Overexpressed microRNA-103a-3p inhibits acute lower-extremity deep venous thrombosis via inhibition of CXCL12.

Studies have shown that microRNAs (miRNAs) participate in almost all pathological and physiological processes including acute lower-extremity deep venous thrombosis (LEDVT). Here, this study was designed to elucidate the possible function of miR-103a-3p in acute LEDVT. Expression of miR-103a-3p and chemokine C-X-C motif ligand 12 (CXCL12) was initially quantified in plasma collected from 81 LEDVT patients. Then LEDVT mouse models were established by injection with 3% sodium pentobarbital. The interaction between miR-103a-3p and CXCL12 was identified by dual-luciferase reporter gene assay. After gain- and loss-of-function studies, interleukin-6 (IL-6) and IL-8 and tissue factor (TF) levels, and expression of plasminogen activator inhibitors (PAIs), von Willebrand factor (vWF), thromboxane A2 (TH-A2), F4/80, IL-12, Arginase-1 (Arg-1) and CD206 were determined using enzyme-linked immunosorbent assay (ELISA), reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analysis, respectively. miR-103a-3p was downregulated, while CXCL12 was upregulated in patients and mice with LEDVT. miR-103a-3p targets CXCL12 and inhibited its expression. Overexpressed miR-103a-3p or downregulated CXCL12 decreased expression of IL-6, IL-8, TF, PAIs, vWF, TH-A2, M1 markers (IL-6 and IL-12), yet increased expression of M2 markers (Arg-1 and CD206) in LEDVT mice. Additionally, upregulated miR-103a-3p or silencing CXCL12 suppressed thrombosis in LEDVT mice. However, overexpression of CXCL12 reversed the tendency mentioned above. Altogether, miR-103a-3p can potentially downregulate CXCL12 expression to disrupt inflammatory response and thrombosis, ultimately preventing the development of LEDVT. Our findings underscore a possible alternative therapeutic strategy to limit LEDVT.

The primary growth of laryngeal squamous cell carcinoma cells in vitro is effectively supported by paired cancer-associated fibroblasts alone.

Most primarily cultured laryngeal squamous cell carcinoma cells are difficult to propagate in vitro and have a low survival rate. However, in our previous work to establish a laryngeal squamous cell carcinoma cell line, we found that laryngeal cancer-associated fibroblasts appeared to strongly inhibit the apoptosis of primarily cultured laryngeal squamous cell carcinoma cells in vitro. In this study, we investigated whether paired laryngeal cancer-associated fibroblasts alone can effectively support the growth of primarily cultured laryngeal squamous cell carcinoma cells in vitro. In all, 29 laryngeal squamous cell carcinoma specimens were collected and primarily cultured. The laryngeal squamous cell carcinoma cells were separated from cancer-associated fibroblasts by differential trypsinization and continuously subcultured. Morphological changes of the cultured laryngeal squamous cell carcinoma cells were observed. Immunocytofluorescence was used to authenticate the identity of the cancer-associated fibroblasts and laryngeal squamous cell carcinoma cells. Flow cytometry was used to quantify the proportion of apoptotic cells. Western blot was used to detect the protein levels of caspase-3. Enzyme-linked immunosorbent assay was used to detect the levels of chemokine (C-X-C motif) ligand 12, chemokine (C-X-C motif) ligand 7, hepatocyte growth factor, and fibroblast growth factor 1 in the supernatants of the laryngeal squamous cell carcinoma and control cells. AMD3100 (a chemokine (C-X-C motif) receptor 4 antagonist) and an anti-chemokine (C-X-C motif) ligand 7 antibody were used to block the tumor-supporting capacity of cancer-associated fibroblasts. Significant apoptotic changes were detected in the morphology of laryngeal squamous cell carcinoma cells detached from cancer-associated fibroblasts. The percentage of apoptotic laryngeal squamous cell carcinoma cells and the protein levels of caspase-3 increased gradually in subsequent subcultures. In contrast, no significant differences in the proliferation capacity of laryngeal squamous cell carcinoma cells cocultured with cancer-associated fibroblasts were detected during subculturing. High level of chemokine (C-X-C motif) ligand 12 was detected in the culture supernatant of cancer-associated fibroblasts. The tumor-supporting effect of cancer-associated fibroblasts was significantly inhibited by AMD3100. Our findings demonstrate that the paired laryngeal cancer-associated fibroblasts alone are sufficient to support the primary growth of laryngeal squamous cell carcinoma cells in vitro and that the chemokine (C-X-C motif) ligand 12/chemokine (C-X-C motif) receptor 4 axis is one of the major contributors.

