FOSL1-mediated LINC01566 negatively regulates CD4+ T-cell activation in myasthenia gravis.

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.

Autophagy Inhibition by ATG3 Knockdown Remits Oxygen-Glucose Deprivation/Reoxygenation-Induced Injury and Inflammation in Brain Microvascular Endothelial Cells.

Autophagy participates in the development of cerebral ischemia stroke. Autophagy-related 3 (ATG3), an important autophagy regulator, was reported to be upregulated in a rat model of cerebral ischemia/reperfusion (CI/R) injury and an oxygen-glucose deprivation/reoxygenation (OGD/R) cell model. However, the detailed role of ATG3 in CI/R injury remains elusive. An in vitro cellular model was established to mimic CI/R injury by exposing hBMECs and bEnd.3 cells to OGD/R. OGD/R-induced injury were evaluated by cell counting kit-8 (CCK-8), LDH release assay, caspase-3 activity assay and TUNEL assay. Inflammation was assessed by detecting mRNA expression and concentrations of interleukin-1ß (IL-1ß), IL-6 and tumor necrosis factor-α (TNF-α) using qRT-PCR and ELISA, respectively. The protein levels of ATG3, light chain 3 (LC3)-I, LC3-II, p62, protein kinase B (Akt), and phosphorylated Akt (p-Akt) were determined by western blot analysis. We successfully established an in vitro OGD/R injury model using hBMECs and bEnd.3 cells. ATG3 was time-dependently upregulated and ATG3 knockdown inhibited autophagy in OGD/R-challenged brain microvascular endothelial cells. Moreover, autophagy inhibition by ATG3 interference attenuated OGD/R-induced viability inhibition and increase of LDH release, caspase-3 activity, programmed cell death, and production of IL-1ß, IL-6 and TNF-α. Inhibition of autophagy by ATG3 silencing activated the phosphoinositide 3-kinase (PI3K)/Akt pathway in OGD/R-challenged brain microvascular endothelial cells. Furthermore, inhibition of the PI3K/Akt pathway reversed the protective effects of ATG3 silencing on OGD/R-induced injury and inflammation. In conclusion, autophagy inhibition by ATG3 knockdown remitted OGD/R-induced injury and inflammation in brain microvascular endothelial cells via activation of the PI3K/Akt pathway.