LncRNA H19 induces immune dysregulation of BMMSCs, at least partly, by inhibiting IL-2 production.

BACKGROUND: Systemic lupus erythematosus (SLE) is a representative systemic autoimmune disease. LncRNA H19 has been identified to participate in various biological processes in human diseases. However, the role of H19 in SLE remains unclear. METHODS: In this study, we first examined H19 expression in SLE patients by RT-qPCR and found that H19 expression was significantly upregulated in the serum and bone marrow-derived mesenchymal stem cells (BMMSCs) of SLE patients and positively associated with SLE disease activity index. We then performed gain-of-function and loss-of-function using mimic-H19 (H19-OE) and inhibitor-H19 (H19-KD) to examine the effects of H19 on BMMSC differentiation, proliferation, migration, and apoptosis using flow cytometry, DAPI staining, and migration and apoptosis assays. RESULTS: The results showed that H19 inhibited proliferation and migration but promoted apoptosis of BMMSCs, interfered with BMMSCs-mediated Treg cell proliferation and differentiation, and regulated BMMSCs-mediated Tfh/Treg cell balance. Dual-luciferase reporter assay confirmed the in silico prediction of interaction between H19 and IL-2. Furthermore, RT-qPCR showed that H19 directly inhibited IL-2 transcription in BMMSCs. ELISA showed that both active and total IL-2 protein levels were significantly lower in SLE BMMSCs. More importantly, we found that IL-2 significantly enhanced H19-OE-induced Treg cell differentiation and migration of BMMSCs, and these effects were reversed by anti-IL-2 antibody. CONCLUSION: Overall, our study indicates that LncRNA H19 induces immune dysregulation of BMMSCs, at least partly, by inhibiting IL-2 production and might be a novel therapeutic target for SLE.

LncRNA XIST promotes adjuvant-induced arthritis by increasing the expression of YY1 via miR-34a-5p.

Objectives: This study aims to explore the mechanism by which long non-coding ribonucleic acids (lncRNA) X-inactive specific transcript (XIST) affects the progression of adjuvant-induced arthritis (AIA). Materials and methods: Freund's complete adjuvant was used to induce arthritis in rats. The polyarthritis, spleen and thymus indexes were calculated to evaluate AIA. Hematoxylin-eosin (H&E) staining was used to reveal the pathological changes in the synovium of AIA rats. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the expression of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6 and IL-8 in the synovial fluid of AIA rats. The cell continuing kit (CCK)-8, flow cytometry, and Transwell assays were used to assess the proliferation, apoptosis, migration and invasion of transfected fibroblast-like synoviocytes (FLS) isolated from AIA rats (AIA-FLS). Dual-luciferase reporter assay was performed to verify the binding sites between XIST and miR-34b-5p or between YY1 mRNA and miR-34b-5p. Results: The XIST and YY1 were highly expressed, and miR-34a-5p was lowly expressed in the synovium of AIA rats and in AIA-FLS. Silencing of XIST impaired the function of AIA-FLS in vitro and inhibited the progression of AIA in vivo. The XIST promoted the expression of YY1 by competitively binding to miR-34a-5p. Inhibition of miR-34a-5p strengthened the function of AIA-FLS by upregulating XIST and YY1. Conclusion: The XIST controls the function of AIA-FLS and may promote the progression of rheumatoid arthritis via the miR-34a-5p/YY1 axis.

Evolution of viral RNA in a Chinese patient to interferon/ribavirin therapy for hepatitis C.

OBJECTIVE: The combination of interferon (IFN) and ribavirin (RBV) is the standard therapy for hepatitis C virus (HCV) infection. HCV genotype 2a has proved more amenable to the therapy, but its efficacy is yet limited. This study aimed to investigate the mechanism of the poor response in a case of HCV genotype 2a infection. METHODS: We analyzed dynamic change of HCV RNA from a patient, infected with HCV genotype 2a, showing a poor virological response to IFN/RBV as judged 12 weeks after initiation of the therapy by HCV clone sequencing. Then we constructed subgenomic Japanese fulminant hepatitis-1 (JFH1) replicon and different chimeric replicons with humanized Gaussia luciferase gene. The chimeric replicons were derived from subgenomic JFH1 replicon, in which the NS5A region was replaced by the patient's sequence from the pre/post-treatment, and the chimeric replicons' susceptibility to IFN were evaluated by relative Gausia Luciferase activity. RESULTS: The pretreatment HCV sequences appeared almost uniform, and the quasispecies variation was further more simplified after 12 weeks of therapy. Besides, the quasispecies variation seemed to be more diversified in the NS5A, relatively, a region crucial for IFN response, and each of chimeric replicons exhibited distinct response to IFN. CONCLUSIONS: During the course of the chronic infection, HCV population seems to be adapted to the patient's immunological system, and further to be selected by combination of IFN/RBV therapy, indicating quasispecies may completely eliminated by addition of other drugs with targets different from those of IFN. In addition, each different response of chimeric replicon to IFN is most likely related to amino acid changes in or near the IFN-sensitivity determining region (ISDR) of NS5A during chronic infection and IFN/RBV therapy.

