficiency on PI3K/AKT signaling pathway and cell proliferation in clear cell renal cell carcinoma 786-O cells. Methods: Crispr-Cas9 system was used to construct the ARRDC3 deficient 786-O cell line. The effect of ARRDC3 deficiency on AXL protein was detected by Western Blotting analysis. The effect of ARRDC3 deficiency on the ubiquitination of AXL was investigated by Immunoprecipitation. Effects of ARRDC3 and AXL expression on PI3K/AKT signaling pathway were detected by Western Blotting, shRNA interference and established ARRDC3 deficient cells. CCK-8 technology was utilized to discovered the effect of ARRDC3 and AXL expression on proliferation in RCC cells. Results: Compared with wild type 786-O RCC cells, the protein of AXL was elevated. Further examination of ubiquitination indicated that ARRDC3 deficiency significantly reduced the ubiquitination of AXL in 786-O cells. The results of Western blotting and CCK-8 analyses showed that ARRDC3 deficiency increased the phosphorylation of AXL/PI3K/AKT signaling pathway and cell proliferation ability. Moreover, knocking down AXL protein expression in ARRDC3-deficient cells leaded the activity of AXL/PI3K/AKT signaling pathway and cell proliferation back to the levels similar to wild type, which suggested the effects of ARRDC3 deficiency on the activity of AXL/PI3K/AKT signaling pathway and proliferation of RCC cells is AXL-dependent. Conclusion: ARRDC3 deficiency in 786-O cells up-regulates AXL protein level and activity of the PI3K/AKT signaling pathway and promotes proliferation of cells via reducing the AXL ubiquitination mediated by ARRDC3, which may provide a kind of potential therapeutic taget and new idea for individualized treatment of RCC. |
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