MicroRNA-19a-3p promotes rheumatoid arthritis fibroblast-like synoviocytes via targeting SOCS3

Rheumatoid arthritis (RA) is a common chronic autoimmune disease and effective treatment for RA is still lacking. In this study, the regulatory role of miR-19a-3p in RA was investigated. Quantitative polymerase chain reaction analysis of human blood samples showed that the level of miR-19a-3p was significantly lower in the RA patients compared with that in healthy patients (P < 0.05). In RA fibroblast-like synoviocytes (RAFLS), miR-19a-3p and suppressor of cytokine signaling 3 (SOCS3) were also downregulated and upregulated, respectively, compared with those of normal FLS. Transfection of miR-19a-3p mimic in RAFLS inhibited cell proliferation and promoted cell apoptosis. TargetScan identified SOCS3 as a target of miR-19a-3p, which was confirmed by dual-luciferase assay. Western blot indicated that SOCS3 protein level was significantly decreased after miR-19a-3p overexpression. Moreover, SOCS3 silencing through siRNA transfection also enhanced cell proliferation, meanwhile inhibiting RAFLS apoptosis. In addition, SOCS3 overexpression abrogated the effects of miR-19a-3p overexpression on cell proliferation and apoptosis, corroborating that SOCS3 acts as a downstream effector in the miR-19a-3p-mediated function of RAFLS. These findings suggest that miR-19a-3p plays an important role in RA, and the miR-19a-3p/SOCS3 axis may become a potential therapeutic target for RA.     

LncRNA H19 Drives Proliferation of Cardiac Fibroblasts and Collagen Production via Suppression of the miR-29a-3p/miR-29b-3p-VEGFA/TGF-β Axis

The aim of this study was to investigating whether lncRNA H19 promotes myocardial fibrosis by suppressing the miR-29a-3p/miR-29b-3p-VEGFA/TGF-β axis. Patients with atrial fibrillation (AF) and healthy volunteers were included in the study, and their biochemical parameters were collected. In addition, pcDNA3.1-H19, si-H19, and miR-29a/b-3p mimic/inhibitor were transfected into cardiac fibroblasts (CFs), and proliferation of CFs was detected by MTT assay. Expression of H19 and miR-29a/b-3p were detected using real-time quantitative polymerase chain reaction, and expression of α-smooth muscle actin (α-SMA), collagen I, collagen II, matrix metalloproteinase-2 (MMP-2), and elastin were measured by western blot analysis. The dual luciferase reporter gene assay was carried out to detect the sponging relationship between H19 and miR-29a/b-3p in CFs. Compared with healthy volunteers, the level of plasma H19 was significantly elevated in patients with AF, while miR-29a-3p and miR-29b-3p were markedly depressed (P < 0.05). Serum expression of lncRNA H19 was negatively correlated with the expression of miR-29a-3p and miR-29b-3p among patients with AF (r_s = –0.337, r_s = –0.236). Moreover, up-regulation of H19 expression and down-regulation of miR-29a/b-3p expression facilitated proliferation and synthesis of extracellular matrix (ECM)-related proteins. SB431542 and si-VEGFA are able to reverse the promotion of miR-29a/b-3p on proliferation of CFs and ECM-related protein synthesis. The findings of the present study suggest that H19 promoted CF proliferation and collagen synthesis by suppressing the miR-29a-3p/miR-29b-3p-VEGFA/TGF-β axis, and provide support for a potential new direction for the treatment of AF.     

B lymphocytes and B-cell activating factor promote collagen and profibrotic markers expression by dermal fibroblasts in systemic sclerosis

Introduction

B lymphocytes might play a pathogenic role in dermal fibrosis in systemic sclerosis (SSc). B-cell activating factor (BAFF), a key cytokine for B-cell activation, is increased in the serum and the skin of patients with SSc. However, the ability of B cells directly to stimulate dermal fibroblasts and the role of BAFF are not fully understood. We therefore investigated the involvement of B cells and BAFF in the expression of collagen and profibrotic markers by dermal fibroblasts.

Methods

Cocultures of blood B cells from healthy blood donors and normal or SSc dermal fibroblasts stimulated with anti-IgM and BAFF were performed. Alpha-SMA, TIMP1, MMP9, COL1A1, COL1A2, and COL3A1 mRNA expression were determined by quantitative RT-PCR. Soluble collagen, BAFF, IL-6, IL-1β, TGF-β1, and CCL2 protein secretion were assessed.

