LncNFYB promotes the proliferation of rheumatoid arthritis fibroblast-like synoviocytes via LncNFYB/ANXA2/ERK1/2 axis.

Long noncoding RNAs (lncRNAs) are specifically expressed in different diseases and regulate disease progression. To explore the functions of rheumatoid arthritis (RA)-specific lncRNA, we determined the lncRNA expression profile of fibroblast-like synoviocytes (FLS) obtained from patients with RA and osteoarthritis (OA) using a LncRNA microarray and identified up-regulated LncNFYB in RA as a potential therapeutic target. Using gain- and loss-of-function studies, LncNFYB was proven to promote FLS proliferation and cell cycle progress but not affect their invasion, migration, and apoptotic abilities. Further investigation discovered that LncRNA could combine with annexin A2 (ANXA2) and enhance the level of phospho-ANXA2 (Tyr24) in the plasma membrane area, which induced the activation of ERK1/2 to promote proliferation. These findings provide new insights into the biological functions of LncNFYB on modification of FLS, which may be exploited for the therapy of RA.

Genome-based classification of Pedobacter polysacchareus sp. nov., isolated from Antarctic soil producing exopolysaccharide.

A psychrotolerant bacterial strain, designated ZS13-49T, with strong extracellular polysaccharide synthesis ability was isolated from soil collected in Antarctica and subjected to polyphasic taxonomic and comparative genomics. Chemotaxonomic features, including fatty acids, and polar lipid profiles, support the assignment of strain ZS13-49T to the genus Pedobacter. 16S rRNA gene phylogeny demonstrates that strain ZS13-49T forms a well-supported separate branch as a sister clade to Pedobacter gandavensis LMG 31462T and is clearly separated from Pedobacter steynii DSM 19110T and Pedobacter caeni DSM 16990T. Phylogenetic analysis showed strain ZS13-49T shared the highest 16S rRNA gene sequence similarity (99.9%) with P. gandavensis LMG 31462T. However, the digital DNA-DNA hybridization (dDDH), average nucleotide identity (ANI) value and average amino identity (AAI) value between strain ZS13-49T and P. gandavensis LMG 31462T were 26.5%, 83.3%, and 87.5%, respectively. Phylogenomic tree and a comparative genomic analysis indicated distinct characteristics to distinguish strain ZS13-49T from the closely related species. The complete genome sequence of strain ZS13-49T consists of 5 830 353 bp with 40.61% G + C content. Genomic features of strain ZS13-49T adapted to Antarctic environment were also revealed. Based on the phenotypic, chemotaxonomic, and genomic data, strain ZS13-49T could be assigned to a novel species within the genus Pedobacter for which the name Pedobacter polysacchareus sp. nov. is proposed. The type strain is ZS13-49T ( = CCTCC AB 2019394T = KCTC 72824T).

Identification of Circular RNAs Circ_0005008 and Circ_0005198 in Plasma as Novel Biomarkers for New-Onset Rheumatoid Arthritis.

The progression of autoimmune diseases is affected by the differential expression of circular RNAs (circRNAs). However, in the plasma from rheumatoid arthritis (RA), circRNAs have an uncertain role. Herein, microarray analysis was used to determine the plasma expression profile of circRNAs from new-onset patients with RA and healthy controls (HCs). CircRNA expression was verified using quantitative real-time reverse transcription PCR. The correlation between clinical variables and circRNA expression was assessed using Spearman's correlation test. The diagnostic value of plasma circRNAs was evaluated using receiver operating characteristic (ROC) curves. Circ_0005008 and circ_0005198 were confirmed to be elevated significantly in plasma samples from new-onset patients with RA compared with those from HCs and from patients with systemic lupus erythematosus. Among these new-onset patients with RA, we found that the levels of circ_0005008 and circ_0005198 correlated positively with the severity of disease, including the rheumatoid factor, C-reactive protein, the erythrocyte sedimentation rate, and the disease activity score in 28 joints (DAS28). However, their expression levels did not correlate with anti-cyclic citrullinated peptide antibodies. Analysis using ROC curves implied that circ_0005008 and circ_0005198 have significant value in the diagnosis of RA. In addition, we found that compared with that in osteoarthritis fibroblast-like synoviocytes (OA-FLSs), circ_0005198 expression was enhanced in RA-FLSs and correlated positively with DAS28. The level of the miRNA target of circ_0005198, miR-4778-3p, was identified as significantly decreased in RA-FLSs, and the expression levels of circ_0005198 and miR-4778-3p correlated significantly and negatively. The results suggested that in new-onset patients with RA, plasma circ_0005008 and circ_0005198 levels are associated with disease activity and represent possible RA biomarkers.