Pathogenesis of FOLFOX induced sinusoidal obstruction syndrome in a murine chemotherapy model.

BACKGROUND & AIMS: Sinusoidal obstruction syndrome (SOS) following oxaliplatin based chemotherapy can have a significant impact on post-operative outcome following resection of colorectal liver metastases. To date no relevant experimental models of oxaliplatin induced SOS have been described. The aim of this project was to establish a rodent model which could be utilised to investigate mechanisms underlying SOS to aid the development of therapeutic strategies. METHODS: C57Bl/6 mice, maintained on a purified diet, were treated with intra-peritoneal FOLFOX (n=10), or vehicle (n=10), weekly for five weeks and culled one week following final treatment. Sections of the liver and spleen were fixed in formalin and paraffin embedded for histological analysis. The role of oxidative stress on experimental-induced SOS was determined by dietary supplementation with butylated hydroxyanisole and N-acetylcysteine. RESULTS: FOLFOX treatment was associated with the development of sinusoidal dilatation and hepatocyte atrophy on H&E stained sections of the liver in keeping with SOS. Immunohistochemistry for p21 demonstrated the presence of replicative senescence within the sinusoidal endothelium. FOLFOX induced endothelial damage leads to a pro-thrombotic state within the liver associated with upregulation of PAI-1 (p<0.001), vWF (p<0.01) and Factor X (p<0.001), which may contribute to the propagation of liver injury. Dietary supplementation with the antioxidant BHA prevented the development of significant SOS. CONCLUSIONS: We have developed the first reproducible model of chemotherapy induced SOS that reflects the pathogenesis of this disease in patients. It appears that the use of antioxidants alongside oxaliplatin based chemotherapy may be of value in preventing the development of SOS in patients with colorectal liver metastases.

Decreased serum CXCL12/SDF-1 concentrations may reflect disease severity of non-traumatic osteonecrosis of femoral head.

OBJECTIVE: The current study was performed to investigate the potential association of serum CXCL12 with disease severity in non-traumatic ONFH. METHODS: This study enrolled 182 patients with non-traumatic ONFH and 182 age- and gender-matched healthy controls. The CXCL12 levels in serum were measured by enzyme-linked immunosorbent assay. Meanwhile, serum levels of procollagen type I (PINP) and Interleukin-33(IL-33) were also detected. The radiographic severity was determined by FICAT grade. Clinical severity was evaluated by visual analogue scale (VAS), Harris Hip Score (HHS) and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC). Among the non-traumatic ONFH, 90 patients ONFH received total hip arthroplasty, the localization and expression of the CXCL12 protein and mRNA were detected by immunohistochemistry, Western blot analysis, RT-PCR and in necrotic area and adjacent non-necrotic area from lesioned femoral neck from ONFH patients and healthy femoral head from femoral neck fracture patients. Receiver operating characteristic (ROC) curve analysis was carried out to confirm the diagnostic value serum CXCL12, PINP and IL-33 with regard to the FICAT grade. RESULTS: Serum CXCL12 levels were significantly lower in non-traumatic ONFH patients compared with healthy controls. CXCL12 mRNA and protein expressions were both significantly decreased in necrotic area in comparison with non-necrotic area and healthy femoral head. Serum CXCL12 concentrations were drastically reduced in patients with FICAT stage 4 compared with stage 3, and CXCL12 concentrations in patients with stage 3 were markedly lower than stage 2. Serum CXCL12 levels were negatively related to FICAT grading. In addition, Serum CXCL12 concentrations were also negatively related to VAS, WOMAC scores and positively correlated with HHS scores. Meanwhile, serum CXCL12 levels were positively correlated with serum PINP and negatively correlated with IL-33 levels. ROC curve analysis implicated that decrease CXCL12 in serum may act as a favorable marker for FICAT grade. CONCLUSIONS: Decreased serum CXCL12 concentrations may reflect disease severity of non-traumatic ONFH.

microRNA-23 inhibits inflammation to alleviate rheumatoid arthritis via regulating CXCL12.

