三甲基胺 N-氧化物介导的 Y 盒结合蛋白-1 核转位通过直接下调慢性肾脏病细胞模型中 Gadd45a 的表达促进细胞周期进程。
Trimethylamine N-oxide mediated Y-box binding protein-1 nuclear translocation promotes cell cycle progression by directly downregulating Gadd45a expression in a cellular model of chronic kidney disease.
机构信息
Department of Nephrology, Shanghai General Hospital, Nanjing Medical University, No.100 Haining Road, Hongkou District, Shanghai, China; Department of Nephrology, Shanghai General Hospital, No.100 Haining Road, Hongkou District, Shanghai, China.
Department of Nephrology, Shanghai General Hospital, No.100 Haining Road, Hongkou District, Shanghai, China.
出版信息
Life Sci. 2021 Apr 15;271:119173. doi: 10.1016/j.lfs.2021.119173. Epub 2021 Feb 5.
AIMS
Cell cycle arrest plays critical roles in preventing renal tubular epithelial cell (RTEC) injury and maladaptation after the onset of chronic kidney disease (CKD), but the underlying mechanism governing this arrest has not been fully elucidated. This study was designed to determine the underlying role of YB-1 in promoting cell cycle progression and nuclear translocation in HK-2 cells induced by trimethylamine N-oxide (TMAO).
MAIN METHODS
YB-1 primarily accumulated in the cytoplasm in HK-2 cells after they were treated with TMAO for 30 min and 6 h. Gene expression was analysed using RNA sequencing in HK-2 cells treated with TMAO. Cell cycle progression was analysed via flow cytometry. Luciferase assay and ChIP-PCR were performed to determine the relationship between transcription factor YB-1 and Gadd45a promoter region. Additionally, mice were fed with TMAO to test renal dysfunction and measure the expression of YB-1, GADD45a and CCNA2 in the kidney sections through immunohistochemistry.
KEY FINDINGS
YB-1 primarily accumulated in the cytoplasm in HK-2 cells after they were treated with TMAO for 30 min and 6 h. RNA sequencing analysis showed that the cell cycle checkpoint genes growth arrest and DNA damage (Gadd)45a, Gadd45g, cyclin (Ccn)a2, Ccnb1, Ccne1 and Ccnf were differentially expressed in HK-2 cells after treated with 400 μM TMAO for 30 min. Flow cytometry results demonstrated that cell cycle progression was blocked at the G2/M checkpoint. In animal models, elevated dietary TMAO directly led to progressive renal tubulointerstitial dysfunction and inhibited the expression of YB-1 in kidney. Moreover, YB-1 was determined to regulate Gadd45a expression by directly binding to its promoter region. YB-1 expression was negatively correlated with the expression of Gadd45a and Gadd45g but positively correlated with Ccna2, Ccnb1, Ccne1 and Ccnf in CKD.
SIGNIFICANCE
YB-1 may be a reliable molecular target and an effective prognostic biomarker for CKD.
目的
细胞周期停滞在预防慢性肾脏病（CKD）发生后肾小管上皮细胞（RTEC）损伤和适应不良方面起着关键作用，但控制这种停滞的潜在机制尚未完全阐明。本研究旨在确定 YB-1 在三甲基胺 N-氧化物（TMAO）诱导 HK-2 细胞周期进展和核转位中的作用。
主要方法
HK-2 细胞用 TMAO 处理 30 分钟和 6 小时后，YB-1 主要在细胞质中积累。用 TMAO 处理的 HK-2 细胞进行 RNA 测序分析基因表达。通过流式细胞术分析细胞周期进展。进行荧光素酶测定和 ChIP-PCR 以确定转录因子 YB-1 与 Gadd45a 启动子区域之间的关系。此外，用 TMAO 喂养小鼠以测试肾功能，并通过免疫组织化学测量肾脏切片中 YB-1、GADD45a 和 CCNA2 的表达。
主要发现
HK-2 细胞用 TMAO 处理 30 分钟和 6 小时后，YB-1 主要在细胞质中积累。RNA 测序分析显示，用 400μM TMAO 处理 30 分钟后，HK-2 细胞中的细胞周期检查点基因生长停滞和 DNA 损伤（Gadd）45a、Gadd45g、周期蛋白（Ccn）a2、Ccnb1、Ccne1 和 Ccnf 差异表达。流式细胞术结果表明，细胞周期停滞在 G2/M 检查点。在动物模型中，饮食中 TMAO 水平升高直接导致进行性肾小管间质功能障碍，并抑制肾脏中 YB-1 的表达。此外，YB-1 被确定通过直接结合其启动子区域来调节 Gadd45a 的表达。在 CKD 中，YB-1 表达与 Gadd45a 和 Gadd45g 的表达呈负相关，与 Ccna2、Ccnb1、Ccne1 和 Ccnf 的表达呈正相关。
意义
YB-1 可能是 CKD 的可靠分子靶标和有效的预后生物标志物。
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