miRNA expression profiling uncovers a role of miR-302b-3p in regulating skin fibroblasts senescence
miRNA expression profiling uncovers a role of miR-302b-3p in regulating skin fibroblasts senescence
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miRNA表达谱揭示miR-302b-3p在调节皮肤成纤维细胞衰老中的作用
DOI：
10.1002/jcb.28862
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发表时间：
2020-01-01
影响因子：
4
通讯作者：
Li, Jing
中科院分区：
文献类型：
--
作者：
Tan, Jingyong;Hu, Longyuan;Li, Jing
大量新出现的证据表明，可变的microRNA（miRNA）表达促进了衰老过程。本研究旨在区分衰老皮肤中miRNA的异常表达，并探讨miR-302 b-3 p上调的生物学功能及其可能机制。首先，利用miRNA微阵列技术研究了D-半乳糖（d-gal）致衰老小鼠皮肤组织中miRNA的表达谱。我们在老年小鼠皮肤中鉴定了29种异常miRNAs。接下来，使用DIANA-miPath v3.0进行KEGG富集分析，其揭示富集途径涉及细胞外基质-受体相互作用、MAPK信号通路和雷帕霉素的哺乳动物靶（mTOR）信号通路等过程。从四种生物信息学算法（miRDB、Targetscan、miRwalk和Tarbase）预测失调的miRNA的靶基因。使用Cytoscape软件可视化miRNA及其靶标的相互作用网络。因此，我们发现一些枢纽基因（包括JNK 2，AKT 1/2/3，PAK 7，TRPS 1，BCL 2L 11和IKZF 2）被12种潜在的miRNA（包括miR-302 b-3 p，miR-291 a-5 p，miR-139- 3 p，miR-467 c-3 p，miR-186- 3 p等）靶向。随后，我们通过定量聚合酶链反应鉴定了五种上调的miRNA，并且与年轻对照组织相比，它们在老年皮肤组织中均被证实显著增加。其中，miR-302 b-3 p在衰老皮肤组织和衰老成纤维细胞中都有高表达。此外，miR-302 b-3 p模拟物可加速皮肤成纤维细胞衰老并抑制长寿相关基因Sirt 1的表达，而miR-302 b-3 p抑制剂可延缓皮肤成纤维细胞衰老并促进Sirt 1的表达。此外，我们通过荧光素酶报告基因分析证明了c-Jun N-末端激酶2（JNK 2）是miR-302 b-3 p的直接靶点。在成纤维细胞中证实了miR-302 b-3 p和JNK 2之间的负相关性。最重要的是，siRNA JNK 2证实JNK 2的低表达可以加速成纤维细胞衰老。总之，我们的研究结果表明过表达的miR-302 b-3 p通过直接靶向JNK 2基因在加速皮肤衰老过程中发挥重要的生物学作用。
Numbers of emerging evidence suggest that variable microRNA (miRNA) expression facilitates the aging process. In this study, we distinguished aberrant miRNA expression in aged skin and explored the biological functions and potential mechanism of upregulated miR-302b-3p. At first, miRNA microarray analysis was examined to explore miRNA expression profiling in the skin of aging mice model by D-galactose (d-gal) injection. We identified 29 aberrant miRNAs in aged mice skin. Next, KEGG enrichment analysis was conducted with DIANA-miPath v3.0, which was revealed that enrichment pathways involved in such processes as extracellular matrix-receptor interaction, MAPK signaling pathway, and mammalian target of rapamycin (mTOR) signaling pathway. The target genes of deregulated miRNAs were predicted from four bioinformatic algorithms (miRDB, Targetscan, miRwalk, and Tarbase). The interaction network of miRNAs and their targets were visualized using Cytoscape software. As a result, we found that some hub genes (including JNK2, AKT1/2/3, PAK7, TRPS1, BCL2L11, and IKZF2) were targeted by 12 potential miRNAs (including miR-302b-3p, miR-291a-5p, miR-139-3p, miR-467c-3p, miR-186-3p, etc.). Subsequently, we identified five upregulated miRNA via quantitative polymerase chain reaction and all of them were confirmed increased significantly in aged skin tissues compared with young control tissues. Among them, high expression of miR-302b-3p was verified in both aged skin tissues and senescence fibroblasts. Furthermore, miR-302b-3p mimic accelerated skin fibroblast senescence and suppressed the longevity-associated gene Sirtuin 1(Sirt1) expression, whereas miR-302b-3p inhibitor could delay skin fibroblast senescence and contribute Sirt1 expression. In addition, we demonstrated that c-Jun N-terminal kinase 2(JNK2) is a direct target of miR-302b-3p by a luciferase reporter assay. An inverse correlation was verified in fibroblasts between miR-302b-3p and JNK2. Most importantly, siRNA JNK2 confirmed that low expression of JNK2 could accelerate fibroblasts senescence. In conclusion, our results indicated that overexpressed miR-302b-3p plays an important biological role in accelerating skin aging process via directly targeting JNK2 gene.