肾阳虚大鼠肾组织中microRNA差异性表达与信号通路分析

宣玲; 包玉婷; 周小杰; 姬丽婷; 杨元宵; 李昌煜

doi:10.13422/j.cnki.syfjx.20191837

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肾阳虚大鼠肾组织中microRNA差异性表达与信号通路分析

药理 | 更新时间：2021-02-09

- 肾阳虚大鼠肾组织中microRNA差异性表达与信号通路分析
- Differential Expression and Signal Transduction Analysis of MicroRNA Related to Kidney Yang Deficiency in Kidney Tissues of Rats
- 中国实验方剂学杂志 2019年25卷第17期页码：57-63
- 作者机构：
  
  1.浙江中医药大学　药学院，杭州　 310053；
  2.杭州医学院　基础医学与法医学院，杭州　 310013
- 作者简介：
  
  宣玲，在读硕士，从事中药药理及新药开发研究，E-mail：15375682006@163.com
  李昌煜，硕士，教授，从事中药药理及新药开发研究，E-mail：lcyzcmu@sina.com
- 基金信息：
  
  国家自然科学基金项目(81673839;81373507);浙江省中医药科技计划项目(2019ZQ014);大学生创新创业孵化项目(2018R410051)
- DOI：10.13422/j.cnki.syfjx.20191837
  中图分类号： R2-0; R285.5; R289
- 收稿日期：2019-01-03，
  
  网络出版日期：2019-06-06，
  
  纸质出版日期：2019-09-05
- 稿件说明：
移动端阅览
宣玲, 包玉婷, 周小杰, 等. 肾阳虚大鼠肾组织中microRNA差异性表达与信号通路分析[J]. 中国实验方剂学杂志, 2019,25(17):57-63.

Ling XUAN, Yu-ting BAO, Xiao-jie ZHOU, et al. Differential Expression and Signal Transduction Analysis of MicroRNA Related to Kidney Yang Deficiency in Kidney Tissues of Rats[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(17): 57-63.
宣玲, 包玉婷, 周小杰, 等. 肾阳虚大鼠肾组织中microRNA差异性表达与信号通路分析[J]. 中国实验方剂学杂志, 2019,25(17):57-63. DOI： 10.13422/j.cnki.syfjx.20191837.

Ling XUAN, Yu-ting BAO, Xiao-jie ZHOU, et al. Differential Expression and Signal Transduction Analysis of MicroRNA Related to Kidney Yang Deficiency in Kidney Tissues of Rats[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(17): 57-63. DOI： 10.13422/j.cnki.syfjx.20191837.

摘要

目的：

采用基因芯片检测腺嘌呤造模诱导肾阳虚证大鼠肾组织中显著差异表达的小分子核糖核酸(miRNA)，并用生物信息学方法分析其意义。

方法：

模型组大鼠灌胃150 mg·kg

－1

腺嘌呤诱导肾阳虚肾损伤模型，正常组灌服等量生理盐水。麻醉处死后取部分肾组织病理切片做苏木素-伊红(HE)染色并检测血液中血尿素氮(BUN)，血肌酐(SCr)的含量，尿液中24 h尿蛋白(24U-TP)含量；trizol法提取肾脏组织中RNA，RNA经检测质量合格后用于后续芯片分析。μParaflo®微流体芯片分析肾组织中差异表达的miRNA；通过实时荧光定量聚合酶链式反应(Real-time PCR)进一步验证芯片结果；利用生物信息学网站分析差异表达miRNA的靶基因及其功能。

结果：

基因芯片结果显示造模后共有50个miRNA差异表达，与正常组比较，模型组只有9个miRNA在肾组织中高表达且具有显著性差异，其中，与正常组比较，模型组中rno-miR-21-5p，rno-let-7i-5p，rno-miR-146b-5p和rno-miR-15b-5p表达明显升高(

0.05，

0.01)；rno-miR-6215，rno-miR-192-5p，rno-miR-378b，rno-miR-378a-3p和rno-miR-194-5p表达明显降低(

0.05)。经PCR验证表明，与正常组比较，rno-miR-21-5p，rno-miR-146b-5p表达显著上升(

0.01)；rno-miR-192-5p，rno-miR-378b，rno-miR-378a-3p，rno-miR-194-5p表达显著下降(

0.01)。其中，miR-192，miR-21，miR-378与上皮细胞-间充质转化/间充质-上皮细胞转化(EMT/MET)平衡相关，miR-192和miR-378可作为抗纤维化因子保护肾脏，而miR-21可作为促纤维化因子诱导肾脏损伤。miR-194可通过靶向调控脑Ras同源蛋白(Rheb)拮抗缺氧再灌注诱导的人肾近端肾小管上皮细胞HK-2损伤。这些差异miRNA的靶基因主要富集在Wnt和丝裂原活化蛋白激酶(MAPK)信号通路上。这两条信号通路与EMT/MET平衡密切相关。

结论：

通过基因芯片表达谱，发现4个参与肾间质纤维化(RIF)作用调节和2个未知功能miRNA表达。为进一步深入分析肾阳虚的调控网络提供了一个新的线索。

Abstract

Objective:

Microarray chip was used to detect the differentially expressed microRNA (miRNA) in kidney tissues of rats with kidney-Yang deficiency induced by adenine

and its significance was analyzed by bioinformatics method.

Method:

Rats with kidney-Yang deficiency were induced by intragastric administration of 150 mg·kg

-1

adenine in model group

while rats in normal group were given the same amount of saline.Kidney tissues were taken for hematoxylin-eosin (HE) staining pathological sections after anesthesia and blood urea nitrogen (BUN)

creatinine (SCr) in blood and 24-hour urinary protein (24U-TP) in urine were measured. μParaflo® microfluidic chip technology was used to investigate differential expression miRNA in kidney tissues

and microarray results were verified by Real-time PCR. Bioinformatics database was used to analyze the target genes and functions of differential expression miRNAs.

Result:

Gene chip results showed that there were 50 differentially expressed microRNAs after modeling. Compared with control group

only 9 miRNAs were highly expressed in kidney tissues with significant difference were detected in model group. Compared with the normal group

the expression of rno-miR-21-5p

rno-let-7i-5p

rno-miR-146b-5p and rno-miR-15b-5p in model group increased significantly(

0.05

0.01)

the expression of rno-miR-6215

rno-miR-192-5p

rno-miR-378b

rno-miR-378a-3p and rno-miR-194-5p decreased significantly (

0.05). Verification by Real-time PCR showed that

compared with the normal group

the expression of rno-miR-21-5p and rno-miR-146b-5p increased significantly (

0.01)

while the expression of rno-miR-192-5p

rno-miR-378b

rno-miR-378a-3p and rno-miR-194-5p decreased significantly (

0.01). In which

miR-192

miR-21 and miR-378 are associated with epithelial-mesenchymal transition/mesenchymal-epithelial transition(EMT/MET) balance.miR-192 and miR-378 can be used as anti-fibrosis factors to protect the kidney

while miR-21 can be used as fibrosis factors to induce kidney injury. miR-194 can antagonize the damage of human proximal renal tubular epithelial cells (HK-2) induced by hypoxia-reperfusion by targeting brain Ras homologous protein (Rheb). Target genes of differentially expressed miRNAs were predominantly involved in Wnt and mitogen-activated protein kinase(MAPK)signaling pathway.

Conclusion:

This experiment found 4 miRNAs involved in the regulation of renal interstitial fibrosis (RIF) and 2miRNAs with unknown functions

which provided a new clue for further analysis of the regulatory network of kidney-yang deficiency.

关键词

Keywords

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