Circ_0088194 Promotes the Invasion and Migration of Rheumatoid Arthritis Fibroblast-Like Synoviocytes via the miR-766-3p/MMP2 Axis.

Dysregulation of circular RNAs (circRNAs) is involved in various human diseases. Fibroblast-like synoviocytes (FLSs), which form the lining of the joint, are epigenetically imprinted with an aggressive phenotype and contribute to joint destruction in rheumatoid arthritis (RA). In the present study, we identified a novel circRNA, Circ_0088194, which was upregulated in RA fibroblast-like synoviocytes (RA-FLSs) and correlated with the disease activity score in 28 joints. Overexpression of Circ_0088194 promoted RA-FLS migration and invasion, while inhibition of Circ_0088194 had the opposite effect. Mechanistically, Circ_0088194 acted as a miR-766-3p sponge to relieve the repressive effect of miR-766-3p on its target, MMP2 (encoding matrix metalloproteinase 2), thereby promoting migration and invasion. The expression level of Circ_0088194 was inversely correlated with that of miR-766-3p in RA-FLSs. Importantly, overexpression of miR-766-3p partially blocked the migration and invasion induced by Circ_0088194 overexpression. Collectively, this study identified a novel circRNA Circ_0088194 that promotes RA-FLS invasion and migration via the miR-766-3p/MMP2 axis. Circ_0088194 might represent a novel therapeutic target to prevent and treat RA.

Hsa_circ_0088036 promotes the proliferation and migration of fibroblast-like synoviocytes by sponging miR-140-3p and upregulating SIRT 1 expression in rheumatoid arthritis.

Circular RNAs (circRNAs) have been demonstrated to play crucial roles in the development and progression of various types of cancers by serving as microRNA sponges to regulate the expression of target genes. Although in-depth studies of circRNAs have been conducted, their functional and pathological significance in autoimmune diseases, including rheumatoid arthritis (RA), remains unclear. Our previous study verified that hsa_circ_0088036 (circ0088036) is significantly elevated in peripheral blood mononuclear cells from patients with RA. The present study aimed to explore the roles of circ0088036 in the pathogenesis of RA. The circ0088036/miR-140-3p/silent information regulator 1 (SIRT 1) axis was predicted by bioinformatics tools. Circ0088036 was found to be aberrantly upregulated in fibroblast-like synoviocytes (FLSs) in RA compared with FLSs in osteoarthritis (OA). Functionally, upregulated circ0088036 promoted the proliferation and migration of RA-FLSs. Mechanistically, circ0088036 acted as a miR-140-3p sponge to upregulate SIRT 1 expression, subsequently promoting RA progression. In conclusion, this study revealed that circ0088036 may play an essential role in promoting synovial pathogenesis via the circ0088036/miR-140-3p/SIRT 1 axis in RA, providing new insight into circRNAs during RA progression.

Co3O4 nanoparticles/graphitic carbon nitride heterojunction for photoelectrochemical aptasensor of oxytetracycline.

As a broad-spectrum tetracycline antibiotic, the overuse of oxytetracycline (OTC) causes antibiotics residues in the environment and seriously threats to human health owing to effective antibacterial properties. Thus, it is particularly important to design a photoelectrochemical (PEC) aptasensor to detect OTC with excellent performance. Herein, we developed a selective and stable PEC aptasensor of OTC on the basis of Co3O4 nanoparticles (Co3O4 NPs)/graphitic carbon nitride (g-CN) heterojunction, used as PEC active materials. The Co3O4 NPs were successfully grown on the g-CN via grinding and calcining mixture of Co3O4 precursors and bulk g-CN. The Co3O4/g-CN heterojunction with improved light utilization and promoted electrons/holes separation capability can exhibit higher PEC signal than that of g-CN. In order to implement the purpose of specific recognition, OTC-aptamer was introduced into modified electrode to construct highly selective PEC aptasensor for OTC determination, which can possess wide linear range (0.01-500 nM) with low detection limit (3.5 pM, S/N = 3). This PEC aptasensor platform with excellent selectivity and high stability can provide a practical application in the field of water monitoring.