Results

Coculture of blood B cells and dermal fibroblasts isolated from SSc patients induced IL-6, TGF-β1, CCL2, and collagen secretion, as well as Alpha-SMA, TIMP1, and MMP9 expression in dermal fibroblasts. Transwell assays demonstrated that this induction was dependent on cell-cell contact. Addition of anti-IgM and BAFF to the coculture increased IL-6, CCL2, TGF-β1, and collagen secretion. B cell- and BAFF-induced collagen secretion was highly reduced by anti-TGF-β1 antibodies.

Conclusions

Our results showed for the first time a direct role of B cells on the production of collagen by dermal fibroblasts, which is further enhanced by BAFF. Thus, these results demonstrate a new pathogenic role of B cells and BAFF in fibrosis and systemic sclerosis.     

MiR-133a-3p inhibits scar formation in scalded mice and suppresses the proliferation and migration of scar derived-fibroblasts by targeting connective tissue growth factor

Excessive scar formation post burn injury can cause great pain to the patients. MiR-133a-3p has been demonstrated to be anti-fibrotic in some fibrosis-related diseases. However, its possible role in scar formation has not been elucidated yet. In present study, the effect of miR-133a-3p on scar formation was investigated in a scalded model of mice. Moreover, the function of miR-133a-3p on proliferation and migration of scar-derived fibroblasts (SFs) was studied in vitro. It was found that miR-133a-3p was dramatically downregulated in scar tissue of scalded mice. Upregulation of miR-133a-3p by miR-133a-3p agomir obviously inhibited the scar formation in scalded mice. Histological staining showed that upregulation of miR-133a-3p attenuated the excessive deposition of collagen in scar tissue of scalded mice. In vitro study showed that upregulation of miR-133a-3p effectively suppressed the proliferation and migration of SFs. Besides, upregulation of miR-133a-3p attenuated the protein levels of α-smooth muscle actin (α-SMA) and collagen I, indicating that miR-133a-3p could suppress the activation of SFs. The expression of connective tissue growth factor (CTGF), a critical mediator in cell proliferation, migration and extracellular matrix (ECM) synthesis, was also downregulated by the upregulation of miR-133a-3p. Luciferase reporter assay validated that CTGF was directly targeted by miR-133a-3p. In addition, overexpression of CTGF abolished the effect of miR-133a-3p on inhibiting the proliferation, migration and activation of SFs, indicating that miR-133a-3p functioned by targeting CTGF. Therefore, miR-133a-3p might be a promising target for treating pathological scars.     

MiR-125a-3p inhibits cell proliferation and inflammation responses in fibroblast-like synovial cells in rheumatoid arthritis by mediating the Wnt/β-catenin and NF-κB pathways via targeting MAST3

Objectives:To explore the role and mechanism of miR-125a-3p in rheumatoid arthritis (RA) progression.Methods:The RA-tissues and fibroblast-like synovial cells in rheumatoid arthritis (RA-FLS) were used in this study. qRT-PCR, western blot and ELISA assay were performed to detect the expression levels of IL-6, IL-β and ΤΝF-α. Dual-luciferase reporter gene assay was used to observe the binding effect of miR-125a-3p and MAST3, and CCK-8 was used to observe the effect of miR-125a-3p on the proliferation of RA-FLS.Results:miR-125a-3p was significantly downregulated in the RA-tissues and RA-FLS, and miR-125a-3p could inhibit the proliferation and reduce the inflammation response of RA-FLS. Besides, MAST3 was found as a target of miR-125a-3p, and increased MAST3 could reverse the effects of miR-125a-3p on RA-FLS including decreased proliferation, reduced inflammation level and the inactivation of Wnt/β-catenin and NF-κB pathways.Conclusions:This study suggests that miR-125a-3p could inactivate the Wnt/β-catenin and NF-κB pathways to reduce the proliferation and inflammation response of RA-FLS via targeting MAST3.     

MicroRNA-30e* Suppresses Dengue Virus Replication by Promoting NF-κB–Dependent IFN Production

MicroRNAs have been shown to contribute to a repertoire of host-pathogen interactions during viral infection. Our previous study demonstrated that microRNA-30e* (miR-30e*) directly targeted the IκBα 3′-UTR and disrupted the NF-κB/IκBα negative feedback loop, leading to hyperactivation of NF-κB. This current study investigated the possible role of miR-30e* in the regulation of innate immunity associated with dengue virus (DENV) infection. We found that DENV infection could induce miR-30e* expression in DENV-permissive cells, and such an overexpression of miR-30e* upregulated IFN-β and the downstream IFN-stimulated genes (ISGs) such as OAS1, MxA and IFITM1, and suppressed DENV replication. Furthermore, suppression of IκBα mediates the enhancing effect of miR-30e* on IFN-β-induced antiviral response. Collectively, our findings suggest a modulatory role of miR-30e* in DENV induced IFN-β signaling via the NF-κB-dependent pathway. Further investigation is needed to evaluate whether miR-30e* has an anti-DENV effect in vivo.     

