来源于脐带间充质干细胞的外泌体环状 RNA HIPK3 通过阻断 miR-20b-5p/ULK1/Atg13 轴促进皮肤成纤维细胞自噬。

Exosomal circHIPK3 derived from umbilical cord-derived mesenchymal stem cells enhances skin fibroblast autophagy by blocking miR-20b-5p/ULK1/Atg13 axis.

机构信息

Department of Burn & Skin Repair Surgery, Hainan General Hospital (Hainan Affiliated Hospital of Hainan Medical University), Haikou, Hainan, China.

出版信息

J Diabetes Investig. 2023 Dec;14(12):1344-1355. doi: 10.1111/jdi.14077. Epub 2023 Sep 8.

DOI:10.1111/jdi.14077

PMID:37688345

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10688123/

Abstract

BACKGROUND

Umbilical cord-derived mesenchymal stem cells (UCMSCs) could alleviate diabetes-induced injury. Hence, this investigation aimed to explore the role and mechanism of UCMSCs-derived exosomal circHIPK3 (exo-circHIPK3) in diabetes mellitus (DM).

METHODS

HFF-1 cells were cultured in high glucose (HG) medium or normal medium, and treated with UCMSCs-derived exo-circHIPK3 or miR-20b-5p mimics or Unc-51-like autophagy activating kinase 1 (ULK1) overexpression vector. The surface markers of UCMSCs were analyzed using a flow cytometer. The differentiation potential of UCMSCs was evaluated using oil red O staining, alizarin red staining and alkaline phosphatase (ALP) staining. Cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The miRNA expressions were analyzed by reverse transcription-quantitative polymerase chain reaction (qRT-PCR). Protein levels were quantified by western blot. An immunofluorescence staining was used for observing LC3 expression. The interaction between miR-20b-5p and circHIPK3, and between miR-20b-5b and ULK1 were identified by a RNA immunoprecipitation (RIP) assay and a luciferase reporter assay.

RESULTS

Up-regulation of circHIPK3 was found in UCMSCs-derived exosomes. Exo-circHIPK3 decreased the miR-20b-5p level while increasing the contents of ULK1 and autophagy-related gene 13 (Atg13) in HG-induced fibroblasts. In addition, exo-circHIPK3 activated HG-induced fibroblast autophagy and proliferation. Overexpressed miR-20b-5p promoted fibroblast injury by inhibiting cell autophagy via the ULK1/Atg13 axis in HG conditions of high glucose. Moreover, exo-circHIPK3 enhanced autophagy and cell viability in HG-induced fibroblasts through the miR-20b-5p/ULK1/Atg13 axis.

CONCLUSION

UCMSCs-derived exosomal circHIPK3 promoted cell autophagy and proliferation and accelerated the fibroblast injury repair by the miR-20b-5p/ULK1/Atg13 axis.

摘要

背景

脐带间充质干细胞（UCMSCs）可减轻糖尿病引起的损伤。因此，本研究旨在探讨 UCMSCs 衍生的外泌体环状 HIPK3（exo-circHIPK3）在糖尿病（DM）中的作用和机制。

方法

将 HFF-1 细胞在高糖（HG）培养基或正常培养基中培养，并分别用 UCMSCs 衍生的外泌体 circHIPK3 或 miR-20b-5p 模拟物或 Unc-51 样自噬激活激酶 1（ULK1）过表达载体处理。用流式细胞仪分析 UCMSCs 的表面标志物。用油红 O 染色、茜素红染色和碱性磷酸酶（ALP）染色评估 UCMSCs 的分化潜能。通过 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）测定细胞增殖。采用逆转录定量聚合酶链反应（qRT-PCR）分析 miRNA 表达。用 Western blot 定量蛋白水平。用免疫荧光染色观察 LC3 的表达。通过 RNA 免疫沉淀（RIP）测定和荧光素酶报告测定鉴定 miR-20b-5p 与 circHIPK3 以及 miR-20b-5p 与 ULK1 之间的相互作用。

结果

发现 UCMSCs 衍生的外泌体中 circHIPK3 上调。外泌体 circHIPK3 降低了 HG 诱导的成纤维细胞中 miR-20b-5p 的水平，同时增加了 ULK1 和自噬相关基因 13（Atg13）的含量。此外，外泌体 circHIPK3 激活了 HG 诱导的成纤维细胞自噬和增殖。在高糖的 HG 条件下，过表达的 miR-20b-5p 通过抑制细胞自噬来促进成纤维细胞损伤，从而抑制细胞自噬。此外，外泌体 circHIPK3 通过 miR-20b-5p/ULK1/Atg13 轴增强 HG 诱导的成纤维细胞自噬和细胞活力。