Cancer-associated fibroblast-secreted exosomal miR-423-5p promotes chemotherapy resistance in prostate cancer by targeting GREM2 through the TGF-β signaling pathway

Therapeutic failure in prostate cancer (PC) is believed to result from its unusually invasive and metastatic nature. Cancer-associated fibroblasts (CAFs) are essential in the tumor microenvironment. We intended to study the role of CAF-derived exosomes in the context of PC and the potential regulatory mechanism associated with miR-423-5p and GREM2. CAF-derived exosomes decreased the chemosensitivity of parental PC cells and enhanced the drug resistance of drug-resistant cells. PC-associated fibroblast-derived exosomes carrying miR-423-5p increased the resistance of PC to taxane by inhibiting GREM2 through the TGF-β pathway. Inhibition of the TGF-β pathway partially reversed the increased drug resistance in PC cells induced by CAF-derived exosomes. Inhibition of miR-423-5p enhanced the drug sensitivity of PC cells in vivo. We showed that CAF-secreted exosomal miR-423-5p promoted chemotherapy resistance in PC by targeting GREM2 through the TGF-β pathway. This study may allow the development of novel approaches for PC.Subject terms: Cancer, Cancer     

Serum exosomal microRNAs as novel biomarkers for hepatocellular carcinoma

Recent studies have shown that circulating microRNAs are a potential biomarker in various types of malignancies. The aim of this study was to investigate the feasibility of using serum exosomal microRNAs as novel serological biomarkers for hepatocellular carcinoma (HCC) in patients with chronic hepatitis B (CHB). We measured the serum exosomal microRNAs and serum circulating microRNAs in patients with CHB (n=20), liver cirrhosis (LC) (n=20) and HCC (n=20). Serum exosomal microRNA was extracted from 500 μl of serum using an Exosome RNA Isolation kit. The expression levels of microRNAs were quantified by real-time PCR. The expression levels of selected microRNAs were normalized to Caenorhabditis elegans microRNA (Cel-miR-39). The serum levels of exosomal miR-18a, miR-221, miR-222 and miR-224 were significantly higher in patients with HCC than those with CHB or LC (P<0.05). Further, the serum levels of exosomal miR-101, miR-106b, miR-122 and miR-195 were lower in patients with HCC than in patients with CHB (P=0.014, P<0.001, P<0.001 and P<0.001, respectively). There was no significant difference in the levels of miR-21 and miR-93 among the three groups. Additionally, the serum levels of circulating microRNAs showed a smaller difference between HCC and either CHB or LC. This study suggests that serum exosomal microRNAs may be used as novel serological biomarkers for HCC.     

Dynamic synovial fibroblasts are modulated by NBCn1 as a potential target in rheumatoid arthritis

Rheumatoid arthritis (RA) is an autoimmune disease characterized by aggressive fibroblast-like synoviocytes (FLSs) and pannus formation. Various therapeutic strategies have been developed against inflammatory cytokines in RA in recent decades. Based on the migratory features of FLSs, we examined whether modulation of the migratory module attenuates RA severity. In this study, inflamed synovial fluid-stimulated FLSs exhibited enhanced migration and migratory apparatus expression, and sodium bicarbonate cotransporter n1 (NBCn1) was identified in primary cultured RA-FLSs for the first time. The NBC inhibitor S0859 attenuated the migration of FLSs induced with synovial fluid from patients with RA or with TNF-α stimulation. Inhibition of NBCs with S0859 in a collagen-induced arthritis (CIA) mouse model reduced joint swelling and destruction without blood, hepatic, or renal toxicity. Primary FLSs isolated from the CIA-induced mouse model also showed reduced migration in the presence of S0859. Our results suggest that inflammatory mediators in synovial fluid, including TNF-α, recruit NBCn1 to the plasma membrane of FLSs to provide dynamic properties and that modulation of NBCn1 could be developed into a therapeutic strategy for RA.Subject terms: Chemotaxis, Bone, Ion channel signalling, Rheumatoid arthritis, Drug development     