Rac Inhibition Reverses the Phenotype of Fibrotic Fibroblasts

Background

Fibrosis, the excessive deposition of scar tissue by fibroblasts, is one of the largest groups of diseases for which there is no therapy. Fibroblasts from lesional areas of scleroderma patients possess elevated abilities to contract matrix and produce α−smooth muscle actin (α-SMA), type I collagen and CCN2 (connective tissue growth factor, CTGF). The basis for this phenomenon is poorly understood, and is a necessary prerequisite for developing novel, rational anti-fibrotic strategies.

Methods and Findings

Compared to healthy skin fibroblasts, dermal fibroblasts cultured from lesional areas of scleroderma (SSc) patients possess elevated Rac activity. NSC23766, a Rac inhibitor, suppressed the persistent fibrotic phenotype of lesional SSc fibroblasts. NSC23766 caused a decrease in migration on and contraction of matrix, and α−SMA, type I collagen and CCN2 mRNA and protein expression. SSc fibroblasts possessed elevated Akt phosphorylation, which was also blocked by NSC23766. Overexpression of rac1 in normal fibroblasts induced matrix contraction and α−SMA, type I collagen and CCN2 mRNA and protein expression. Rac1 activity was blocked by PI3kinase/Akt inhibition. Basal fibroblast activity was not affected by NSC23766.

Conclusion

Rac inhibition may be considered as a novel treatment for the fibrosis observed in SSc.     

MicroRNA-27a-3p inhibits lung and skin fibrosis of systemic sclerosis by negatively regulating SPP1

ObjectiveTo investigate the role and mechanism of microRNAs (miRNAs) in fibrotic processes involved in the pathology of systemic sclerosis (SSc).MethodsR language and bioinformatics methods were used to identify differential miRNAs and mRNAs and analyze their biological functions. Transfection experiments were performed to evaluate the function and regulatory mechanism of miR-27a-3p in vitro. Levels of fibrosis-related genes, SPP1 and cell proliferation were assessed.ResultsMiR-27a-3p is reduced both in SSc lung and skin tissues. Overexpression of miR-27a-3p significantly inhibited fibrosis-related genes expression and protein abundance and cell proliferation, whereas inhibition of miR-27a-3p significantly enhanced these phenomena. Moreover, miR-27a-3p exerts its anti-fibrosis effect by negatively regulating SPP1 and ERK signal, more prominent in fibroblasts.ConclusionsOur findings show that miR-27a-3p regulates a common mechanism in the process of SSc skin and lung fibrosis. MiR-27a-3p/SPP1/ERK1/2 axis may be an important target for delaying the progression of SSc fibrosis.     

Exosomal miR-146a-5p and miR-155-5p promote CXCL12/CXCR7-induced metastasis of colorectal cancer by crosstalk with cancer-associated fibroblasts

C-X-C motif chemokine receptor 7 (CXCR7) is a newly discovered atypical chemokine receptor that binds to C-X-C motif chemokine ligand 12 (CXCL12) with higher affinity than CXCR4 and is associated with the metastasis of colorectal cancer (CRC). Cancer-associated fibroblasts (CAFs) have been known to promote tumor progression. However, whether CAFs are involved in CXCR7-mediated metastasis of CRC remains elusive. We found a significant positive correlation between CXCR7 expression and CAF activation markers in colonic tissues from clinical specimens and in villin-CXCR7 transgenic mice. RNA sequencing revealed a coordinated increase in the levels of miR-146a-5p and miR-155-5p in CXCR7-overexpressing CRC cells and their exosomes. Importantly, these CRC cell-derived miR-146a-5p and miR-155-5p could be uptaken by CAFs via exosomes and promote the activation of CAFs through JAK2–STAT3/NF-κB signaling by targeting suppressor of cytokine signaling 1 (SOCS1) and zinc finger and BTB domain containing 2 (ZBTB2). Reciprocally, activated CAFs further potently enhanced the invasive capacity of CRC cells. Mechanistically, CAFs transfected with miR-146a-5p and miR-155-5p exhibited a robust increase in the levels of inflammatory cytokines interleukin-6, tumor necrosis factor-α, transforming growth factor-β, and CXCL12, which trigger the epithelial–mesenchymal transition and pro-metastatic switch of CRC cells. More importantly, the activation of CAFs by miR-146a-5p and miR-155-5p facilitated tumor formation and lung metastasis of CRC in vivo using tumor xenograft models. Our work provides novel insights into CXCR7-mediated CRC metastasis from tumor–stroma interaction and serum exosomal miR-146a-5p and miR-155-5p could serve as potential biomarkers and therapeutic targets for inhibiting CRC metastasis.Subject terms: Cancer microenvironment, Colon cancer     