Rheumatoid arthritis (RA) is a common systemic, inflammatory and autoimmune disorder. MicroRNAs (miRs) are strongly associated with the initiation and progression of RA. However, the functions and mechanisms underlying miR-23 in RA are not completely understood. Therefore, the present study aimed to investigate the molecular mechanisms underlying miR-23 in RA. A bioinformatics tool (StarBase) and a wide range of experimental assays, including reverse transcription-quantitative PCR, western blotting, luciferase reporter assays and ELISAs, were performed to investigate the biological role of miR-23 in RA. The results indicated that miR-23 was downregulated and chemokine C-X-C motif ligand 12 (CXCL12) was upregulated in RA samples compared with healthy samples. Furthermore, miR-23 overexpression suppressed inflammation via reducing TNF-α, IL-1ß and IL-8 expression levels compared with the NC mimic group. Regarding the underlying mechanism, compared with NC mimic, miR-23 mimic decreased CXCL12 mRNA expression by binding to its 3'-untranslated region. Additionally, CXCL12 overexpression reversed miR-23 mimic-mediated effects on inflammation. NF-κB signaling is associated with inflammation. Therefore, the present study indicated that CXCL12 promoted inflammation by activating NF-κB signaling. In conclusion, miR-23 inhibited inflammation to alleviate RA by regulating CXCL12 via the NF-κB signaling pathway, which may serve as a potential target for the diagnosis and treatment of RA.

Relationship between osteoporosis and expression of Bcl-2 and CXCL12.

The changes of expression of anti-apoptotic factor B-cell lymphoma 2 (Bcl-2) and chemokine C-X-C motif ligand 12 (CXCL12) in the pathological process of osteoporosis (OP) were investigated, to provide new ideas for the diagnosis and treatment of OP. A total of 60 postmenopausal women who needed to undergo hip replacement surgery were enrolled and divided into osteoporosis group (OP, n=32) and control group (CK, n=28) according to the results of dual-energy X-ray bone density measure; after operation, cancellousbone from the femoral head or femoral neck was removed, and osteoblasts and osteoclasts were isolated and cultured in vitro. The proliferation and apoptosis in the two groups of osteoblasts and osteoclasts were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetry and Annexin V/PI double staining method, respectively. The expression levels of Bcl-2 and CXCL12 mRNA and protein in the two groups of osteoblasts and osteoclasts were determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. The analysis of cell proliferation and apoptosis showed that compared with the CK group, osteoblast proliferation was significantly inhibited and apoptosis rate was distinctly increased in the OP group, compared with the CK group, osteoclast proliferation was distinctly enhanced and apoptosis rate was remarkably reduced in the OP group. The results of RT-qPCR and western blot analysis displayed that Bcl-2 and CXCL12 mRNA and protein levels in osteoblasts of the OP group were significantly lower than those of the CK group, while mRNA and protein levels of Bcl-2 and CXCL12 in osteoclast of the OP group were distinctly increased compared to those in the CK group. The incidence of OP is closely associated with the bone balance maintained by osteoblasts and osteoclasts, and this mechanism may be achieved by inhibiting osteoblast proliferation and osteoclast apoptosis via regulating Bcl-2 and CXCL12 gene expression changes.

Lysozyme association with circulating RNA, extracellular vesicles, and chronic stress.