Blockade of translationally controlled tumor protein attenuated the aggressiveness of fibroblast-like synoviocytes and ameliorated collagen-induced arthritis

Histamine releasing factor/translationally controlled tumor protein (HRF/TCTP) stimulates cancer progression and allergic responses, but the role of HRF/TCTP in rheumatoid arthritis (RA) remains undefined. In this study, we explored the pathogenic significance of HRF/TCTP and evaluated the therapeutic effects of HRF/TCTP blockade in RA. HRF/TCTP transgenic (TG) and knockdown (KD) mice with collagen-induced arthritis (CIA) were used to determine the experimental phenotypes of RA. HRF/TCTP levels in the sera of RA patients were measured and compared to those from patients with osteoarthritis (OA), ankylosing spondylitis, Behçet’s disease, and healthy controls. HRF/TCTP expression was also assessed in the synovium and fibroblast-like synoviocytes (FLSs) obtained from RA or OA patients. Finally, we assessed the effects of HRF/TCTP and dimerized HRF/TCTP-binding peptide-2 (dTBP2), an HRF/TCTP inhibitor, in RA-FLSs and CIA mice. Our clinical, radiological, histological, and biochemical analyses indicate that inflammatory responses and joint destruction were increased in HRF/TCTP TG mice and decreased in KD mice compared to wild-type littermates. HRF/TCTP levels in the sera, synovial fluid, synovium, and FLSs were higher in patients with RA than in control groups. Serum levels of HRF/TCTP correlated well with RA disease activity. The tumor-like aggressiveness of RA-FLSs was exacerbated by HRF/TCTP stimulation and ameliorated by dTBP2 treatment. dTBP2 exerted protective and therapeutic effects in CIA mice and had no detrimental effects in a murine tuberculosis model. Our results indicate that HRF/TCTP is a novel biomarker and therapeutic target for the diagnosis and treatment of RA.Subject terms: Translational research, Autoimmunity     

Chemokine Receptor 5 Antagonism Causes Reduction in Joint Inflammation in a Collagen-Induced Arthritis Mouse Model

Rheumatoid arthritis (RA) is a chronic inflammatory disease mainly affecting the synovial joints. A highly potent antagonist of C-C chemokine receptor 5 (CCR5), maraviroc (MVC), plays an essential role in treating several infectious diseases but has not yet been evaluated for its potential effects on RA development. This study focused on evaluating the therapeutic potential of MVC on collagen-induced arthritis (CIA) in DBA/1J mice. Following CIA induction, animals were treated intraperitoneally with MVC (50 mg/kg) daily from day 21 until day 35 and evaluated for clinical score and histopathological changes in arthritic inflammation. We further investigated the effect of MVC on Th9 (IL-9, IRF-4, and GATA3) and Th17 (IL-21R, IL-17A, and RORγT) cells, TNF-α, and RANTES in CD8+ T cells in the spleen using flow cytometry. We also assessed the effect of MVC on mRNA and protein levels of IL-9, IL-17A, RORγT, and GATA3 in knee tissues using RT-PCR and western blot analysis. MVC treatment in CIA mice attenuated the clinical and histological severity of inflammatory arthritis, and it substantially decreased IL-9, IRF4, IL-21R, IL-17A, RORγT, TNF-α, and RANTES production but increased GATA3 production in CD8+ T cells. We further observed that MVC treatment decreased IL-9, IL-17A, and RORγt mRNA and protein levels and increased those of GATA3. This study elucidates the capacity of MVC to ameliorate the clinical and histological signs of CIA by reducing pro-inflammatory responses, suggesting that MVC may have novel therapeutic uses in the treatment of RA.     

miR-19b enhances osteogenic differentiation of mesenchymal stem cells and promotes fracture healing through the WWP1/Smurf2-mediated KLF5/β-catenin signaling pathway

