Composition of EVs markers under normoxic and hypoxic conditions depends on the expression level of adaptor protein Ruk/CIN85 in mouse renal carcinoma Renca cells

Q4 Biochemistry, Genetics and Molecular Biology

Biopolymers and Cell Pub Date : 2021-10-30 DOI:10.7124/bc.000a5e

A. Zhyvolozhnyi, I. Horak, T. Skaterna, O. V. Khudiakova, S. Vainio, A. Samoylenko, L. Drobot

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Abstract

Aim. To isolate and characterize extracellular vesicles (EVs) produced by mouse renal carcinoma Renca cells with different expression levels of the adaptor protein Ruk/CIN85 under normoxia and hypoxia conditions. Methods. The density gradient centrifugation was used to isolate EVs from the conditioned medium of Renca cells cultured under normoxia and hypoxia conditions. Further characterization of EVs was performed by using nanoparticle tracking analysis (NTA), electron microscopy and Western Blot analysis. Results. Significant differences in average particle size between EVs produced by sublines studied under experimen-tal conditions were not found. At the same time, concentration of particles produced by Ruk/ CIN85 overexpressing cells turned out to be an order of magnitude higher in hypoxia in comparison to normoxia conditions. It was shown that under normoxia conditions the content of both Ruk/CIN85 and EVs’ markers Alix and CD81 was increased in vesicles produced by Renca cells with Ruk/CIN85 overexpression in comparison with those from control mock-transfected cells. Under hypoxia conditions, the content of studied proteins decreased by more than two orders of magnitude in EVs secreted by Renca cells with up-regulation of adaptor protein whereas the content of Ruk/CIN85 and CD81 increased in EVs from mock-transfec ted cells. Conclusions. It has been demonstrated that the adaptor protein Ruk/CIN85 is a novel component of EVs produced by tumor cells that may play a role in the control of EV composition under normoxia and hypoxia.

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常氧和缺氧条件下EVs标志物的组成取决于小鼠肾癌Renca细胞中衔接蛋白Ruk/CIN85的表达水平

的目标。目的:分离和表征常氧和缺氧条件下不同受体蛋白Ruk/CIN85表达水平的小鼠肾癌Renca细胞产生的细胞外囊泡(EVs)。方法。采用密度梯度离心法从常氧和缺氧条件下培养的Renca细胞的条件培养基中分离出ev。利用纳米颗粒跟踪分析(NTA)、电子显微镜和Western Blot分析对ev进行进一步表征。结果。在实验条件下，不同亚系生产的电动汽车的平均粒径没有显著差异。同时，在低氧条件下，Ruk/ CIN85过表达细胞产生的颗粒浓度比常氧条件下高一个数量级。结果表明，在常氧条件下，Ruk/CIN85过表达的Renca细胞产生的囊泡中Ruk/CIN85和ev标记物Alix和CD81的含量均高于对照模拟转染细胞。在缺氧条件下，Renca细胞分泌的EVs中所研究的蛋白质含量下降了两个数量级以上，而模拟转染细胞的EVs中Ruk/CIN85和CD81的含量增加。结论。研究表明，受体蛋白Ruk/CIN85是肿瘤细胞产生的EV的新成分，可能在常氧和缺氧条件下控制EV的组成中起作用。

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来源期刊

Biopolymers and Cell Biochemistry, Genetics and Molecular Biology-Biochemistry, Genetics and Molecular Biology (all)

CiteScore

1.10

自引率

0.00%

发文量

期刊介绍： “Biopolymer and cell” is published since 1985 at the Institute of Molecular Biology and Genetics NAS of Ukraine under the supervision of the National Academy of Sciences of Ukraine. Our journal covers a wide scope of problems related to molecular biology and genetics including structural and functional genomics, transcriptomics, proteomics, bioinformatics, biomedicine, molecular enzymology, molecular virology and immunology, theoretical bases of biotechnology, physics and physical chemistry of proteins and nucleic acids and bioorganic chemistry.