FOXC1­mediated TRIM22 regulates the excessive proliferation and inflammation of fibroblast­like synoviocytes in rheumatoid arthritis via NF­κB signaling pathway.

Rheumatoid arthritis (RA) is a common systemic autoimmune disorder of unknown etiology, which threatens public health. The regulatory role of tripartite motif­containing 22 (TRIM22) has been reported in multiple types of cancers and disease, but not in RA. The aim of the present study was therefore to elucidate the potential roles and underlying mechanisms of TRIM22 in fibroblast­like synoviocytes (FLSs) in RA. The Gene Expression Omnibus database was used to examine TRIM22 mRNA expression levels in synovial tissue samples of patients with RA and healthy controls. TRIM22 and forkhead box C1 (FOXC1) mRNA and protein expression levels in normal FLSs and RA­FLSs were assessed using reverse transcription­quantitative PCR (RT­qPCR) and western blotting, respectively. The Cell Counting Kit­8 assay was used to assess cell proliferation. Cell apoptosis was analyzed using flow cytometry. The migratory and invasive abilities of RA­FLSs were assessed using Transwell assays. Western blotting was used to analyze the protein expression levels of apoptosis­related factors, MMP2, MMP9 and NF­κB signaling pathway­related proteins. Inflammatory factors levels were assessed via ELISA and RT­qPCR. Furthermore, the JASPAR database, chromatin immunoprecipitation and the dual­luciferase reporter assays were used to determine the interaction between FOXC1 and the TRIM22 promoter. The results of the present study demonstrated that TRIM22 expression levels were significantly elevated in the synovial tissue samples of patients with RA and RA­FLSs. Moreover, FOXC1 was also significantly overexpressed in RA­FLSs. TRIM22 knockdown significantly reduced cell proliferation, migration, invasion and the inflammatory response, whereas cell apoptosis was significantly increased. Furthermore, the results demonstrated that FOXC1 may have positively mediated TRIM22 expression via binding to the TRIM22 promoter. Moreover, FOXC1 overexpression significantly reversed the outcome of TRIM22 knockdown on the proliferation, apoptosis, migration, invasion and inflammation of RA­FLSs. FOXC1 overexpression also significantly reversed the inactivation of the NF­κB signaling pathway caused by TRIM22 knockdown. In summary, the present study demonstrated that TRIM22 was potentially activated via FOXC1, which contributed to the progression of RA via the NF­κB signaling pathway.

RND3 Transcriptionally Regulated by FOXM1 Inhibits the Migration and Inflammation of Synovial Fibroblasts in Rheumatoid Arthritis Through the Rho/ROCK Pathway.

Rheumatoid arthritis (RA) is a systemic immune disease. Rho family GTPase 3 (RND3) has been reported to play an important role in inflammatory diseases. In this study, the expression of RND3 in RA was analyzed by gene chips. After RND3 was overexpressed, cell counting kit-8 assay was to detect the viability of fibroblast-like synovial cells (RA-FLSs). Transwell assays were to appraise the migratory and invasive capacities of RA-FLSs. Enzyme-linked immunosorbent assay (ELISA) and Western blot analysis were to estimate inflammatory response. In addition, MMP3 and MMP9 levels were also tested by ELISA analysis. After forkhead box M1 (FOXM1) was overexpressed, RND3 expression was detected by Western blot. The transcriptional relationship between FOXM1 and RND3 was predicted by HumanTFDB and JASPAR databases. Luciferase reporter and chromatin immunoprecipitation assays verified the binding ability of FOXM1 and RND3. The role of FOXM1/RND3 axis in RA was detected again by functional experiments. Western blot detected the expression of Rho/ROCK pathway-related proteins. RND3 expression was downregulated in RA. Overexpression of RND3 reduced the proliferation, migration, invasion, and inflammation of RA-FLSs. RND3 was inhibited by FOXM1 transcription, and upregulated FOXM1 reduced the inhibitory effect of RND3 overexpression on cell growth and inflammation, which might be associated with the Rho/ROCK pathway. RND3 transcriptionally regulated by FOXM1 inhibited the migration and inflammation of RA-FLSs in RA through the Rho/ROCK pathway.