Bone marrow mesenchymal stem cell (BMSC)-derived exosomes have been found to enhance fracture healing. In addition, microRNAs contributing to the healing of various bone fractures have attracted widespread attention in recent years, but knowledge of the mechanisms by which they act is still very limited. In this study, we clarified the function of altered microRNA-19b (miR-19b) expression in BMSCs in fracture healing. We modulated miR-19b expression via mimics/inhibitors in BMSCs and via agomirs in mice to explore the effects of these changes on osteogenic factors, bone cell mineralization and the healing status of modeled fractures. Through gain- and loss-of function assays, the binding affinity between miR-19b and WWP1/Smurf2 was identified and characterized to explain the underlying mechanism involving the KLF5/β-catenin signaling pathway. miR-19b promoted the differentiation of human BMSCs into osteoblasts by targeting WWP1 and Smurf2. Overexpression of WWP1 or Smurf2 degraded the target protein KLF5 in BMSCs through ubiquitination to inhibit fracture healing. KLF5 knockdown delayed fracture healing by modulating the Wnt/β-catenin signaling pathway. Furthermore, miR-19b enhanced fracture healing via the KLF5/β-catenin signaling pathway by targeting WWP1 or Smurf2. Moreover, miR-19b was found to be enriched in BMSC-derived exosomes, and treatment with exosomes promoted fracture healing in vivo. Collectively, these results indicate that mesenchymal stem cell-derived exosomal miR-19b represses the expression of WWP1 or Smurf2 and elevates KLF5 expression through the Wnt/β-catenin signaling pathway, thereby facilitating fracture healing.Subject terms: Diseases, Biotechnology     

Myeloid differentiation primary response protein 88 blockade upregulates indoleamine 2,3-dioxygenase expression in rheumatoid synovial fibroblasts

Indoleamine 2,3-dioxygenase (IDO) is a key negative regulator of immune responses and has been implicated in tumor tolerance, autoimmune disease and asthma. IDO was detected in the joint synovial tissue in the inflammatory microenvironment of rheumatoid arthritis (RA), but IDO expression in joint synovial tissue is not sufficient to overcome the inflamed synovial environment. This study aimed to unravel the mechanisms involving the failure to activate tolerogenic IDO in the inflamed joint. We demonstrate that both poly (I:C) and lipopolysaccharide (LPS) induce expression of IDO in synovial fibroblasts. However, inflammatory cytokines such as IL-17, TNF-alpha, IL-12, IL-23 and IL-16 did not induce IDO expression. Poly (I:C) appeared to induce higher IDO expression than did LPS. Surprisingly, toll-like receptor (TLR)4-mediated IDO expression was upregulated after depletion of myeloid differentiation primary response protein 88 (MyD88) in synovial fibroblasts using small interfering RNA (siRNA). IDO, TLR3 and TLR4 were highly expressed in synovial tissue of RA patients compared with that of osteoarthritis patients. In addition, RA patients with severe disease activity had higher levels of expression of IDO, TLR3 and TLR4 in the synovium than patients with mild disease activity. These data suggest that upregulation of IDO expression in synovial fibroblasts involves TLR3 and TLR4 activation by microbial constituents. We showed that the mechanisms responsible for IDO regulation primarily involve MyD88 signaling in synovial fibroblasts, as demonstrated by siRNAmediated knockdown of MyD88.     

Simultaneous and multiplex detection of exosomal microRNAs based on the asymmetric Au@Au@Ag probes with enhanced Raman signal

Simultaneous and quantitative detection of multiple exosomal micro RNAs(miRNAs) was successfully performed by a surface-enhanced Raman scattering(SERS) assay consisting of Raman probes and capture probes. In this design, the asymmetric core-shell structured Au@Au@Ag nanoparticles were first synthesized by layer-by-layer self-assembly method and modified with different Raman molecules and recognition sequences(poly A-DNA) to prepare the surface-enhanced Raman probes. Then, the streptavidinmodifie...     

