Zhu Kun, Sun Shirui, Li Zepeng, Deng Ge, Guo Yingcong, Zheng Bingxuan, He Qi, Zhao Zhenting, Ding Chenguang
Department of Neurology, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China.
Department of Kidney Transplantation, Nephropathy Hospital, the First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China.
FASEB J. 2025 Feb 15;39(3):e70320. doi: 10.1096/fj.202402443R.
Renal ischemia-reperfusion injury (IRI) is a prevalent cause of acute kidney injury, however, the regulatory mechanisms of miR-374b-5p in renal IRI remain poorly understood. We established hypoxia/reoxidation (H/R)-induced renal injury models using HK-2 and TCMK-1 cells, as well as an ischemia-reperfusion (I/R)-induced mouse model. Renal tubular epithelial cells (RTECs) viability and apoptosis were assessed using CCK-8, flow cytometry, and TUNEL assays. The targeting relationship between miR-374b-5p and SRSF7 was analyzed using dual luciferase reporter assays. The interaction between METTL3 and miR-374b-5p was confirmed through methylated RNA immunoprecipitation (MeRIP) and co-immunoprecipitation (Co-IP) assays. We found that miR-374b-5p levels were significantly upregulated in H/R-induced HK-2 and TCMK-1 cells. Furthermore, miR-374b-5p promoted H/R-induced RTEC injury by suppressing cell viability and exacerbating apoptosis. SRSF7 was identified as a downstream target of miR-374b-5p, inhibition of SRSF7 reversed the inhibitory effects of miR-374b-5p inhibitors on RTEC injury. Additionally, METTL3 interacted with the microprocessor protein DGCR8 and modulated the processing of pri-miR-374b-5p in an m6A-dependent manner. In the renal IRI model, METTL3 and miR-374b-5p levels were upregulated, and knockdown of METTL3 inhibited apoptosis in H/R-induced HK-2 and TCMK-1 cells. Conversely, miR-374b-5p reversed the protective effects of METTL3 knockdown on renal IRI. Our findings provide novel insights into the role of m6A methylation in the development of renal IRI, demonstrating that METTL3 promotes renal IRI by modulating the miR-374b-5p/SRSF7 axis.
肾缺血再灌注损伤（IRI）是急性肾损伤的常见原因，然而，miR-374b-5p在肾IRI中的调控机制仍知之甚少。我们使用HK-2和TCMK-1细胞建立了缺氧/复氧（H/R）诱导的肾损伤模型，以及缺血再灌注（I/R）诱导的小鼠模型。使用CCK-8、流式细胞术和TUNEL检测评估肾小管上皮细胞（RTECs）的活力和凋亡。使用双荧光素酶报告基因检测分析miR-374b-5p与SRSF7之间的靶向关系。通过甲基化RNA免疫沉淀（MeRIP）和免疫共沉淀（Co-IP）检测证实METTL3与miR-374b-5p之间的相互作用。我们发现，在H/R诱导的HK-2和TCMK-1细胞中，miR-374b-5p水平显著上调。此外，miR-374b-5p通过抑制细胞活力和加剧凋亡促进H/R诱导的RTEC损伤。SRSF7被确定为miR-374b-5p的下游靶点，抑制SRSF7可逆转miR-374b-5p抑制剂对RTEC损伤的抑制作用。此外，METTL3与微处理器蛋白DGCR8相互作用，并以m6A依赖的方式调节pri-miR-374b-5p的加工。在肾IRI模型中，METTL3和miR-374b-5p水平上调，敲低METTL3可抑制H/R诱导的HK-2和TCMK-1细胞中的凋亡。相反，miR-374b-5p可逆转METTL3敲低对肾IRI的保护作用。我们的研究结果为m6A甲基化在肾IRI发生发展中的作用提供了新的见解，表明METTL3通过调节miR-374b-5p/SRSF7轴促进肾IRI。
