通过整合生物信息学和实验分析鉴定 lncRNA HCG18 在重症肌无力中的调控作用。
Identification of the regulatory role of lncRNA HCG18 in myasthenia gravis by integrated bioinformatics and experimental analyses.
机构信息
Department of Neurology, The Second Affiliated Hospital of Harbin Medical University, Harbin, 150081, Heilongjiang, China.
College of Bioinformatics Science and Technology, Harbin Medical University, Harbin, 150081, Heilongjiang, China.
出版信息
J Transl Med. 2021 Nov 18;19(1):468. doi: 10.1186/s12967-021-03138-0.
BACKGROUND
Long non-coding RNAs (lncRNAs), functioning as competing endogenous RNAs (ceRNAs), have been reported to play important roles in the pathogenesis of autoimmune diseases. However, little is known about the regulatory roles of lncRNAs underlying the mechanism of myasthenia gravis (MG). The aim of the present study was to explore the roles of lncRNAs as ceRNAs associated with the progression of MG.
METHODS
MG risk genes and miRNAs were obtained from public databases. Protein-protein interaction (PPI) network analysis and module analysis were performed. A lncRNA-mediated module-associated ceRNA (LMMAC) network, which integrated risk genes in modules, risk miRNAs and predicted lncRNAs, was constructed to systematically explore the regulatory roles of lncRNAs in MG. Through performing random walk with restart on the network, HCG18/miR-145-5p/CD28 ceRNA axis was found to play important roles in MG, potentially. The expression of HCG18 in MG patients was detected using RT-PCR. The effects of HCG18 knockdown on cell proliferation and apoptosis were determined by CCK-8 assay and flow cytometry. The interactions among HCG18, miR-145-5p and CD28 were explored by luciferase assay, RT-PCR and western blot assay.
RESULTS
Based on PPI network, we identified 9 modules. Functional enrichment analyses revealed these modules were enriched in immune-related signaling pathways. We then constructed LMMAC network, containing 25 genes, 50 miRNAs, and 64 lncRNAs. Through bioinformatics algorithm, we found lncRNA HCG18 as a ceRNA, might play important roles in MG. Further experiments indicated that HCG18 was overexpressed in MG patients and was a target of miR-145-5p. Functional assays illustrated that HCG18 suppressed Jurkat cell apoptosis and promoted cell proliferation. Mechanistically, knockdown of HCG18 inhibited the CD28 mRNA and protein expression levels in Jurkat cells, while miR-145-5p inhibitor blocked the reduction of CD28 expression induced by HCG18 suppression.
CONCLUSION
We have reported a novel HCG18/miR-145-5p/CD28 ceRNA axis in MG. Our findings will contribute to a deeper understanding of the molecular mechanism of and provide a novel potential therapeutic target for MG.
背景
长链非编码 RNA（lncRNA）作为竞争性内源性 RNA（ceRNA），已被报道在自身免疫性疾病的发病机制中发挥重要作用。然而，lncRNA 在重症肌无力（MG）发病机制中的调控作用知之甚少。本研究旨在探讨 lncRNA 作为 ceRNA 与 MG 进展相关的作用。
方法
从公共数据库中获取 MG 风险基因和 miRNAs。进行蛋白质-蛋白质相互作用（PPI）网络分析和模块分析。构建一个 lncRNA 介导的模块相关 ceRNA（LMMAC）网络，该网络整合了模块中的风险基因、风险 miRNAs 和预测 lncRNAs，以系统地探讨 lncRNA 在 MG 中的调控作用。通过在网络上进行随机游走再启动，发现 HCG18/miR-145-5p/CD28 ceRNA 轴可能在 MG 中发挥重要作用。使用 RT-PCR 检测 MG 患者中 HCG18 的表达。通过 CCK-8 测定和流式细胞术测定，检测 HCG18 敲低对细胞增殖和凋亡的影响。通过荧光素酶报告基因测定、RT-PCR 和 Western blot 测定，探讨 HCG18、miR-145-5p 和 CD28 之间的相互作用。
结果
基于 PPI 网络，我们鉴定了 9 个模块。功能富集分析表明，这些模块富集在免疫相关信号通路中。我们随后构建了 LMMAC 网络，包含 25 个基因、50 个 miRNAs 和 64 个 lncRNAs。通过生物信息学算法，我们发现 lncRNA HCG18 作为 ceRNA，可能在 MG 中发挥重要作用。进一步的实验表明，HCG18 在 MG 患者中过度表达，是 miR-145-5p 的靶点。功能测定表明，HCG18 抑制 Jurkat 细胞凋亡并促进细胞增殖。机制上，HCG18 的敲低抑制 Jurkat 细胞中 CD28 的 mRNA 和蛋白表达水平，而 miR-145-5p 抑制剂阻断 HCG18 抑制诱导的 CD28 表达降低。
结论
我们报道了 MG 中的一个新的 HCG18/miR-145-5p/CD28 ceRNA 轴。我们的发现将有助于深入了解发病机制，并为 MG 提供新的潜在治疗靶点。
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