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- 中山大学学报（医学科学版） 2022年43卷第6期 页码：928-937
- 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：
张春敏，硕士，主治医师，研究方向：脓毒症相关急性肾损伤，E-mail： min_135@163.com
- 基金信息：广州市卫生健康科技项目(20211A011038);广东省自然科学基金(2022A1515010556);西藏自治区自然科学基金［XZ2022ZR-ZY44（Z）］
DOI：10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).2022.0608
中图分类号： R394.2纸质出版日期：2022-11-20，
收稿日期：2022-07-21，
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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
2
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
2
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
2
与对照组相比，LPS诱导的HK2细胞中
miR-4321
（
n
=3，
t
=7.154，
P
=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
2
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
2
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
2
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
2
Compared with the control group， the
miR-4321
expression was increased in LPS-induced HK2 cell model （
n
=3，
t
=7.154，
P
=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
2
Inhibition of
miR-4321
significantly alleviates SA-AKI， which may be achieved by increasing the expression of mTOR.
关键词
脓毒症相关急性肾损伤脂多糖miR-4321mTOR
Keywords
sepsis associated acute kidney injurylipopolysaccharidemiR-4321mTOR
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