Plasma Exosomes of Patients with Breast and Ovarian Tumors Contain an Inactive 20S Proteasome

Exosomes are directly involved in governing of physiological and pathological conditions of an organism through the transfer of information from producing to receiving cells. It can be assumed that exosomes are one of the key players of tumor dissemination since they are very stable and small enough to penetrate from various tissues into biological fluids and then back, thus interacting with tissue target cells. We evaluated the enzymatic activity and the level of 20S proteasome in tissue and exosomes of healthy females (n = 39) and patients with ovarian (n = 50) and breast (n = 108) tumors to reveal the critical role of exosomal cargo in the mediation of different types of metastases. Exosomes from plasma and ascites were isolated and characterized in according to International Society for Extracellular Vesicles guidelines. The level of 20S proteasome in tissue and exosomes was determined using Western blot analysis. Chymotrypsin- and caspase-like (ChTL and CL, respectively) peptidase activities of the proteasomes were determined using fluorogenic Suc-LLVY-AMC and Cbz-LLG-AMC substrates, respectively. We observed increased levels of 20S proteasome in ovarian cancer tissue and luminal B subtype breast cancer tissue as well as in plasma exosomes from cancer patients. Moreover, the level of the 20S proteasome in plasma exosomes and ascites exosomes in patients with ovarian tumors is comparable and higher in ovarian cancer patients with low volume ascites than in patients with moderate and high-volume ascites. We also found increased ChTL and CL activities in breast cancer and ovarian cancer tissues, as well as in peritoneal metastases in ovarian cancer, while proteasomal activity in exosomes from plasma of healthy females and all patients, as well as from ascites of ovarian tumor patients were lower than detection limit of assay. Thus, regardless of the type of tumor metastasis (lymphogenous or peritoneal), the exosomes of cancer patients were characterized by an increased level of 20S proteasome, which do not exhibit enzymatic activity.     

Obesity aggravates the joint inflammation in a collagen-induced arthritis model through deviation to Th17 differentiation

White fat cells secrete adipokines that induce inflammation and obesity has been reported to be characterized by high serum levels of inflammatory cytokines such as IL-6 and TNF-α. Rheumatoid arthritis (RA) is a prototype of inflammatory arthritis, but the relationship between RA and obesity is controversial. We made an obese inflammatory arthritis model: obese collagen-induced arthritis (CIA). C57BL/6 mice were fed a 60-kcal high fat diet (HFD) from the age of 4 weeks and they were immunized twice with type II collagen (CII). After immunization, the obese CIA mice showed higher arthritis index scores and histology scores and a more increased incidence of developing arthritis than did the lean CIA mice. After treatment with CII, mixed lymphocyte reaction also showed CII-specific response more intensely in the obese CIA mice than lean CIA. The anti-CII IgG and anti-CII IgG2a levels in the sera of the obese CIA mice were higher than those of the lean CIA mice. The number of Th17 cells was higher and the IL-17 mRNA expression of the splenocytes in the obese CIA mice was higher than that of the lean CIA mice. Obese CIA mice also showed high IL-17 expression on synovium in immunohistochemistry. Although obesity may not play a pathogenic role in initiating arthritis, it could play an important role in amplifying the inflammation of arthritis through the Th1/Th17 response. The obese CIA murine model will be an important tool when we investigate the effect of several therapeutic target molecules to treat RA.     

The arachidonic acid metabolite 11,12-epoxyeicosatrienoic acid alleviates pulmonary fibrosis

Epoxyeicosatrienoic acids (EETs) are metabolites of arachidonic acid that are rapidly metabolized into diols by soluble epoxide hydrolase (sEH). sEH inhibition has been shown to increase the biological activity of EETs, which are known to have anti-inflammatory properties. However, the role of EETs in pulmonary fibrosis remains unexplored. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) was used to analyze EETs in the lung tissues of patients with idiopathic pulmonary fibrosis (IPF, n = 29) and controls (n = 15), and the function of 11,12-EET was evaluated in in vitro and in vivo in pulmonary fibrosis models. EET levels in IPF lung tissues, including those of 8,9-EET, 11,12-EET, and 14,15-EET, were significantly lower than those in control tissues. The 11,12-EET/11,12-DHET ratio in human lung tissues also differentiated IPF from control tissues. 11,12-EET significantly decreased transforming growth factor (TGF)-β1-induced expression of α-smooth muscle actin (SMA) and collagen type-I in MRC-5 cells and primary fibroblasts from IPF patients. sEH-specific siRNA and 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU; sEH inhibitor) also decreased TGF-β1-induced expression of α-SMA and collagen type-I in fibroblasts. Moreover, 11,12-EET and TPPU decreased TGF-β1-induced p-Smad2/3 and extracellular-signal-regulated kinase (ERK) expression in primary fibroblasts from patients with IPF and fibronectin expression in Beas-2B cells. TPPU decreased the levels of hydroxyproline in the lungs of bleomycin-induced mice. 11,12-EET or sEH inhibitors could inhibit pulmonary fibrosis by regulating TGF-β1-induced profibrotic signaling, suggesting that 11,12-EET and the regulation of EETs could serve as potential therapeutic targets for IPF treatment.Subject terms: Respiratory tract diseases, Chronic inflammation     