Influence mechanism of structure on shear mechanical deformation characteristics of loess-steel interface.

The mechanical properties of loess-steel interface are of great significance for understanding the residual strength and deformation of loess. However, the undisturbed loess has significant structural properties, while the remolded loess has weak structural properties. There are few reports on the mechanical properties of loess-steel interface from the structural point of view. This paper focused on the ring shear test between undisturbed loess as well as its remolded loess and steel interface under the same physical mechanics and test conditions (water content, shear rate and vertical pressure), and explored the influence mechanism of structure on the mechanical deformation characteristics of steel-loess interface. The results show that the shear rate has little effect on the residual strength of the undisturbed and remolded loess-steel interface. However, the water content has a significant influence on the residual strength of the loess-steel interface, moreover, the residual internal friction angle is the dominant factor supporting the residual strength of the loess-steel interface. In general, the residual strength of the undisturbed loess-steel interface is greater than that of the remolded loess specimen (for example, the maximum percentage of residual strength difference between undisturbed and remolded loess specimens under the same moisture content is 6.8%), which is because that compared with the mosaic arrangement structure of the remolded loess, the overhead arrangement structure of the undisturbed loess skeleton particles makes the loess particles on the loess-steel interface re-adjust the arrangement direction earlier and reach a stable speed relatively faster. The loess particles with angular angles in the undisturbed loess make the residual internal friction between the particles greater than the smoother particles of the remolded loess (for example, the maximum percentage of residual cohesion difference between undisturbed and remolded loess specimens under the same vertical pressure is 4.29%), and the intact cement between undisturbed loess particles brings stronger cohesion than the remolded loess particles with destroyed cement (for example, the maximum difference percentage of residual cohesion between undisturbed and remolded soil specimens under the same vertical pressure is 33.80%). The test results provide experimental basis for further revealing the influence mechanism of structure, and parameter basis for similar engineering construction.

Microstructure of unsaturated loess and its influence on strength characteristics.

Previous studies have shown that structure has a significant influence on the mechanical deformation of unsaturated loess, but there is little published information focused on the influence mechanism of microstructure and mesostructure on the mechanical properties of loess. In this paper, the unsaturated undisturbed loess and its remolded loess under the same physical condition were taken as the research objects. The unsaturated triaxial shear tests with constant suction and net confining pressure were carried out, and the microstructure differences between the two are compared by using SEM and CT scanning to reveal the influence of structure on strength characteristics. The test results show that the cohesion and internal friction angle of undisturbed loess are greater than those of remolded loess. The angle of undisturbed soil particles is obvious, and the particles are bracket contact with good cementation. The remolded loess particles are close to round shape, and the particles are inlaid contact with destroyed cementation. The average radius of undisturbed soil is higher than that of remolded soil, indicating that there are bracket pores in undisturbed soil, but the bracket structure and macropores are deformed during shear deformation, and good structural and cementation ensure the strength of loess specimens.

SPTBN1 attenuates rheumatoid arthritis synovial cell proliferation, invasion, migration and inflammatory response by binding to PIK3R2.