Regulation of NF-κB signaling by oxidized glycerophospholipid and IL-1β induced miRs-21-3p and -27a-5p in human aortic endothelial cells

Exposure of endothelial cells (ECs) to agents such as oxidized glycerophospholipids (oxGPs) and cytokines, known to accumulate in atherosclerotic lesions, perturbs the expression of hundreds of genes in ECs involved in inflammatory and other biological processes. We hypothesized that microRNAs (miRNAs) are involved in regulating the inflammatory response in human aortic endothelial cells (HAECs) in response to oxGPs and interleukin 1β (IL-1β). Using next-generation sequencing and RT-quantitative PCR, we characterized the profile of expressed miRNAs in HAECs pre- and postexposure to oxGPs. Using this data, we identified miR-21-3p and miR-27a-5p to be induced 3- to 4-fold in response to oxGP and IL-1β treatment compared with control treatment. Transient overexpression of miR-21-3p and miR-27a-5p resulted in the downregulation of 1,253 genes with 922 genes overlapping between the two miRNAs. Gene Ontology functional enrichment analysis predicted that the two miRNAs were involved in the regulation of nuclear factor κB (NF-κB) signaling. Overexpression of these two miRNAs leads to changes in p65 nuclear translocation. Using 3′ untranslated region luciferase assay, we identified 20 genes within the NF-κB signaling cascade as putative targets of miRs-21-3p and -27a-5p, implicating these two miRNAs as modulators of NF-κB signaling in ECs.     

IFN-α Regulates Blimp-1 Expression via miR-23a and miR-125b in Both Monocytes-Derived DC and pDC

Type I interferon (IFN-I) have emerged as crucial mediators of cellular signals controlling DC differentiation and function. Human DC differentiated from monocytes in the presence of IFN-α (IFN-α DC) show a partially mature phenotype and a special capability of stimulating CD4+ T cell and cross-priming CD8+ T cells. Likewise, plasmacytoid DC (pDC) are blood DC highly specialized in the production of IFN-α in response to viruses and other danger signals, whose functional features may be shaped by IFN-I. Here, we investigated the molecular mechanisms stimulated by IFN-α in driving human monocyte-derived DC differentiation and performed parallel studies on peripheral unstimulated and IFN-α-treated pDC. A specific miRNA signature was induced in IFN-α DC and selected miRNAs, among which miR-23a and miR-125b, proved to be negatively associated with up-modulation of Blimp-1 occurring during IFN-α-driven DC differentiation. Of note, monocyte-derived IFN-α DC and in vitro IFN-α-treated pDC shared a restricted pattern of miRNAs regulating Blimp-1 expression as well as some similar phenotypic, molecular and functional hallmarks, supporting the existence of a potential relationship between these DC populations. On the whole, these data uncover a new role of Blimp-1 in human DC differentiation driven by IFN-α and identify Blimp-1 as an IFN-α-mediated key regulator potentially accounting for shared functional features between IFN-α DC and pDC.     

Differential regulation of cell functions by CSD peptide subdomains

Background

In fibrotic lung diseases, expression of caveolin-1 is decreased in fibroblasts and monocytes. The effects of this deficiency are reversed by treating cells or animals with the caveolin-1 scaffolding domain peptide (CSD, amino acids 82–101 of caveolin-1) which compensates for the lack of caveolin-1. Here we compare the function of CSD subdomains (Cav-A, Cav-B, Cav-C, Cav-AB, and Cav-BC) and mutated versions of CSD (F92A and T90A/T91A/F92A).

Methods

Migration toward the chemokine CXCL12 and the associated expression of F-actin, CXCR4, and pSmad 2/3 were studied in monocytes from healthy donors and SSc patients. Fibrocyte differentiation was studied using PBMC from healthy donors and SSc patients. Collagen I secretion and signaling were studied in fibroblasts derived from the lung tissue of healthy subjects and SSc patients.

