南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (12): 2035-2042.doi: 10.12122/j.issn.1673-4254.2023.12.07

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甲基化酶WTAP通过调控FOXO1的表达参与肾缺血再灌注损伤

赵刚刚，李华锋，张鸿毅，肖克兵，杨 辉，李子峰，傅崇德   

  1. 西安医学院第一附属医院泌尿外科，陕西 西安 710000；西安航天总医院泌尿外科，陕西 西安 710100
  • 出版日期:2023-12-20 发布日期:2023-12-29

m6A methylase WTAP participates in renal ischemia-reperfusion injury by regulating FOXO1 expression

ZHAO Ganggang, LI Huafeng, ZHANG Hongyi, XIAO Kebing, YANG Hui, LI Zifeng, FU Chongde   

  1. Department of Urology, First Affiliated Hospital of Xi'an Medical University, Xi'an 710000, China; Department of Urology, Xi'an Aerospace General Hospital, Xi'an 710000, China
  • Online:2023-12-20 Published:2023-12-29

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摘要： 目的 探讨m6A甲基化酶WTAP在小鼠肾缺血再灌注（I/R）损伤中的表达情况及对肾小管上皮细胞生物学行为的影响，阐明其可能的作用机制。方法 C57BL/6小鼠随机分为假手术组（sham组）、I/R组，8只/组，建立急性缺血肾损伤模型；全自动生化分析仪检测血中尿素氮（BUN）及肌酐（Scr）水平评估肾功能；HE染色检测肾组织病理和形态改变；IHC检测肾组织中WTAP和FOXO1蛋白的表达；人肾小管上皮细胞（HK-2）分为Control、H/R、si-NC、si-WTAP、si-NC+H/R、si-WTAP+H/R组；Western blot检测蛋白表达；qRT-PCR检测mRNA水平；CCK8检测细胞活性；TUNEL染色法检测细胞凋亡；试剂盒分别检测LDH的释放水平和Caspase-3活性；SRAMP（http://www.cuilab.cn/sramp/）网站预测FOXO1的m6A修饰位点；RIP实验检测WTAP与FOXO1mRNA的相互作用；MeRIP-qPCR检测m6A修饰FOXO1的水平。结果 与sham组相比，I/R组Scr、BUN水平显著升高（P<0.001），WTAP mRNA和蛋白水平都显著升高（P<0.001）。与正常HK-2细胞相比，H/R组细胞活性显著下降（P<0.01），LDH的释放水平显著升高（P<0.001），WTAP的mRNA和蛋白水平都显著升高（P<0.001）。抑制WTAP表达，上述细胞损伤减弱，FOXO1的mRNA和蛋白水平都显著下降（P<0.01）。WTAP与FOXO1 mRNA结合，抑制WTAP表达能够显著降低FOXO1m6A水平（P<0.001）。结论 WTAP在体内外肾缺血再灌注损伤模型中表达均上调；体外抑制WTAP能够抵抗H/R诱导的凋亡损伤，这可能与其介导的FOXO1 m6A调控有关。

关键词: WTAP；m6A；FOXO1；肾缺血再灌注损伤；细胞凋亡

Abstract: Objective To investigate the expression of WTAP, a m6A methylase, in a mouse model of renal ischemia-reperfusion (I/R) injury and the effect of WTAP knockdown on biological behavior of renal tubular epithelial cells exposed to I/R injury. Methods Sixteen C57BL/6 mice with renal I/R injury or sham operation (n=8) were examined for blood urea nitrogen (BUN) and creatinine (Scr) levels to assess renal function, and renal pathologies were observed with HE staining. The expressions of WTAP and FOXO1 proteins in the kidneys of the mice were detected using immunohistochemistry. Human renal tubular epithelial cells (HK-2) were transfected with si-WTAP or si-NC followed by hypoxia-reoxygenation (H/R) exposure, Protein and mRNA expression were assessed by Western blot and qRT-PCR, and changes and changes in cell viability and apoptosis were assessed using CCK8 assay and TUNEL staining, respectively; LDH release level and caspase-3 activity of the cells were measured using commercial assay kits. FOXO1 m6A modification sites were predicted using SRAMP website (http://www.cuilab.cn/sramp/), and the interaction between WTAP and FOXO1 mRNA was analyzed with RIP experiment; the level of FOXO1 modified by m6A was detected by MeRIP-qPCR. Results Compared with sham-operated mice, the mice with renal I/R injury showed significantly increased Scr and BUN levels (P<0.001) and renal expressions of WTAP mRNA and protein (P<0.001). In cultured HK-2 cells, H/R exposure significantly decreased the cell viability (P<0.001) and increased cellular LDH release (P<0.001) and expressions of WTAP mRNA and protein (P<0.001). WTAP knockdown obviously reduced the cell damage induced by I/R injury and significantly decreased the mRNA and protein levels of FOXO1 in the cells (P<0.001). RIP experiment confirmed WTAP binding to FOXO1 mRNA, and inhibition of WTAP expression significantly reduced FOXO1 m6A level in HK-2 cells (P<0.001). Conclusion WTAP expression is up-regulated in the kidneys of mice with renal I/R injury and in HK-2 cells with H/R exposure. Inhibition of WTAP alleviates H/R-induced apoptotic damage in HK-2 cells possibly by inhibiting FOXO1 expression.

Key words: WTAP; m6A; FOXO1; renal ischemia-reperfusion injury; apoptosis