背景:重症肌无力(MG)是一种自身免疫性疾病,其特征是针对神经肌肉接头结构的致病性抗体。证据表明,由转录因子(TF)介导的长链非编码RNA(lncRNA)的调节在MG的病理生理学中起着关键作用。然而,MG中lncRNAs的详细分子机制仍未确定。
方法:使用微阵列分析,我们分析了MG中的lncRNA水平。通过生物信息学分析,发现LINC01566可能在MG中起重要作用。首先,进行qRT-PCR以验证LINC1566在MG患者中的表达。然后,进行荧光原位杂交以确定LINC01566在CD4+T细胞中的定位。最后,我们还使用流式细胞术和CCK-8试验分析了LINC01566敲低或过表达对CD4+T细胞功能的影响.使用双荧光素酶报告基因测定来验证TFFOSL1与LINC01566启动子的结合。
结果:基于lncRNA微阵列和差异表达分析,我们鉴定了563个差异表达(DE)lncRNAs,MG中450个DEmRNA和19个DETFs。然后我们构建了一个lncRNA-TF-mRNA网络。通过网络分析,我们发现LINC01566可能通过调节T细胞相关通路在MG中发挥关键作用.进一步的实验表明LINC01566在MG患者中以低水平表达。功能上,LINC01566主要分布在细胞核中,可以促进CD4+T细胞凋亡,抑制细胞增殖。机械上,我们假设LINC01566可能负向调节DUSP3、CCR2、FADD、SIRPB1,LGALS3和SIRPB1参与T细胞活化途径,进一步影响MG细胞增殖和凋亡。此外,我们发现LINC01566对MG中CD4+T细胞的作用是由TFFOSL1介导的,体外实验表明FOSL1可以与LINC01566的启动子区结合。
结论:总之,我们的研究揭示了LINC01566在临床样本和细胞实验中的保护作用,说明FOSL1/LINC01566负调节MG中CD4+T细胞活化的潜在作用和机制。
BACKGROUND: Myasthenia gravis (MG) is an autoimmune disease characterized by pathogenic antibodies that target structures of the neuromuscular junction. The evidence suggests that the regulation of long noncoding RNAs (lncRNAs) that is mediated by transcription factors (TFs) plays a key role in the pathophysiology of MG. Nevertheless, the detailed molecular mechanisms of lncRNAs in MG remain largely undetermined.
METHODS: Using microarray analysis, we analyzed the lncRNA levels in MG. By bioinformatics analysis, LINC01566 was found to potentially play an important role in MG. First, qRT‒PCR was performed to verify the LINC1566 expressions in MG patients. Then, fluorescence in situ hybridization was conducted to determine the localization of LINC01566 in CD4 + T cells. Finally, the impact of LINC01566 knockdown or overexpression on CD4 + T-cell function was also analyzed using flow cytometry and CCK-8 assay. A dual-luciferase reporter assay was used to validate the binding of the TF FOSL1 to the LINC01566 promoter.
RESULTS: Based on the lncRNA microarray and differential expression analyses, we identified 563 differentially expressed (DE) lncRNAs, 450 DE mRNAs and 19 DE TFs in MG. We then constructed a lncRNA-TF-mRNA network. Through network analysis, we found that LINC01566 may play a crucial role in MG by regulating T-cell-related pathways. Further experiments indicated that LINC01566 is expressed at low levels in MG patients. Functionally, LINC01566 is primarily distributed in the nucleus and can facilitate CD4 + T-cell apoptosis and inhibit cell proliferation. Mechanistically, we hypothesized that LINC01566 may negatively regulate the expressions of DUSP3, CCR2, FADD, SIRPB1, LGALS3 and SIRPB1, which are involved in the T-cell activation pathway, to further influence the cellular proliferation and apoptosis in MG. Moreover, we found that the effect of LINC01566 on CD4 + T cells in MG was mediated by the TF FOSL1, and in vitro experiments indicated that FOSL1 can bind to the promoter region of LINC01566.
CONCLUSIONS: In summary, our research revealed the protective roles of LINC01566 in clinical samples and cellular experiments, illustrating the potential roles and mechanism by which FOSL1/LINC01566 negatively regulates CD4 + T-cell activation in MG.