Results

Cav-BC and CSD at concentrations as low as 0.01 μM inhibited the hypermigration of SSc monocytes and TGFβ-activated Normal monocytes and the differentiation into fibrocytes of SSc and Normal monocytes. While CSD also inhibited the migration of poorly migrating Normal monocytes, Cav-A (and other subdomains to a lesser extent) promoted the migration of Normal monocytes while inhibiting the hypermigration of TGFβ-activated Normal monocytes. The effects of versions of CSD on migration may be mediated in part via their effects on CXCR4, F-actin, and pSmad 2/3 expression. Cav-BC was as effective as CSD in inhibiting fibroblast collagen I and ASMA expression and MEK/ERK signaling. Cav-C and Cav-AB also inhibited collagen I expression, but in many cases did not affect ASMA or MEK/ERK. Cav-A increased collagen I expression in scleroderma lung fibroblasts. Full effects on fibroblasts of versions of CSD required 5 μM peptide.

Conclusions

Cav-BC retains most of the anti-fibrotic functions of CSD; Cav-A exhibits certain pro-fibrotic functions. Results obtained with subdomains and mutated versions of CSD further suggest that the critical functional residues in CSD depend on the cell type and readout being studied. Monocytes may be more sensitive to versions of CSD than fibroblasts and endothelial cells because the baseline level of caveolin-1 in monocytes is much lower than in these other cell types.     

Role of MicroRNA Modulation in the Interferon-α/Ribavirin Suppression of HIV-1 In Vivo

Background

Interferon-α (IFN-α) treatment suppresses HIV-1 viremia and reduces the size of the HIV-1 latent reservoir. Therefore, investigation of the molecular and immunologic effects of IFN-α may provide insights that contribute to the development of novel prophylactic, therapeutic and curative strategies for HIV-1 infection. In this study, we hypothesized that microRNAs (miRNAs) contribute to the IFN-α-mediated suppression of HIV-1. To inform the development of novel miRNA-based antiretroviral strategies, we investigated the effects of exogenous IFN-α treatment on global miRNA expression profile, HIV-1 viremia, and potential regulatory networks between miRNAs and cell-intrinsic anti-HIV-1 host factors in vivo.

Methods

Global miRNA expression was examined in longitudinal PBMC samples obtained from seven HIV/HCV-coinfected, antiretroviral therapy-naïve individuals before, during, and after pegylated interferon-α/ribavirin therapy (IFN-α/RBV). We implemented novel hybrid computational-empirical approaches to characterize regulatory networks between miRNAs and anti-HIV-1 host restriction factors.

Results

miR-422a was the only miRNA significantly modulated by IFN-α/RBV in vivo (p<0.0001, paired t test; FDR<0.037). Our interactome mapping revealed extensive regulatory involvement of miR-422a in p53-dependent apoptotic and pyroptotic pathways. Based on sequence homology and inverse expression relationships, 29 unique miRNAs may regulate anti-HIV-1 restriction factor expression in vivo.

Conclusions

The specific reduction of miR-422a is associated with exogenous IFN-α treatment, and likely contributes to the IFN-α suppression of HIV-1 through the enhancement of anti-HIV-1 restriction factor expression and regulation of genes involved in programmed cell death. Moreover, our regulatory network analysis presents additional candidate miRNAs that may be targeted to enhance anti-HIV-1 restriction factor expression in vivo.     

miR-21a-5p Promotes Inflammation following Traumatic Spinal Cord Injury through Upregulation of Neurotoxic Reactive Astrocyte (A1) Polarization by Inhibiting the CNTF/STAT3/Nkrf Pathway

Reactive astrocytes are implicated in traumatic spinal cord injury (TSCI). Interestingly, naïve astrocytes can easily transform into neurotoxic reactive astrocytes (A1s) with inflammatory stimulation. Previous studies demonstrated that microRNA(miR)-21a-5p was up-regulated in spinal cord tissue after TSCI; however, it is not clear whether this affected reactive astrocyte polarization. Here, we aim to detect the effects of miR-21a-5p on the induction of A1 formation and the underlying mechanisms. Our study found that the expression of miR-21a-5p was significantly increased while that of Cntfr α was decreased, since naïve astrocytes transformed into A1s 3 days post-TSCI; the binding site between miR-21a-5p and Cntfr α was further confirmed in astrocytes. After treatment with CNTF, the levels of A1 markers decreased while that of A2 increased. The expression of A1 markers significantly decreased with the downregulation of miR-21a-5p, while Cntfr α siRNA treatment caused the opposite both in vitro and in vivo. To summarize, miR-21a-5p/Cntfr α promotes A1 induction and might enhance the inflammatory process of TSCI; furthermore, we identified, for the first time, the effect and potential mechanism by which CNTF inhibits naïve astrocytes transformation into A1s. Collectively, our findings demonstrate that targeting miR-21a-5p represents a prospective therapy for promoting the recovery of TSCI.