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LncRNA-PCAT1 通过靶向 miR-145-5p 促进脂肪来源干细胞 TLR4 相关成骨分化。

LncRNA-PCAT1 targeting miR-145-5p promotes TLR4-associated osteogenic differentiation of adipose-derived stem cells.

机构信息

Department of Orthopaedic Surgery, Peking Union Medical College Hospital, Peking Union Medical College and Chinese Academy of Medical Science, Dongcheng District, Beijing, China.

Department of Orthopaedic Surgery, Beijing Jishuitan Hospital, Fourth Clinical Medical College of Peking University, Xicheng District, Beijing, China.

出版信息

J Cell Mol Med. 2018 Dec;22(12):6134-6147. doi: 10.1111/jcmm.13892. Epub 2018 Oct 19.

DOI:10.1111/jcmm.13892
PMID:30338912
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6237555/
Abstract

This study was aimed to explore the differential expression of long noncoding RNAs (lncRNA)-PCAT1, miR-145-5p and TLR4 in osteogenic differentiation via the Toll-like receptor (TLR) signalling pathway and consequently determine the potential molecular mechanism. The mRNAs and pathways related to the osteogenic differentiation in human adipose-derived stem cells (hADSCs) were analysed by bioinformatics. The MiRanda and TargetScan database were employed to detect the potential binding sites of miRNAs on lncRNAs and mRNAs. The differential expression of lncRNA-PCAT1, miR-145-5p and TLR4 were detected by qRT-PCR. Rrelated protein expression was analysed by Western blot. The targeted relationships between lncRNA-PCAT1, miR-145-5p and TLR4 were verified by dual-luciferase reporter assay. Alkaline phosphatase (ALP) activity and ARS staining assays were used to measure the impacts exerted by lncRNA PCAT1, miR-145-5p and TLR4 mRNA on osteogenic differentiation. After the induction of osteoblast differentiation, the expression of lncRNA-PCAT1 and TLR4 increased, while the expression of miR-145-5p decreased. Dual-luciferase reporter assay confirmed the targeted relationship between lncRNA-PCAT1, miR-145-5p, and TLR4. LncRNA-PCAT1 negatively regulated miR-145-5p and positively regulated TLR4. Knockdown of lncRNA-PCAT1 or TLR4 decreased the expression of osteogenic differentiation-related proteins, reduced the ALP and ARS levels and the activity of the TLR signalling pathway. MiR-145-5p could reverse the effects of PCAT1 and TLR4 in hADSCs osteogenic differentiation. LncRNA-PCAT1 negatively regulated miR-145-5p, which promoted TLR4 expression to promote osteogenic differentiation by activating the TLR signalling pathway.

摘要

本研究旨在通过 Toll 样受体（TLR）信号通路探索长链非编码 RNA（lncRNA）-PCAT1、miR-145-5p 和 TLR4 在成骨分化中的差异表达，进而确定潜在的分子机制。通过生物信息学分析人脂肪间充质干细胞（hADSCs）成骨分化相关的 mRNAs 和通路。利用 MiRanda 和 TargetScan 数据库检测 miRNA 与 lncRNA 和 mRNAs 的潜在结合位点。通过 qRT-PCR 检测 lncRNA-PCAT1、miR-145-5p 和 TLR4 的差异表达。通过 Western blot 分析相关蛋白表达。通过双荧光素酶报告基因实验验证 lncRNA-PCAT1、miR-145-5p 和 TLR4 之间的靶向关系。通过碱性磷酸酶（ALP）活性和 ARS 染色实验检测 lncRNA PCAT1、miR-145-5p 和 TLR4 mRNA 对成骨分化的影响。在诱导成骨分化后，lncRNA-PCAT1 和 TLR4 的表达增加，而 miR-145-5p 的表达减少。双荧光素酶报告基因实验证实了 lncRNA-PCAT1、miR-145-5p 和 TLR4 之间的靶向关系。lncRNA-PCAT1 负调控 miR-145-5p，正调控 TLR4。敲低 lncRNA-PCAT1 或 TLR4 降低了成骨分化相关蛋白的表达，降低了 ALP 和 ARS 水平以及 TLR 信号通路的活性。miR-145-5p 可逆转 lncRNA-PCAT1 和 TLR4 在 hADSCs 成骨分化中的作用。lncRNA-PCAT1 负调控 miR-145-5p，促进 TLR4 表达，通过激活 TLR 信号通路促进成骨分化。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9a5b/6237555/ca5001e78686/JCMM-22-6134-g009.jpg
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