抑制miR-4321减轻脓毒症相关急性肾脏损伤的作用和机制研究

Effect and Mechanism of miR-4321 Inhibition Alleviates Sepsis Associated Acute Kidney Injury

【目的】本研究旨在探讨抑制miR-4321在改善脓毒症相关急性肾损伤(Sepsis associated acute kidney injury,SA-AKI)中的作用和机制。【方法】采用RT-qPCR方法检测LPS诱导的HK2细胞模型和CLP模型小鼠肾脏组织中miR-3165、miR-4270和miR-4321的表达水平。利用miR-4321-Inhibitor抑制LPS诱导的HK2细胞损伤模型中miR-4321的表达,分别通过CCK-8分析、EdU染色分析和western blotting评估miR-4321对细胞增殖、细胞活力、细胞因子和凋亡相关蛋白水平的影响。利用miR-4321-Antago干预CLP模型小鼠肾组织中miR-4321的表达,通过H&E染色检测肾组织结构变化,比色法检测血清中肌酐和血尿氮水平,ELISA检测血清中白细胞介素6(IL-6)、白细胞介素1β(IL-1β)和肿瘤坏死因子-α(TNF-α)的表达,IHC染色检测组织中mTOR水平,western blot-ting检测肾组织中凋亡相关蛋白表达水平。机制上,使用Targetscan软件预测miR-4321的靶基因,并通过双荧光素酶报告基因实验对靶基因进行验证。【结果】与对照组相比,LPS诱导的HK2细胞中miR-4321(n=3,t=7.154,P=0.0013)表达升高;miR-4321-Inhibitor可促进HK2细胞增殖和细胞活力,并降低LPS诱导的IL-1β,IL-6和TNFα水平的表达。体内实验证明miR-4321-Antago能够抑制CLP小鼠血清中肌酐和血尿氮水平,改善肾组织损伤,降低血清中IL-1β,IL-6和TNF-α水平,促进肾组织mTOR表达,抑制凋亡相关蛋白的表达。此外,mTOR是miR-4321的靶基因之一。【结论】抑制miR-4321可明显减轻脓毒症相关急性肾损伤,这一作用可能是通过增加mTOR的表达实现的。

【Objective】Sepsis associated acute kidney injury(SA-AKI)is a critical clinical disease.The purpose of this study was to investigate the role and molecular mechanism of miR-4321 in the HK2 cellular damage induced by lipo-polysaccharides(LPS).【Methods】RT-qPCR was conducted to detect the expression of miR-3165,miR-4270 and miR4321 in LPS-induced HK2 cell model and cecal ligation and puncture(CLP)-induced renal injury model.In LPS-in-duced HK2 cell model,miR-4321-inhibitor was used to inhibit the miR-4321 expression.The effects of miR-4321 on cell proliferation,cell viability,cytokines and apoptosis-related protein levels were evaluated by CCK-8 analysis,EdU staining analysis and western blotting analysis,respectively.In CLP-induced renal injury model,miR-4321-Antago was used to intervene the miR-4321 expression.The changes of renal tissue structure were examined by H&E staining.The lev-els of serum creatinine and blood urea nitrogen(BUN)were measured by colorimetric method.ELISA was employed to as-sess the expression of interleukin-6(IL-6),interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)in serum.IHC staining and western blotting were performed to determine the mTOR level and apoptosis-related protein expression in kidney tissues.Target genes of miR-4321 were predicted by Targetscan software and verified by dual-luciferase reporter assay.【Results】Compared with the control group,the miR-4321 expression was increased in LPS-induced HK2 cell mod-el(n=3,t=7.154,P=0.0013).miR-4321 inhibitor promoted the proliferation and viability of HK2 cells,decreased the expression of LPS-induced IL-6,IL-1βand TNF-αand apoptosis-related proteins.In vivo experiments showed that miR-4321-Antago inhibited serum creatinine and BUN levels in CLP mice,improved renal injury,reduced levels of IL1β,IL-6 and TNF-α,promoted the mTOR expression in renal tissues and inhibited the apoptosis-related protein expres-sion.mTOR signaling pathway was believed the target gene of miR-4321【.Conclusion】Inhibition of miR-4321 significant-ly alleviates SA-AKI,which may be achieved by increasing the expression of mTOR.