BAG3 通过靶向INTS7 调节骨髓间充质干细胞增殖。

BAG3 regulates bone marrow mesenchymal stem cell proliferation by targeting INTS7.

机构信息

Department of Orthopaedics, Suzhou Municipal Hospital, The Affiliated Suzhou Hospital of Nanjing Medical University, Gusu School, Nanjing Medical University, Suzhou, Jiangsu, China.

State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing, Jiangsu, China.

出版信息

PeerJ. 2023 Aug 9;11:e15828. doi: 10.7717/peerj.15828. eCollection 2023.

DOI:10.7717/peerj.15828

PMID:37576499

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10422954/

Abstract

BACKGROUND

BAG3 is an essential regulator of cell survival and has been investigated in the context of heart disease and cancer. Our previous study used immunoprecipitation-liquid chromatography-tandem mass spectrometry to show that BAG3 might directly interact with INTS7 and regulate bone marrow mesenchymal stem cell (BMMSCs) proliferation. However, whether BAG3 bound INTS7 directly and how it regulated BMMSCs expansion was unclear.

METHODS

expression was detected by quantitative real-time PCR in BMMSCs after siRNA-mediated knockdown. BMMSC proliferation was determined using the CCK-8 and colony formation assays. The transwell migration, flow cytometry and TUNEL assays were performed to measure BMMSC migration, cell cycle and apoptosis, respectively. Moreover, co-immunoprecipitation, protein half-life assay and western blotting analyses were used to determine the regulatory mechanism underlying the BAG3-mediated increase in BMMSC proliferation.

RESULTS

The results showed that knocking down BAG3 in BMMSCs markedly decreased their proliferative activity, colony formation and migratory capacity, and induced cell apoptosis as well as cell cycle arrest. Meanwhile, overexpression of BAG3 had the opposite effect. Bioinformatics and BAG3-INTS7 co-immunoprecipitation analyses revealed that BAG3 directly interacted with INTS7. Moreover, the downregulation of inhibited the expression of INTS7 and promoted its ubiquitination. We also observed that knockdown increased the levels of reactive oxygen species and the extent of DNA damage in BMMSCs. Notably, the upregulation of or the addition of an antioxidant scavenger could rescue the BMMSC phenotype induced by downregulation.

CONCLUSIONS

BAG3 directly interacts with INTS7 and promotes BMMSC expansion by reducing oxidative stress.

摘要

背景

BAG3 是细胞存活的重要调节剂，在心脏病和癌症的研究中已有涉及。我们之前的研究使用免疫沉淀-液相色谱-串联质谱法表明，BAG3 可能直接与 INTS7 相互作用并调节骨髓间充质干细胞（BMMSCs）的增殖。然而，BAG3 是否直接与 INTS7 结合以及它如何调节 BMMSCs 的扩增尚不清楚。

方法

用 siRNA 介导的敲低检测 BMMSCs 中 BAG3 的表达。用 CCK-8 和集落形成实验检测 BMMSC 的增殖。用 Transwell 迁移、流式细胞术和 TUNEL 实验分别检测 BMMSC 的迁移、细胞周期和凋亡。此外，还进行了 co-immunoprecipitation、蛋白质半衰期测定和 Western blot 分析，以确定 BAG3 介导的 BMMSC 增殖增加的调节机制。

结果

结果表明，在 BMMSCs 中敲低 BAG3 会显著降低其增殖活性、集落形成和迁移能力，并诱导细胞凋亡和细胞周期停滞。同时，过表达 BAG3 则产生相反的效果。生物信息学和 BAG3-INTS7 co-immunoprecipitation 分析表明，BAG3 直接与 INTS7 相互作用。此外，下调抑制了 INTS7 的表达并促进其泛素化。我们还观察到，敲低增加了 BMMSCs 中的活性氧水平和 DNA 损伤程度。值得注意的是，下调后，上调或添加抗氧化剂清除剂可以挽救由下调引起的 BMMSC 表型。