Transforming growth factor ß1 promotes fibroblast-like synoviocytes migration and invasion via TGF-ß1/Smad signaling in rheumatoid arthritis.

Migration and invasion are important characteristics of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs), which are involved in joint damage and contribute to rheumatoid arthritis (RA) pathology. However, the underlying mechanisms remain unclear. Because epithelial-mesenchymal transition (EMT) is a key mechanism related to migration and invasion in cancer cells, we investigated the relationship between EMT and RA-FLSs and explored whether the transforming growth factor ß1 (TGF-ß1)/Smad signaling pathway is involved. In vivo, fibroblast-like synoviocytes (FLSs) were isolated from the synovium of RA or osteoarthritis (OA) patients and cultured for 4-8 passages. EMT markers were detected by immunofluorescence and Western blotting. RA-FLSs were treated with TGF-ß1 or Smad2/3 small interfering RNA (siRNA), EMT markers were detected, and migration and invasion were assessed by Transwell assays. EMT markers could be detected in FLSs; when compared with osteoarthritis fibroblast-like synoviocytes (OA-FLSs), E-cadherin and vimentin decreased, while N-cadherin and α-smooth muscle actin (α-SMA) increased in RA-FLSs. Furthermore, TGF-ß1 enhanced migration and invasion by inducing EMT via activating Smad2/3 in RA-FLSs. Phosphorylation of Smad2/3 was accompanied by degradation of Smad3. Silencing Smad2/3 blocked EMT and inhibited the migration and invasion induced by TGF-ß1. Matrix metalloproteinase 9 (MMP9) and vimentin were not affected when cells were treated with TGF-ß1 or Smad2/3 siRNA. The TGF-ß1/Smad signaling pathway is involved in EMT and contributes to migration and invasion in RA-FLSs. Interestingly, vimentin decreased in RA-FLSs, but there is no correlation between vimentin and TGF-ß1/Smad signaling pathway. Thus, further research on vimentin should be conducted.

Long-term prognosis and quality of life in patients with early rheumatoid arthritis treated according to the 2015 ACR guideline (LELAND): protocol for a multicentre prospective observational study in Southern China.

INTRODUCTION: Rheumatoid arthritis (RA) is a chronic systemic disease and one of the most disabling diseases for patients. The American College of Rheumatology (ACR) issued a new guideline in 2015 for the treatment of RA based on the treat-to-target strategy to achieve better outcomes. This study will focus on the real-world rates of remission and low disease activity of patients with early RA in China, who will be treated according to the 2015 ACR guideline. Additionally, factors influencing treat-to-target outcomes will be analysed, and long-term prognosis and quality of life will be assessed. METHOD AND ANALYSIS: Two-hundred patients with early RA will be enrolled, treated and followed up once every 3 months for 48 months. These patients should fulfil the 2010 RA classification criteria of the ACR/European League Against Rheumatism with a disease course of no more than 6 months and should also fulfil other eligibility criteria. The patients will be treated following the 2015 ACR guideline. Their disease activity will be assessed, and they will be instructed to complete several questionnaires once every 3 months. The primary outcomes are the Disease Activity Score on 28 joints and Health Assessment Questionnaire Disability Index. The secondary outcome variables are the Simplified Disease Activity Index, Clinical Disease Activity Index and Routine Assessment of Patient Index Data 3 results, imaging data and personal medical costs. The data will be analysed using appropriate statistical analyses. ETHICS AND DISSEMINATION: This research was approved by the Nanfang Hospital Ethics Committee (NFEC-2017-192). The results of the study will be published in international peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT03508713; Pre-results.

