Page 20 - 南京医科大学学报自然科学版

南京医科大学学报（自然科学版） 第42卷第1期
· 14 · Journal of Nanjing Medical University（Natural Sciences） 2022年1月

·基础研究·

miR⁃296⁃5p在糖尿病肾病db/db小鼠血浆外泌体中的表达及功能

刘 苏 ，徐巍龙，查 敏，李 楠，周静波，叶丽芳
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南京中医药大学附属医院内分泌科，江苏 南京 210029

［摘 要］ 目的：明确miR⁃296⁃5p在糖尿病肾病（diabetic nephropathy，DN）db/db小鼠血浆外泌体中的表达并初探其在转录因
子 Snail1 介导的上皮⁃间充质转化（epithelial⁃mesenchymal transition，EMT）中的作用。方法：先后采集 12 只 12 周龄 db/db 小鼠
（模型组）和 12 只 12 周龄 db/m 小鼠（对照组）血浆，提取外泌体 RNA 进行测序，利用生物信息学软件筛选靶向 Snail1 的差异
miRNA；利用透视电镜、纳米颗粒跟踪分析鉴定外泌体；并通过小鼠肾组织及血浆外泌体验证获得目标 miRNA，双荧光素酶
实验明确其与Snail1的靶向关系，RT⁃qPCR 和免疫组化检测db/db 小鼠和db/m 小鼠肾组织中Snail1、钙黏附蛋白（E⁃cadherin）
和α⁃平滑肌肌动蛋白（α⁃smooth muscle actin，α⁃SMA）基因和蛋白的表达。结果：miR⁃296⁃5p在db/db小鼠血浆外泌体及肾组
织中表达下调（P=0.011，P=0.001）；双荧光素酶实验证实 miR⁃296⁃5p 与 Snail1 的靶向关系；db/db 小鼠肾组织中 Snail1 mRNA
和蛋白表达显著升高（P < 0.001，P=0.005），E⁃cadherin mRNA 和蛋白表达降低（P=0.012，P=0.020），α⁃SMA mRNA 表达增加
（P=0.042）。结论：miR⁃296⁃5p在db/db小鼠血浆外泌体中低表达，进而可能通过血浆外泌体递送，靶向上调肾小管上皮细胞
Snail1，诱导肾小管上皮细胞EMT，加速DN肾间质纤维化。
［关键词］ 血浆外泌体；糖尿病肾病；Snail1；上皮细胞⁃间充质转化
［中图分类号］ R587.2 ［文献标志码］ A ［文章编号］ 1007⁃4368（2022）01⁃014⁃09
doi：10.7655/NYDXBNS20220103

Expression and function of miR⁃296⁃5p in plasma exosomes of db/db mice with diabetic
nephropathy

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LIU Su ，XU Weilong，ZHA Min，LI Nan，ZHOU Jingbo，YE Lifang
Department of Endocrinology，Affiliated Hospital of Nanjing University of Chinese Medicine，Nanjing 210029，China

［Abstract］ Objective： To investigate the expression of miR⁃296⁃5p in plasma exosomes of db/db mice with diabetic nephropathy
（DN）and explore its role in Snail1⁃mediated epithelial⁃mesenchymal transition. Methods： The plasma of 12 12⁃week⁃old db/db mice
（Model group）and 12 12⁃week⁃old db/m mice（Control group）were collected successively，and plasma exosomal RNA was extracted
and sequenced. The differential miRNAs targeting Snail1 were summarized by bioinformatics software. Exosomes were identified with
transmission electron microscopy and nanoparticle tracking analyzer. The objective miRNA was obtained by screening using kidney
tissues and plasma exosomes in db/db mice. The targeting relationship between miR ⁃ 296 ⁃ 5p and Snail1 was verified by double
luciferase experiment. The expression of Snail1，E⁃cadherin（E⁃cadherin），and α⁃smooth muscle actin（α⁃SMA）were detected in
kidney tissues of db/db mice and db/m mice by RT⁃qPCR and immunohistochemistry. Results： The miR⁃296⁃5p was significantly down⁃
regulated in plasma exosomes and kidney of db/db mice，respectively（P=0.011；P=0.001）. The miR⁃296⁃5p and Snail1 showed a
targeted negative regulation relationship in double luciferase experiment. The gene and protein expression of Snail1 in kidney tissue of
db/db mice increased significantly respectively（P < 0.001；P=0.005）. However，the mRNA and protein expression of E ⁃ cadherin
decreased significantly（P=0.012；P=0.020），while the mRNA expression of α ⁃ SMA up ⁃ regulated（P=0.042）. Conclusion： The
expression of miR⁃296⁃5p is down⁃regulated in plasma exosomes of db/db mice，which may be delivered to renal tubular epithelial cells
by plasma exosomes，targeting up⁃regulated expression of Snail1，and inducing EMT of renal tubular epithelial cells and accelerating
renal interstitial fibrosis of DN.
［Key words］ plasma exosomes；diabetic nephropathy；Snail1；epithelial⁃mesenchymal transition
［J Nanjing Med Univ，2022，42（01）：014⁃022］

［基金项目］ 国家自然科学基金青年基金（81804027）；国家自然科学基金面上项目（81774117）
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通信作者（Corresponding author），E⁃mail： nfmkls@163.com