BACKGROUND: As an autoimmune systemic disorder, rheumatoid arthritis (RA) features chronic inflammation as well as synovial infiltration of immune cells. This study was designed with the purpose of discussing the hidden mechanism of SPTBN1 and exploring favorable molecular-targeted therapies. METHODS: With the application of RT-qPCR and western blot, the expressions of SPTBN1 and PIK3R2 before or after transfection were estimated. Besides, Cell Counting Kit-8, Edu, wound healing, transwell, enzyme-linked immunosorbent assay, and TUNEL were adopted for the evaluation of the viability, proliferation, migration, invasion, inflammatory response, and apoptosis of fibroblast-like synoviocyte (FLS). In addition, the interaction of SPTBN1 and PIK3R2 was testified by applying immunoprecipitation (IP) and western blot was utilized for the assessment of migration-, apoptosis-, and PI3K/AKT signal-related proteins. RESULTS: It was discovered that SPTBN1 declined in RA synovial cells and its overexpression repressed the proliferation, migration, invasion, and inflammation of RA-FLSs but promoted apoptosis. IP confirmed that SPTBN1 could bind to PIK3R2 in FLSs. To further figure out the hidden mechanism of SPTBN1 in RA, a series of functional experiments were carried out and the results demonstrated that the reduced expressions of MMP2, MMP9, IL-8, IL-1ß, IL-6, and Bcl2 as well as increased levels of Bax and cleaved caspase3 in SPTBN1-overexpressed RA-FLSs were reversed by PIK3R2 depletion, revealing that SPTBN1 repressed the migration and inflammation and promoted the apoptosis of RA-FLSs via binding to PIK3R2. Results obtained from western blot also revealed that PIK3R2 interference ascended the contents of p-PI3K and p-AKT in SPTBN1-overexpressed RA-FLSs, implying that SPTBN1 repressed PI3K/AKT signal in RA via PIK3R2. DISCUSSION: SPTBN1 alleviated the proliferation, migration, invasion, and inflammation in RA via interacting with PIK3R2.

Metallothionein-1 is Positively Correlated with Inflammation and Ankylosing Spondylitis Activity.

Introduction: Ankylosing spondylitis (AS) is a common form of chronic inflammatory rheumatic disease. Metallothionein-1 (MT-1) has been known to play an immunosuppressive role in various noninfectious inflammatory diseases, especially osteoarthritis and rheumatoid arthritis, thus inhibiting inflammation and pathogenesis in various diseases. However, whether MT-1 is related to AS is unclear. Here, we examined the levels of MT-1 in patients with AS and its correlation with the disease activity, complication, clinical indexes, and inflammatory cytokines and attempted to explain the effect of MT-1 on inflammation in AS. Methods: The messenger RNA (mRNA) and protein expression of MT-1 in patients with AS were detected through real-time polymerase chain reaction and enzyme-linked immunosorbent assay. The associations between serum MT-1 protein level and clinical indexes or proinflammatory cytokines in AS were analyzed using the Spearman correlation test. Results: The mRNAs and serum protein levels of MT-1 were significantly higher in patients with AS, especially in patients with active AS and patients with osteoporosis (OP) than in healthy controls (HCs), and no difference was observed between patients with inactive AS and HCs. Serum MT-1 levels positively correlated with disease activity, proinflammatory cytokines, and clinical indexes Ankylosing Spondylitis Disease Activity Score with C-Reactive Protein, C-reactive protein level, and erythrocyte sedimentation rate in patients with AS. Conclusion: MT-1 expression was upregulated in patients with active AS but not in those with inactive AS and positively correlated with clinical indexes, especially in OP, as well as with proinflammatory cytokines tumor necrosis factor-alpha, interleukin (IL)-1ß, and IL-6 in patients with AS.

[Construction of the fusion gene of Hsp65 of Mycobacterium tuberculosis and EGFP and preparation of dendritic cell vaccine against tuberculosis].

AIM: To construct the fusion gene of Hsp65 of Mycobacterium tuberculosis H37Rv and enhanced green fluorescence protein (EGFP) and prepare dendritic cell (DC) vaccine. METHODS: Hsp65 DNA amplified by PCR was cloned into eukaryotic expression vector EGFP-C1. The recombinant plasmid pEGHsp65 was subsequently transfected into Hela cells, and the transfection rate was determined under confocal laser scanning fluorescence microscope at different times. RT-PCR was used to detect the expression of Hsp65 mRNA in Hela cells. The GM-CSF and IL-4 induced DCs from mouse bone marrow cells were transfected with recombinant plasmid pEGHsp65. Proliferation of unprimed splenocytes activated by transfected DCs was detected by MTT colorimetry. RESULTS: Restrictive enzyme digestion analysis (EcoR I, Bgl II) confirmed that Hsp65 DNA had been inserted into the vector pEGFP-C1. The recombinant plasmid pEGHsp65 was transfected into Hela cells and the expression of the fusion gene reached peak at the 48 hours after transfection. Expression of Hsp65 mRNA was detected in Hela cells by RT-PCR. DCs transfected with pEGHsp65 could stimulate the proliferation of unprimed splenocytes. CONCLUSION: The pEGHsp65 fusion gene was successfully constructed and DCs transfected with the pEGHsp65 might be a candidate vaccine for tuberculosis.