MicroRNA (miR)-489-3p contributes to lipopolysaccharide-induced HK-2 cell injury by targeting RIPK4 and inactivating the β-catenin signaling

Acute kidney injury (AKI) involves a sudden loss of renal function and is associated with high mortality. MicroRNA (miR)-489-3p may serve as a critical molecule in AKI according to previous studies. The study aimed to explore the functional roles of miR-489-3p in AKI and underlying mechanisms. An in vitro model of sepsis-induced cell injury was established by treating human proximal tubular epithelial cells (HK-2) with lipopolysaccharide (LPS). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed to examine expression levels of kidney injury molecule-1 (KIM-1), miR-489-3p, and downstream mRNAs. After silencing miR-489-3p, receptor-interacting protein kinase 4 (RIPK4), or β-catenin signaling, HK-2 cell viability was measured using cell counting kit-8 assays. Apoptosis was assessed by TdT-mediated dUTP nick-end labeling (TUNEL) assay and flow cytometry analysis. Western blot analysis was performed to examine protein levels of apoptotic markers, RIPK4, and β-catenin. Luciferase reporter assays were conducted to explore the interaction between miR-489-3p and RIPK4. Immunofluorescence staining was performed to evaluate the distribution of β-catenin in LPS-stimulated HK-2 cells. MiR-489-3p was significantly upregulated in LPS-treated HK-2 cells. Its inhibition attenuated the pro-apoptotic effects induced by LPS, increased cell viability, and reduced KIM-1 level. MiR-489-3p directly targeted 3′ untranslated region of RIPK4 and suppressed its expression. Knockdown of RIPK4 rescued the suppressive effect of miR-489-3p inhibition on cell apoptosis. Moreover, miR-489-3p was found to inactivate the β-catenin signaling by reducing RIPK4 expression. IWR-1 treatment similarly rescued the suppressive effect of miR-489-3p inhibition on cell apoptosis. In conclusion, miR-489-3p promotes LPS-induced HK-2 cell damage by targeting RIPK4 and inactivating β-catenin signaling.急性肾损伤（Acute kidney injury, AKI）指肾功能突发丧失，且与高死亡率密切相关。既往研究表明，微小核糖核酸（microRNA, miR）-489-3p可能是AKI发生发展中的关键分子。本研究旨在探讨miR-489-3p在AKI中的功能作用及其潜在分子机制。 本研究采用脂多糖（lipopolysaccharide, LPS）处理人近端肾小管上皮细胞（HK-2），构建脓毒症诱导的细胞损伤体外模型。通过逆转录定量聚合酶链反应（reverse transcription quantitative polymerase chain reaction, RT-qPCR）检测肾损伤分子-1（kidney injury molecule-1, KIM-1）、miR-489-3p及下游mRNA的表达水平。 分别沉默miR-489-3p、受体相互作用蛋白激酶4（receptor-interacting protein kinase 4, RIPK4）或阻断β-连环蛋白（β-catenin）信号通路后，采用细胞计数试剂盒-8（cell counting kit-8, CCK-8）实验检测HK-2细胞活力。通过TdT介导的dUTP缺口末端标记（TdT-mediated dUTP nick-end labeling, TUNEL）实验及流式细胞术分析细胞凋亡情况；采用蛋白质印迹法（Western blot）检测凋亡标志物、RIPK4及β-连环蛋白的蛋白表达水平。 采用荧光素酶报告基因实验探究miR-489-3p与RIPK4的相互作用；通过免疫荧光染色评估LPS刺激后HK-2细胞中β-连环蛋白的分布特征。 实验结果显示，LPS处理后的HK-2细胞中miR-489-3p表达显著上调。抑制miR-489-3p可减轻LPS诱导的促凋亡效应，提升细胞活力，并降低KIM-1的表达水平。 miR-489-3p可直接靶向RIPK4的3'非翻译区（3' untranslated region, UTR）并抑制其表达。敲低RIPK4可逆转miR-489-3p抑制对细胞凋亡的抑制作用。 此外，miR-489-3p可通过降低RIPK4的表达水平失活β-连环蛋白信号通路；IWR-1处理同样可逆转miR-489-3p抑制对细胞凋亡的抑制效应。 综上，miR-489-3p通过靶向RIPK4并失活β-连环蛋白信号通路，促进LPS诱导的HK-2细胞损伤。