Proteomics comparison of exosomes from serum and plasma between ultracentrifugation and polymer‐based precipitation kit methods

Exosomes are vesicles with sizes ranging from 30 to 150 nm. The analysis and detection of blood exosomes offers an effective route for cancer diagnosis, prognosis assessment, and therapeutic evaluation of diseases. Due to the difference in separation procedure, collection method and the usage of anticoagulants, serum and plasma samples show diversity test results. In order to evaluate the isolation effect of exosomes in serum and plasma samples, two commonly used exosomal isolation methods, ultracentrifugation and polymer‐based precipitation kit, were used, respectively. And the isolation effects were evaluated by comparing the composition and abundant of proteins from isolated exosomes based on MS‐based proteomics analysis. The results showed that the plasma exosomes extracted by ultracentrifugation identified more exosome biomarkers, and the concentrations of these biomarkers were higher than others. And plasma exosomes could be a better sample for blood‐based proteomics research of exosomes. It would be more useful for future targeted biomarker discovery.     

The association between the CASP5 rs7939842 polymorphism and the risk of rheumatoid arthritis in Chinese Han individuals

BackgroundThe association between inflammatory cysteinyl aspartate protease-5 (CASP5) and the susceptibility to rheumatoid arthritis (RA) remains unclear. This study examined whether the CASP5 rs7939842 polymorphism affects RA risk in Chinese Han individuals.MethodsThis study recruited 805 RA patients and 1095 healthy controls to investigate the association between the CASP5 rs7939842 polymorphism and RA risk. Genotype was examined using the 48-Plex SNPscan? Kit. Plasma CASP5 levels were determined using enzyme-linked immunosorbent assays, and CASP5 gene expression was detected by quantitative polymerase chain reaction in 40 RA patients and 40 healthy controls.ResultThe CASP5 rs7939842 polymorphism G allele is a putative risk factor for RA. After stratified analyses, this polymorphism increased the risk of RA among CRP-, ACPA-, RF-, and ESR-positive individuals, as well as individuals with DAS28 ≥ 3.20 and functional class III + IV. Furthermore, the plasma CASP5 levels and CASP5 mRNA expression were higher in RA patients than in healthy controls.ConclusionThe CASP5 rs7939842 polymorphism appears to be associated with an elevated risk of RA in Chinese Han individuals. Blood CASP5 protein and mRNA levels were significantly higher in RA patients than in healthy controls.     

基于鸟枪法脂质组学研究环氧酶-2抑制剂对关节炎模型血清脂代谢的干预作用

心血管疾病(CVD)是类风湿关节炎(RA)患者死亡的重要原因之一,脂代谢紊乱与CVD发生有密切关联,因而有必要对RA和药物治疗导致的脂代谢改变进行探讨。该研究采用胶原诱导法(CIA)构建关节炎模型,引入多维质谱鸟枪法开展血清脂质分析,检测了血清中105种脂质分子,发现模型大鼠体内7种磷脂酰肌醇、15种鞘磷脂、5种神经酰胺、10种磷脂酰胆碱和2种溶血磷脂酰胆碱异常上调,环氧酶-2(COX-2)抑制剂可部分修复紊乱的脂代谢,但对5种磷脂酰胆碱和1种溶血磷脂酰胆碱具有异常调控作用。该研究从脂质分子水平探讨了RA及COX-2抑制剂对脂代谢的干预作用,可以为RA的心血管风险研究提供信息。     

