Dan Huang

and 6 more

Objectives:To analyze the function and roles of circRNAs in the peripheral blood mononuclear cells (PBMCs) of ankylosing spondylitis (AS) patients. Methods: The expression of AS-related circRNAs were detected by high-throughput RNA-sequencing within the PBMCs of 5 AS cases and healthy controls. After profiling circRNA expression in these samples, differentially expressed circRNAs (DECs) were identified, and a qPCR-based validation approach was used to confirm the differential expression of six of these DECs in patient samples. Spearman’s correlation tests and ROC curve analyses were further used to assess the relationship between disease severity and the expression of DECs of interest in AS patients, after which a putative circRNA-microRNAs (miRNAs) interaction network was constructed leading to the detection of six validated DECs with competing endogenous RNA (ceRNA) functionality. Besides, cell experiments were also performed to investigate the potential mechanism of key circRNA in AS. Results: 10,441 circRNAs were identified in these 10 PBMC samples, with 131 total DECs including 89 and 42 that were up- and down-regulated, respectively. In qPCR validation assays, patterns of hsa_circ_0000702, hsa_circ_0006209, hsa_circ_0047920, hsa_circ_0001543, hsa_circ_0072697 and hsa_circ_0005076 were confirmed to align well with RNA-seq results. In addition, the expression levels of hsa_circ_0006209, hsa_circ_0047920, and hsa_circ_0072697 were detected to be positively correlated with disease severity. ROC curve analyses suggested that hsa_circ_0072697 may offer value as a diagnostic biomarker for AS. Cell experiments indicated that hsa_circ_0072697 could suppress the progression of AS by targeting NF-κB pathway Conclusions: The identification of six circRNAs with putative ceRNA functionality in AS patients highlights potential molecular mechanisms governing this debilitating disease. However, further research will be necessary to formally confirm the roles of these DECs and their target miRNAs in AS. Further, hsa_circ_0072697 has a good diagnostic value in AS patients, and it could suppress the progression of AS by targeting NF-κB pathwa

LIU FEIFEI

and 3 more

Abstract: Background: The abnormal expression of lncRNA HOTAIR has been associated with synovial angiogenesis in rheumatoid arthritis (RA). The aim of this study is to investigate whether lncRNA HOTAIR can participate in synovial angiogenesis in RA by regulating the PI3K/AKT pathway through the miR-126/PIK3R2 axis. Methods: In this study, we conducted in vitro experiments by designing overexpression plasmids and small interfering RNAs targeting lncRNA HOTAIR and then transfected them into fibroblast-like synoviocytes (FLS) obtained from RA patients. We then co-cultured the modified FLS with human umbilical vein endothelial cells (HUVEC) to establish an RA-FLS-induced HUVEC model. We investigated the effects of lncRNA HOTAIR on the proliferation, migration, and tube formation abilities of HUVECs, as well as the expression of synovial endothelial cell markers, angiogenic factors, and the PI3K/AKT pathway. To validate the interactions between lncRNA HOTAIR, miR-126-3p, and PIK3R2, we used bioinformatics and luciferase reporter experiments. We also used a combination of real-time fluorescence quantitative (RT-qPCR), Western blotting (WB), and immunofluorescence (IF) methods to identify target genes and proteins. Results: LncRNA HOTAIR was highly expressed in HUVEC cells induced by RA-FLS. Overexpression of lncRNA HOTAIR significantly increased the expression of VEGF, bFGF, CD34 and CD105 in HUVEC cells, promoted their proliferation, invasion, and tube formation, while the silencing of lncRNA HOTAIR reversed these effects, and the PI3K/AKT activator also reversed them. Overexpression of lncRNA HOTAIR activated the PI3K/AKT pathway, promoting the expression of PI3K, AKT, and P-AKT proteins. Bioinformatics and dual-luciferase assays verified the targeting relationship between LncRNA HOTAIR, miR-126-3p, and PIK3R2. Conclusion: LncRNA HOTAIR can activate the PI3K/AKT pathway, possibly through the regulatory axis of miR-126-3p/PIK3R2, and thus participate in synovial angiogenesis in rheumatoid arthritis.

shaohong cai

and 3 more

Introduction:This study focused on investigating the effects of LncRNA DANCR regulation of Keap1-Nrf2/ARE pathway on inflammation and oxidative stress in RA. Methods:The levels of LncRNA DANCR/miR-486-3p/ Keap1 in peripheral blood of 30 RA groups and 30 normal subjects were examined, and the association of LncRNA DANCR with inflammatory indicators of rheumatoid arthritis was investigated. We construct overexpression plasmids and small interfering RNAs of LncRNA DANCR to investigate the relationship between LncRNA DANCR and FLSs viability and migration in rheumatoid arthritis, as well as the effects on cellular oxidative stress factors and Keap1-Nrf2/ARE pathway; molecular biology analysis was used to predict microRNAs that can bind LncRNA DANCR, and luciferase verified the binding sites of LncRNA DANCR with Keap1 and miR-486-3p; to further refine the gene and protein expression results, we used RT- qPCR and immunoblotting assays . Results: In both groups of PBMCs, the expression levels of LncRNA DANCR and Keap1 mRNA were higher in the rheumatoid arthritis group than in the normal control group, and the opposite was true for miR-486-3p; LncRNA DANCR was positively correlated with TAOC, IL6, RF, IL17, anti-CCP, MDA, and SOD, but not with ESR, DAS28, IL11, and SOD, DAS28, IL11, ROS, CRP were negatively correlated; overexpression of lncRNA DANCR stimulated the Keap1-Nrf2/ARE pathway, decreased the expression of IL10, SOD, TAOC, and increased the expression levels of MDA, IL11, IL-17, PD-L1, and silencing of lncRNA DANCR was the opposite, but knockdown of miR- 486-3p or overexpression of keap1 reversed the expression of the above-mentioned inflammatory and oxidative factors. In addition, pcDNA-DANCR clearly showed stronger cell invasion and migration ability and exacerbated its inflammatory response, we verified their targeting relationship using dual luciferase. Conclusion: The low-expressed lncRNA DANCR may regulate the Keap1-Nrf2/ARE pathway and suppress the inflammatory and oxidative responses in RA patients.