Ma Hualin, Zhang Shuyan, Xu Ying, Zhang Rongrong, Zhang Xinzhou
Department of Nephrology, Shenzhen People's Hospital, Shenzhen Key Laboratory of Kidney Disease, Second Clinical Medical College, Jinan University, Shenzhen, China.
Department of Hematology, Shenzhen People's Hospital, Second Clinical Medical College, Jinan University, Shenzhen, China.
Arch Med Sci. 2018 Aug;14(5):1102-1111. doi: 10.5114/aoms.2017.70878. Epub 2017 Oct 20.
To analyze the microRNA expression of tumor necrosi factor α (TNF-α) stimulated mesenchymal stem cells (MSCs) and exosomes from their culture supernatant.
TNF-α (20 ng/ml) was used to stimulate MSCs, which were then regarded as TNF-α cells (TC), while unstimulated cells were the normal control cells (NCC). MSCs and their culture supernatant were harvested after 48 h. Subsequently, exosomes were isolated from culture supernatants with ExoQuick-TC and were divided into two groups, TNF-α exosomes (TE) and normal control exosomes (NCE). Then, the microRNAs were measured by high-throughput sequencing and the results were differentially analyzed. Finally, the correlation of the target genes corresponding to differently expressed microRNAs was analyzed by gene ontology (GO) and KEGG pathway analysis.
High-throughput sequencing showed that the cellular compartment (TC vs. NCC) had 280 microRNAs. miR-146a-5p was a uniquely up-regulated microRNA ( < 0.001) and the most significantly down-regulated microRNA among the 279 microRNAs included was miR-150-5p ( < 0.001). There were 180 differentially expressed microRNAs in the exosome compartment (TE vs. NCE), where miR-146-5p ( < 0.001) was one of 176 upregulated microRNAs and miR-203b-5p ( < 0.001) was one of 4 downregulated microRNAs. Coincidentally, bioinformatics analysis showed that IRAK1 was a critical target gene of miR-146-5p related to the Toll-like receptor (TLR) signaling pathway.
In contrast with the control group, there were significantly differentially expressed microRNAs in both MSCs and exosomes. Interestingly, miR-146a-5p was up-regulated in both comparative groups, and its target gene IRAK1 plays a crucial part in the TLR signaling pathway. These investigations demonstrate a new direction for subsequent inflammation mechanistic studies.
分析肿瘤坏死因子α（TNF-α）刺激的间充质干细胞（MSCs）及其培养上清液中微小RNA的表达情况。
用TNF-α（20 ng/ml）刺激MSCs，将其视为TNF-α细胞（TC），未刺激的细胞作为正常对照细胞（NCC）。48小时后收集MSCs及其培养上清液。随后，用ExoQuick-TC从培养上清液中分离出外泌体，并分为两组，即TNF-α外泌体（TE）和正常对照外泌体（NCE）。然后，通过高通量测序测定微小RNA，并对结果进行差异分析。最后，通过基因本体论（GO）和KEGG通路分析对差异表达微小RNA对应的靶基因的相关性进行分析。
高通量测序显示，细胞组（TC与NCC）中有280种微小RNA。miR-146a-5p是唯一上调的微小RNA（<0.001），在所包含的279种微小RNA中，下调最显著的是miR-150-5p（<0.001）。在外泌体组（TE与NCE）中有180种差异表达的微小RNA，其中miR-146-5p（<0.001）是176种上调微小RNA之一，miR-203b-5p（<0.001）是4种下调微小RNA之一。巧合的是，生物信息学分析表明IRAK1是与Toll样受体（TLR）信号通路相关的miR-146-5p的关键靶基因。
与对照组相比，MSCs和外泌体中均有显著差异表达的微小RNA。有趣的是，miR-146a-5p在两个比较组中均上调，其靶基因IRAK1在TLR信号通路中起关键作用。这些研究为后续炎症机制研究指明了新方向。
