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.
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(
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FOXO1 m6A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。
Keywords: FOXO1; WTAP; apoptosis; m6A; renal ischemia-reperfusion injury.