Using plasma circRNA_002453 as a novel biomarker in the diagnosis of lupus nephritis.

This study aimed to determine the expression of circRNAs in plasma from lupus nephritis (LN) patients to identify novel biomarkers for LN screening. We initially performed microarray screening of circRNA changes in plasma from 5 L N patients, 5 systemic lupus erythematosus (SLE) patients without LN, and 5 healthy controls. We then confirmed the selected circRNA changes in plasma from 59 SLE patients (30 with LN and 29 without LN), 26 rheumatoid arthritis (RA) patients, and 27 age- and sex-matched controls using real-time quantitative reverse transcription-polymerase chain reaction. Spearman's correlation test was performed to assess the correlation of circRNAs and clinical variables. The receiver operating characteristic (ROC) curve was created to evaluate the diagnostic value. We confirmed that plasma circRNA_002453 was significantly elevated in LN patients when compared with SLE patients without LN, RA patients, and healthy controls. Plasma circRNA_002453 was also found to be upregulated in SLE patients when compared with RA patients and healthy controls. Among these LN patients, we detected no significant correlation between plasma circRNA_002453 and disease activity, including complement 3 (C3), complement 4 (C4), and SLE disease activity index 2000 (SLEDAI-2 K) score. However, its expression level was significantly and positively correlated with 24-hour proteinuria (r = 0.571, p = 0.001) and renal SLEDAI score (r = 0.640, p < 0.001). ROC analysis showed that plasma circRNA_002453 had an area under the curve of 0.906 (95% CI 0.838-0.974, p < 0.001) to discriminate LN patients from controls (SLE patients without LN, RA patients, and healthy controls) with sensitivity of 0.900 and specificity of 0.841. The highest Youden index was 0.741 and the corresponding optimal cut-off value was 0.001. This study suggests that upregulated plasma circRNA_002453 level in LN patients is associated with the severity of renal involvement and may also serve as a potential biomarker for LN patient diagnosis.

Microarray Expression Profile of Circular RNAs in Peripheral Blood Mononuclear Cells from Rheumatoid Arthritis Patients.

BACKGROUND/AIMS: Circular RNAs (circRNAs) compose a large class of RNAs that can be used as biomarkers in clinical blood samples. This study aimed to determine the expression of circRNAs in peripheral blood mononuclear cells (PBMCs) from rheumatoid arthritis (RA) patients to identify novel biomarkers for RA screening. METHODS: We started with a microarray screening of circRNA changes in PBMCs from 5 RA patients and 5 healthy controls. We then confirmed the selected circRNA changes in PBMCs from 30 RA patients and 25 age- and sex-matched controls using the real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Spearman correlation test was performed to assess the correlation of circRNAs and clinical variables. Receiver operating characteristic (ROC) curve was calculated to evaluate the diagnostic value. RESULTS: We identified and verified five circRNAs (092516, 003524, 103047, 104871, 101873) that were significantly elevated in PBMCs from RA patients. Among these RA patients, we detected no significant correlation between the five circRNAs and the disease severity, including disease activity score (DAS28), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and health assessment questionnaire (HAQ). Yet, ROC curve analysis suggested that circRNA_104871 has significant value of RA diagnosis (AUC=0.833, P<0.001), followed by circRNA_003524 (AUC = 0.683, P = 0.020), circRNA_101873 (AUC = 0.676, P = 0.026), and circRNA_103047 (AUC = 0.671, P = 0.030). CONCLUSIONS: This study suggests that increased expression of circRNAs circRNA_104871, circRNA_003524, circRNA_101873 and circRNA_103047 in PBMC from RA patients may serve as potential biomarkers for RA patient diagnosis.

A protocol for the culture and isolation of murine synovial fibroblasts.

The culture of synovial fibroblasts (SFs) is one of the most effective tools for investigating the pathology and physiology of synovial tissues and should prove useful for identifying the importance of SFs in disease as well as for the development of novel therapeutic approaches for several chronic joint diseases, such as rheumatoid arthritis. However, thus far, a detailed protocol for the primary culture and isolation of murine SFs has not been established. Therefore, the present study describes an easy and convenient method for isolating and culturing SFs from C57BL/6 mice. This protocol can be divided into 4 stages: Isolation of synovial tissues, isolation of SFs, seeding of SFs for growth in culture and purity analysis of SFs using the four cell markers, vimentin, cluster of differentiation 90.2 (CD90.2; Thy-1.2), intracellular adhesion molecule 1 (CD54) and vascular cell adhesion molecule 1 (CD106). This method is efficient and a purified population of SFs can be obtained 10 days after the initiation of culture.