BACKGROUND: Stress has demonstrated effects on inflammation though underlying cell-cell communication mechanisms remain unclear. We hypothesize that circulating RNAs and extracellular vesicles (EVs) in patients with chronic stress contain signals with functional roles in cell repair. METHODS: Blood transcriptome from patients with Irritable Bowel Syndrome versus controls were compared to identify signaling pathways and effectors. Plasma EVs were isolated (size-exclusion chromatography) and characterized for effectors' presence (immunogold labelling-electron microscopy). Based on transcriptome pathways and EV-labelling, lysozyme's effects on cell migration were tested in human colon epithelial CRL-1790 cells and compared to the effects of CXCL12, a migration inducer (wound assay). The effect of lysozyme on immune-linked mRNA and protein levels in cells which survived following serum starvation and scratch wound were investigated (NanoString). RESULTS: Blood transcriptomes revealed pyridoxal 5'phosphate salvage, pyrimidine ribonucleotides salvage pathways, atherosclerosis, and cell movement signaling with membrane CD9 and extracellular lysozyme as effectors. Plasma EVs showed labelling with CD9, mucins, and lysozyme. This is the first identification of lysozyme on plasma EVs. In CRL-1790 cells, lysozyme induced migration and repaired scratch wound as well as CXCL12. Immune mRNA and protein expressions were altered in cells which survived following serum starvation and scratch wound, with or without lysozyme in serum-free media post-wounding: CD9, IL8, IL6 mRNAs and CD9, NT5E, PD-L1 proteins. CONCLUSIONS: Repair and inflammatory signals are identified in plasma EVs and circulating RNAs in chronic stress. Registered clinicaltrials.gov #NCT00824941. GENERAL SIGNIFICANCE: This study highlights the role of circulating RNAs and EVs in stress.

Fabrication of bone marrow-like tissue in vitro from dispersed-state bone marrow cells.

A three-dimensional (3D) bone marrow (BM) culture system may facilitate research into the molecular mechanisms involved in hematopoiesis and BM diseases. However, because >90% of BM cells are composed of non-adherent blood cells, it is difficult to organize the dispersed BM cells into 3D multicellular spheroids using conventional aggregation methods such as hanging drop, and rotary shaking culture. The objective of this study was to reproduce BM-like tissue. We reported successful formation of BM aggregates using a 3% methylcellulose (MC) medium. This medium could aggregate even non-adherent materials. In MC medium, BM cells formed tissue-like aggregates within 24 h. Although the cell density of the BM-like tissue is slightly low, sections of the organoids resembled those of intact BM tissue. Cells of the BM-like tissue were approximately 70% viable after 7 days in culture. Staining for CD68, PDGFRα, and CXCL12 indicated that the BM-like tissue contained macrophages, and mesenchymal cells including CXCL12-abundant reticular cells. These results indicated that the method using MC medium effectively reconstitutes the BM-like tissue.

Myeloid derived suppressor cells-An overview of combat strategies to increase immunotherapy efficacy.

Myeloid-derived suppressor cells (MDSCs) contribute to tumor-mediated immune escape and negatively correlate with overall survival of cancer patients. Nowadays, a variety of methods to target MDSCs are being investigated. Based on the intervention stage of MDSCs, namely development, expansion and activation, function and turnover, these methods can be divided into: (I) prevention or differentiation to mature cells, (II) blockade of MDSC expansion and activation, (III) inhibition of MDSC suppressive activity or (IV) depletion of intratumoral MDSCs. This review describes effective mono- or multimodal-therapies that target MDSCs for the benefit of cancer treatment.

Vitamin D deficiency and severe asthma.

Vitamin D has received tremendous amount of attention recently due to the ever-increasing reports of association between vitamin D deficiency and a wide range of conditions, from cancer to fertility to longevity. The fascination of disease association with vitamin D deficiency comes from the relatively easy solution to overcome such a risk factor, that is, either by increase in sun exposure and/or diet supplementation. Many reviews have been written on a protective role of vitamin D in asthma and related morbidities; here, we will summarize the epidemiological evidence supporting a role of vitamin D against hallmark features of severe asthma, such as airway remodeling and asthma exacerbations. Furthermore, we discuss data from in vitro and in vivo studies which provide insights on the potential mechanisms of how vitamin D may protect against severe asthma pathogenesis and how vitamin D deficiency may lead to the development of severe asthma. Approximately 5-15% of asthmatic individuals suffer from the more severe forms of disease in spite of aggressive therapies and they are more likely to have irreversible airflow obstruction associated with airway remodeling. At present drugs commonly used to control asthma symptoms, such as corticosteroids, do not significantly reverse or reduce remodeling in the airways. Hence, if vitamin D plays a protective role against the development of severe asthma, then the most effective therapy may simply be a healthy dose of sunshine.