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
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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)


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

    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.

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刘 苏,徐巍龙,查 敏,李 楠,周静波,叶丽芳. 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[J].南京医科大学学报(自然科学版),2022,42(1):14-22

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  • 收稿日期:2021-05-27
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  • 在线发布日期: 2022-01-27
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