CircTLK1 alleviates oxygen-glucose deprivation/reperfusion induced apoptosis in HK-2 cells through miR-136-5p/Bcl2 signal axis

The biological functions of circTLK1 in acute kidney injury (AKI), which mainly results from renal ischemia-reperfusion (IR), remain largely unknown. HK-2 cell treatment with oxygen and glucose deprivation, reoxygenation, and glucose (OGD/R) was used to simulate an AKI model that was mainly caused by renal IR. Then, the circTLK1 expression level in HK-2 cells treated with OGD/R was assessed by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Functional experiments were performed with circTLK1 knockdown of HK-2 cells <i>via</i> Cell Counting Kit-8 (CCK8), flow cytometry (FCM), RT-qPCR, and western blotting. The circTLK1-miRNAs-mRNAs network was constructed following the ceRNA mechanism and visualized by Cytoscape software to investigate the mechanism of circTLK1 in AKI. RT-qPCR was performed to verify the relationship between circTLK1, miR-136-5p, and Bcl2. The level of miR-136-5p was knocked down to ensure its function in OGD/R-triggered apoptosis through experiments, including CCK8, FCM, RT-qPCR, and western blotting. CircTLK1 was downregulated in HK-2 cells subjected to OGD/R treatment and in mouse kidney tissues after renal IR, but the expression of miR-136-5p was the opposite. Interference with circTLK1 expression accelerated HK-2 cell apoptosis, which was overturned by miR-136-5p inhibitors. CircTLK1 targets miR-136-5p to upregulate Bcl2 expression and attenuate apoptosis in HK-2 cells. These data revealed the possible role of circTLK1 as a new biomarker for diagnosis as well as a target in AKI through the miR-136-5p/Bcl2 signaling axis.

以肾缺血再灌注（renal ischemia-reperfusion, IR）为主要诱因的急性肾损伤（acute kidney injury, AKI）中，环状RNA TLK1（circTLK1）的生物学功能仍未完全阐明。本研究采用氧糖剥夺复糖复氧（oxygen and glucose deprivation, reoxygenation, and glucose, OGD/R）处理HK-2细胞（HK-2 cell），以构建主要由肾缺血再灌注诱导的AKI细胞模型。随后通过定量反转录聚合酶链反应（quantitative reverse transcription polymerase chain reaction, RT-qPCR）检测OGD/R处理后HK-2细胞中circTLK1的表达水平。通过细胞计数试剂盒-8（Cell Counting Kit-8, CCK8）、流式细胞术（flow cytometry, FCM）、RT-qPCR及蛋白质印迹实验，对circTLK1敲低的HK-2细胞开展功能学实验。基于内源竞争RNA（competing endogenous RNA, ceRNA）机制构建circTLK1-微小RNA-信使RNA调控网络，并通过Cytoscape软件进行可视化分析，以探究circTLK1在AKI中的作用机制。采用RT-qPCR验证circTLK1、miR-136-5p与B细胞淋巴瘤因子2（B-cell lymphoma 2, Bcl2）之间的调控关系。通过CCK8、FCM、RT-qPCR及蛋白质印迹实验，敲低miR-136-5p的表达以验证其在OGD/R诱导的细胞凋亡中的功能。在经OGD/R处理的HK-2细胞及肾缺血再灌注后的小鼠肾组织中，circTLK1的表达均呈下调趋势，而miR-136-5p的表达则与之相反。干扰circTLK1的表达会促进HK-2细胞凋亡，而该效应可被miR-136-5p抑制剂逆转。circTLK1通过靶向结合miR-136-5p，上调Bcl2的表达并减轻HK-2细胞的凋亡。本研究结果表明，circTLK1可能通过miR-136-5p/Bcl2信号轴成为AKI诊断的新型生物标志物及治疗靶点。