[IFN-γ stimulates the release of cathepsin S in mouse mast cells].

OBJECTIVE: To investigate the impact of interferon γ (IFN-γ) on cathepsin S (CTSS) expressed by P815 mouse mast cells and mouse bone marrow-derived mast cells (BMMCs). METHODS: IFN-γ of 10, 25 and 50 ng/mL were respectively used to stimulate P815 cells and mouse BMMCs. Real-time PCR and ELISA were applied to detect mRNA and protein levels of CTSS in P815 cells and BMMCs. 25 ng/mL IFN-γ was used to treat P815 cells for 6, 12, 18, 24, 48 and 72 hours and mouse BMMCs for 12, 24 and 48 hours; the above mentioned detection steps were then repeated. RESULTS: IFN-γ induced P815 cells and BMMCs to express CTSS on the mRNA and protein levels in the time- and dose-dependent manners. CONCLUSION: IFN-γ could stimulate mast cells to release CTSS.

[Effects of tumor necrosis factor-α on release of MMP-3, MMP-9, and interleukin-17 in mouse bone marrow-derived mast cells in vitro].

OBJECTIVE: To investigate the effect of tumor necrosis factor-α (TNF-α) on the release of matrix metalloproteinase-3 (MMP-3), MMP-9, and interleukin-17 (IL-17) in cultured mouse bone marrow-derived mast cells (BMMCs) in vitro. METHODS: Primarily cultured mouse BMMCs at 8 weeks were exposed PBS (control) or TNF-α at the concentrations of 2, 10, or 50 ng/mL for 12 or 24 h. Real-time PCR was performed to detect the mRNA expressions of MMP-3, MMP-9, and IL-17 in the exposed cells. RESULTS: A 12-hour exposure of the BMMCs to TNF-α caused significantly increased expressions of MMP-3, MMP-9, and IL-17 in a concentration-dependent manner (P<0.05). Prolonged exposures of the cells to 2 and 10 TNF-α for 24 h further increased MMP-3, MMP-9, and IL-17 mRNA expressions, but exposure to 50 ng/mL TNF-α for 24 h increased only MMP-3 and MMP-9 expressions but not IL-17 mRNA expression. CONCLUSIONS: TNF-α treatment of primarily cultured BMMCs can significantly increase the cellular expressions of MMP-3, MMP-9, and IL-17 mRNA in a time- and dose-dependent manner.

[Effects of bone marrow-derived mast cells on expressions of type II collagen and glycosaminoglycan in co-cultured chondrocytes].

OBJECTIVE: To investigate the influence of the bone marrow-derived mast cells (BMMCs) on the expression of type II collagen and glycosaminoglycan (GAG) in chondrocytes co-cultured with BMMCs. METHODS: Primarily cultured mouse BMMCs at 4 weeks and the second passage of chondrocytes were plated in a Transwell co-cultured system at a ratio of 1:10 in the presence or absence of sodium cromoglycate (DSCG) or compound 48/80 (C48/80). The chondrocytes were harvested and lysed for detecting type II collagen expression with ELISA and Western blotting and GAG expression using 1,9 dimethylmethylene blue (DBM). RESULTS: After a 24-hour culture, the chondrocytes co-cultured with BMMCs showed similar expression levels of type II collagen and GAG to the control group regardless of the presence of DSCG (P>0.05). Compared with chondrocytes cultured alone or with BMMCs, the co-cultured chondrocytes in the presence of C48/80 showed significantly lower expressions of type II collagen and GAG (P<0.01). Such results did not vary significantly as the culture time was extended to 48 h. CONCLUSION: C48/80-activated BMMCs can reduce the expression of type II collagen and GAG in chondrocytes in the co-culture system.