tRF-013354介导阿霉素诱导的足细胞损伤的机制研究

Mechanism of tRF-013354 mediating adriamycin-induced podocyte injury

目的:探究tRNA衍生片段013354(tRF-013354)在阿霉素(ADR)诱导的足细胞损伤中的作用及相关机制。方法:用ADR诱导足细胞损伤模型,tRF-013354 mimic和tRF-013354 NC-mimic分别转染足细胞,Real-time PCR(RT-PCR)验证转染效率,CCK-8检测足细胞存活率,免疫荧光检测足细胞损伤标志物Nephrin、Podocin的定位和表达水平,Western blot检测Nephrin、Podocin和Wnt/β-catenin信号通路相关蛋白非磷酸化β-catenin、Wnt1的蛋白表达水平,RT-PCR检测tRF-013354的表达;用Wnt信号拮抗剂Dickkopf-1(DKK1)阻断Wnt/β-catenin信号通路,Western blot检测非磷酸化β-catenin和Nephrin、Podocin的蛋白表达水平,RT-PCR检测tRF-013354的表达。结果:1μg/mL ADR干预足细胞24 h,可成功构建ADR诱导足细胞损伤模型,Wnt/β-catenin信号通路被激活,tRF-013354下调(P<0.05);足细胞过表达tRF-013354可减轻足细胞损伤;DDK1阻断Wnt/β-catenin信号通路,与ADR组比较,ADR+DKK1组足细胞损伤减轻,tRF-013354表达上调(P<0.05)。结论:tRF-013354可能作为Wnt/β-catenin信号通路的下游效应分子减轻ADR诱导的足细胞损伤。

Objective:To investigate the role and mechanism of tRNA-derived fragment-013354(tRF-013354)in adriamycin(ADR)-induced podocyte injury.Methods:The podocyte injury model was induced by ADR.Podocytes were respectively transfected with tRF-013354 mimic and tRF-013354 NC-mimic,and the transfection efficiency was verified by real-time PCR(RT-PCR).CCK-8 kit was used to detect the survival rate of podocytes.Immunofluorescence was employed to detect the localization and expression levels of podocyte injury markers(nephrin and podocin).The protein expression levels of nephrin,podocin and Wnt/β-catenin signaling pathway-related proteins(non-phosphorylatedβ-catenin and Wnt1)were detected by Western blot,and the expression of tRF-013354 was measured by RT-PCR.After blocking the Wnt/β-catenin signaling pathway with the Wnt signaling antagonist Dickkopf-1(DKK1),the protein expression levels of non-phosphorylatedβ-catenin,nephrin and podocin,and the expression of tRF-013354 were respectively detected with Western blot and RT-PCR.Results:After 24 h intervention with 1ug/ml ADR,adriamycin-induced injury model was successfully constructed,Wnt/β-catenin signaling pathway was activated,and tRF-013354 was significantly down-regulated(P<0.05).Overexpression of tRF-013354 in podocytes could reduce the injury of podocytes.When DDK1 was used to block Wnt/β-catenin signaling pathway,ADR+DKK1 group showed less injury of podocytes and up-regulated expression of tRF-013354 than ADR group(P<0.05).Conclusion:tRF-013354 may act as a downstream effector of Wnt/β-catenin signaling pathway to attenuate ADR-induced podocyte injury.