hnRNPC induces isoform shifts in miR-21-5p leading to cancer development

MicroRNA (miRNA) processing is a critical step in mature miRNA production. Its dysregulation leads to an increase in miRNA isoforms with heterogenous 5′-ends (isomiRs), which can recognize distinct target sites because of their shifted seed sequence. Although some miRNA genes display productive expression of their 5′-isomiRs in cancers, how their production is controlled and how 5′-isomiRs affect tumor progression have yet to be explored. In this study, based on integrative analyses of high-throughput sequencing data produced by our group and publicly available data, we demonstrate that primary miR-21 (pri-miR-21) is processed into the cancer-specific isomiR isomiR-21-5p | ±1, which suppresses growth hormone receptor (GHR) in liver cancer. Treatment with antagomirs against isomiR-21-5p | ±1 inhibited the in vitro tumorigenesis of liver cancer cells and allowed the recovery of GHR, whereas the introduction of isomiR-21-5p | ±1 mimics attenuated these effects. These effects were validated in a mouse model of spontaneous liver cancer. Heterogeneous nuclear ribonucleoprotein C and U2 small nuclear RNA auxiliary factor 2 were predicted to bind upstream of pre-miR-21 via a poly-(U) motif and influence Drosha processing to induce the production of isomiR-21-5p | ±1. Our findings suggest an oncogenic function for the non-canonical isomiR-21-5p | ±1 in liver cancer, and its production was shown to be regulated by hnRNPC.Subject terms: RNAi, Liver cancer     

Mass spectrometric proteome analyses of synovial fluids and plasmas from patients suffering from rheumatoid arthritis and comparison to reactive arthritis or osteoarthritis

Differential proteome analysis is used to study body fluids from patients suffering from rheumatoid arthritis (RA), reactive arthritis (reaA) or osteoarthritis (OA). Mass spectrometric structure characterization of gel-separated proteins provided a detailed view of the protein-processing events that lead to distinct protein species present in the respective body fluids. (i) Fibrin(ogen) beta-chain degradation products, presumably plasmin-derived, appeared solely in synovial fluids (SF) from both patient collectives, (ii) calgranulin B (MRP14) was exclusively identified in SF samples derived from 5 out of 6 patients suffering from RA. Calgranulin B was not observed in synovial fluids from OA patients, nor in plasmas from either patient group. In all cases where calgranulin B was detected, calgranulin C was identified as well. (iii) Serum amyloid A protein spots were determined in plasmas and synovial fluids from patients with RA, but not in patients with OA. In addition to disease-relevant differences, interindividual differences in haptoglobin patterns of the patients under investigation were observed. Hence, in-depth proteome analysis of body fluids has proven effective for identification of multiple molecular markers and determination of associated protein structure modifications, that are thought to play a role for specifically determining a defined pathological state of diseased joints.     

Characterization of Porous Graphitic Carbon LC Phase with some Carbohydrate Derivatives

Stereoselective CE method for investigations of pharmacokinetics of ketoprofen enantiomers (KTP) in patients taking also other drugs is proposed, to establish relation between levels of KTP enantiomers in blood and synovial fluid of patients with rheumatoid arthritis. Resolution of the analytes was obtained in silica capillary filled with chiral selector-heptakis 2,3,6-tri-O-methyl–β-cyclodextrin in triethanolamine-phosphate buffer. Calibration curves for enantiomers in plasma and synovial fluid were linear in the range of 0.25–50.0 mg L⁻¹, but 1.0–250.0 mg L⁻¹ in urine. Concentrations of KTP enantiomers in synovial fluid measured at 4 h after the administration of a tablet with racemic KTP were insignificantly greater [(−)-R = 1.07 ± 0.66; (+)-S-KTP = 1.13 ± 0.65 mg L⁻¹] than in plasma [(−)-R = 0.86 ± 0.37; (+)-S-KTP = 0.96 ± 0.42 mg L⁻¹]. The validated method has been successfully applied for the determination of KTP enantiomers in biological fluids of patients with rheumatoid arthritis.