异常长链非编码 RNA CCAT1/微小 RNA-155/SIRT1 轴促进 LPS 诱导的急性肾损伤中肾小管上皮细胞的炎症反应和凋亡。
Abnormal lncRNA CCAT1/microRNA-155/SIRT1 axis promoted inflammatory response and apoptosis of tubular epithelial cells in LPS caused acute kidney injury.
机构信息
Department of Nephrology, First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan Province, The People's Republic of China.
Department of Nephrology, First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan Province, The People's Republic of China.
出版信息
Mitochondrion. 2020 Jul;53:76-90. doi: 10.1016/j.mito.2020.03.010. Epub 2020 Mar 31.
BACKGROUNDS
Acute kidney injury (AKI) is characterized by excessive inflammatory response and apoptosis in tubular epithelial cells. Recent studies suggested that long non-coding RNAs colon cancer-associated transcript-1 (CCAT-1) and microRNA-155 (miR-155) might regulate cell death and inflammation. We aimed to explore the role of CCAT-1/miRNA-155 axis in the AKI.
METHODS
LPS was applied to establish in vitro and in vivo models of AKI using HK2 cells and pcDNA-CCAT1 transgenic mice, respectively. Gene overexpression or knockdown were performed through plasmids transfection. Apoptosis were determined by qRT-PCR, western blotting (Fas, FasL, Caspase-3), AnnexinV/PI staining and TUNEL assay. Cytokines were assessed by ELISA. Interaction of CCAT1/miR-155 and miR-155/SIRT1 were detected by dual-luciferase reporter assay. RNA immunoprecipitation (RIP) was also performed to determine CCAT1/miR-155 interaction. Pathological changes of AKI were evaluated using H&E staining, blood urine nitrogen (BUN) and serum creatinine (Cr) detection kits. The degree of renal fibrosis was determined by Masson trichrome stain.
RESULTS
LPS administration reduced CCAT1 and SIRT1 expression, but increased miR-155 levels in tubular epithelial cells in vitro. Luciferase assay demonstrated that miR-155 might bind to and regulate CCAT1 and SIRT1. RIP further confirmed the direct interaction of CCAT1 and miR-155. Restoration of CCAT1 attenuated LPS induced inflammation and apoptosis through sequestering miR-155. The anti-inflammation and pro-survival effects of CCAT1 overexpression and miR-155 inhibition were abolished by SIRT1 knockdown, as indicated by the expression of cytokine and apoptotic markers, as well as H&E, BUN and Cr detection. Dysregulated CCAT1/miR-155/SIRT1 pathway regulated disease progression in a murine model of LPS-induced AKI, and NF-κB pathway involved in.
CONCLUSION
CCAT1 restoration sequestered miR-155, leading to upregulation of SIRT1 and alleviated LPS induced renal tubular epithelial cell damage in vitro and in vivo.
背景
急性肾损伤（AKI）的特征是肾小管上皮细胞中过度的炎症反应和细胞凋亡。最近的研究表明，长链非编码 RNA 结肠癌相关转录物 1（CCAT-1）和 microRNA-155（miR-155）可能调节细胞死亡和炎症。我们旨在探讨 CCAT-1/miR-155 轴在 AKI 中的作用。
方法
分别用 LPS 处理 HK2 细胞和 pcDNA-CCAT1 转基因小鼠，建立体外和体内 AKI 模型。通过质粒转染进行基因过表达或敲低。通过 qRT-PCR、western blot（Fas、FasL、Caspase-3）、AnnexinV/PI 染色和 TUNEL 检测测定细胞凋亡。通过 ELISA 测定细胞因子。通过双荧光素酶报告基因检测法检测 CCAT1/miR-155 和 miR-155/SIRT1 的相互作用。还进行了 RNA 免疫沉淀（RIP）以确定 CCAT1/miR-155 相互作用。通过 H&E 染色、血尿氮（BUN）和血清肌酐（Cr）检测试剂盒评估 AKI 的病理变化。通过 Masson 三色染色测定肾纤维化程度。
结果
LPS 处理降低了肾小管上皮细胞中 CCAT1 和 SIRT1 的表达，但增加了 miR-155 的水平。荧光素酶测定表明，miR-155 可能与 CCAT1 和 SIRT1 结合并调节其表达。RIP 进一步证实了 CCAT1 和 miR-155 的直接相互作用。通过结合 miR-155，CCAT1 的恢复减轻了 LPS 诱导的炎症和细胞凋亡。CCAT1 过表达和 miR-155 抑制的抗炎和促生存作用被 SIRT1 敲低所消除，这表明细胞因子和凋亡标志物的表达以及 H&E、BUN 和 Cr 的检测。在 LPS 诱导的 AKI 小鼠模型中，失调的 CCAT1/miR-155/SIRT1 通路调节疾病进展，涉及 NF-κB 通路。
结论
CCAT1 的恢复可结合 miR-155，导致 SIRT1 的上调，并减轻 LPS 诱导的肾小管上皮细胞在体外和体内的损伤。
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