Effect and Mechanism of <italic style="font-style: italic">miR-4321</italic> Inhibition Alleviates Sepsis Associated Acute Kidney Injury

ZHANG Chun-min; CHEN Fei-yan; YANG Wen-min; LIN Yong-min; MAO Bi-tao; CHEN Jie; WU Zhi-yuan; LIANG Yu-feng

doi:10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).2022.0608

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

Basic Research | 更新时间：2024-02-19

- Effect and Mechanism of miR-4321 Inhibition Alleviates Sepsis Associated Acute Kidney Injury
- Journal of Sun Yat-sen University(Medical Sciences) Vol. 43, Issue 6, Pages: 928-937(2022)
- 作者机构：
  
  广州医科大学附属广州市妇女儿童医疗中心儿童重症监护室，广东广州 510120
- 作者简介：
  
  LIANG Yu-feng； E-mail： drliangyufeng@163.com；13710666550@126.com
- 基金信息：
- DOI：10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).2022.0608
  CLC： R394.2
- Received：21 July 2022，
  
  Published：20 November 2022
- 稿件说明：
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张春敏,陈飞燕,杨文敏等.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[J].中山大学学报（医学科学版）,2022,43(06):928-937.

ZHANG Chun-min,CHEN Fei-yan,YANG Wen-min,et al.Effect and Mechanism of miR-4321 Inhibition Alleviates Sepsis Associated Acute Kidney Injury[J].Journal of Sun Yat-sen University(Medical Sciences),2022,43(06):928-937.
张春敏,陈飞燕,杨文敏等.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[J].中山大学学报（医学科学版）,2022,43(06):928-937. DOI： 10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).2022.0608.

ZHANG Chun-min,CHEN Fei-yan,YANG Wen-min,et al.Effect and Mechanism of miR-4321 Inhibition Alleviates Sepsis Associated Acute Kidney Injury[J].Journal of Sun Yat-sen University(Medical Sciences),2022,43(06):928-937. DOI： 10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).2022.0608.

摘要

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 blotting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

（

=3，

=7.154，

=0.001 3）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TNF-α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。

Abstract

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 lipopolysaccharides （LPS）.

Methods

RT-qPCR was conducted to detect the expression of

miR-3165

，

miR-4270

and

miR-4321

in LPS-induced HK2 cell model and cecal ligation and puncture （CLP）-induced renal injury model. In LPS-induced 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 levels of serum creatinine and blood urea nitrogen （BUN） were measured by colorimetric method. ELISA was employed to assess 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 model （

=3，

=7.154，

=0.001 3）.

miR-4321

inhibitor promoted the proliferation and viability of HK2 cells， decreased the expression of LPS-induced IL-6， IL-1β and TNF-α and 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 IL-1β， IL-6 and TNF-α， promoted the mTOR expression in renal tissues and inhibited the apoptosis-related protein expression. mTOR signaling pathway was believed the target gene of

miR-4321

Conclusion

Inhibition of

miR-4321

significantly alleviates SA-AKI， which may be achieved by increasing the expression of mTOR.

关键词

Keywords

references

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Related Institution

Department of Stomatology， The Second Affiliated Hospital of Guangzhou Medical University

Department of Pathology， The Second Affiliated Hospital of Guangzhou Medical University

Institute of Neuroscience， The Second Affiliated Hospital of Guangzhou Medical University

Department of Intensive Care Unit，Division of Huiya，2.Department of Internal Medicine，The Division of Huangpu，3.Department of Emergency，The First Affiliated Hospital，Sun Yat-sen University

Department of Anesthesiology, The Third Affiliated Hospital of Southern Medical University

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

DOI：10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).2022.0608

摘要

Abstract

关键词

Keywords

references