CXCL13 drives spinal astrocyte activation and neuropathic pain via CXCR5

Recent studies have implicated chemokines in microglial activation and pathogenesis of neuropathic pain. C-X-C motif chemokine 13 (CXCL13) is a B lymphocyte chemoattractant that activates CXCR5. Using the spinal nerve ligation (SNL) model of neuropathic pain, we found that CXCL13 was persistently upregulated in spinal cord neurons after SNL, resulting in spinal astrocyte activation via CXCR5 in mice. shRNA-mediated inhibition of CXCL13 in the spinal cord persistently attenuated SNL-induced neuropathic pain. Interestingly, CXCL13 expression was suppressed by miR-186-5p, a microRNA that colocalized with CXCL13 and was downregulated after SNL. Spinal overexpression of miR-186-5p decreased CXCL13 expression, alleviating neuropathic pain. Furthermore, SNL induced CXCR5 expression in spinal astrocytes, and neuropathic pain was abrogated in Cxcr5^–/– mice. CXCR5 expression induced by SNL was required for the SNL-induced activation of spinal astrocytes and microglia. Intrathecal injection of CXCL13 was sufficient to induce pain hypersensitivity and astrocyte activation via CXCR5 and ERK. Finally, intrathecal injection of CXCL13-activated astrocytes induced mechanical allodynia in naive mice. Collectively, our findings reveal a neuronal/astrocytic interaction in the spinal cord by which neuronally produced CXCL13 activates astrocytes via CXCR5 to facilitate neuropathic pain. Thus, miR-186-5p and CXCL13/CXCR5-mediated astrocyte signaling may be suitable therapeutic targets for neuropathic pain.     

Deferoxamine preconditioning potentiates mesenchymal stem cell homing in vitro and in streptozotocin-diabetic rats

Objective: Today, cell therapy is considered a promising alternative in treatment of several diseases such as type 1 diabetes. Loss of transplanted stem cell and more importantly scarcity in the number of cells reaching to target tissue is a major obstacle in cell therapy. There is evidences showing that deferoxamine (DFO), an iron chelator, increases the mobilization and homing of progenitor cells through increasing the stability of hypoxia-inducible factor 1α (HIF-1α) protein. In this study, the effect of DFO on some factors involved in homing of bone marrow-derived mesenchymal stem cell was investigated, and the other objectives of this research were to determine whether DFO is able to increase migration and subsequent homing of mesenchymal stem cell (MSCs) both in vitro and in vivo in streptozotocin-diabetic rats.

Research design and methods: MSCs were treated by DFO in minimal essential medium α (αMEM) for 24 h. The expression and localization of HIF-1α were evaluated by western blotting and immunocytochemistry. The expression of C-X-C chemokine receptor type 4 (CXCR-4) and chemokine receptor 2 (CCR2) were assessed by western blotting and RT-PCR. The activity of matrix metalloproteinases (MMP) -2 and -9 were measured by gelatin zymography. Finally, in vitro migration of MSCs toward different concentrations of stromal cell-derived factor and monocyte chemotactic protein-1 were also evaluated. To demonstrate the homing of MSCs in vivo, DFO-treated chloromethyl-benzamidodialkylcarbocyanine-labeled MSCs were injected into the tail vein of rats, and the number of stained MSCs reaching to the pancreas were determined after 24 h.

Results: In DFO-treated MSCs, expression of HIF-1α (p < 0.001), CXCR4 (p < 0.001), CCR2 (p < 0.001), and the activity of MMP-2 (p < 0.01) and MMP-9 (p < 0.05) were significantly increased compared to control groups. Elevation of HIF-1α, upregulation of CXCR4/CCR2 and higher activity of MMP-2/MMP-9 in DFO-treated MSCs were reversed by 2-methoxyestradiol (2-ME; 5 μmol), a HIF-1α inhibitor. The in vitro migrations as well as in vivo homing of DFO-treated MSCs were also significantly higher than control groups (p < 0.05).

Conclusions: Preconditioning of MSCs by DFO prior to transplantation could increase homing of MSCs through affecting some chemokine receptors as well as proteases involved and eventually improving the efficacy of cell therapy.     

High glucose prevents osteogenic differentiation of mesenchymal stem cells via lncRNA AK028326/CXCL13 pathway

BackgroundHigh glucose (HG) often induces unfavorable effects on proliferation and differentiation of mesenchymal stem cells (MSCs). This study aimed to explore potential molecular pathways underlying HG functional mechanism during osteogenic differentiation of MSCs, involving lncRNA AK028326 and CXCL13.MethodsMurine bone marrow-derived MSCs were cultured in osteogenic-inducing medium supplemented with high glucose level at 25 mM or 5.5 mM as normal control. Expression levels of lncRNA AK028326 and CXCL13 were measured by using real-time PCR. The mineralized nodule formation and alkaline phosphatase (ALP) activity were detected after 21 and 7 days of incubation respectively. Western blot were also performed to determine the expression of CXCL13 and osteogenic gene markers. Plasmid pcDNAs and small interference RNAs were transfected as indicated for functional analysis of AK028326 and CXCL13.ResultsHG suppressed the expression of AK028326 and CXCL13 in MSCs in a time-dependent manner, and also the mineralization, ALP activity, and osteogenic gene expression, which could be reversed by overexpression of AK028326 or CXCL13. CXCL13 expression was positively regulated by AK028326 at both mRNA and protein levels. Moreover, CXCL13 mediated the positive regulation of AK028326 on osteogenic gene expression in MSCs and MC3T3-E1 cells, mineralization and ALP activity in MSCs and also HG-induced inhibitory effects during MSCs differentiation into osteoblast.ConclusionHG could inhibit osteogenic differentiation of MSCs via inhibited expression of CXCL13 mediated by lncRNA AK028326, thereby providing new insights into the molecular mechanism of many osteogenesis-related diseases especially for patients with hyperglycemia.     

CXCL12对人胰腺癌细胞株PANC-1体外侵袭能力的影响

目的 探讨趋化因子CXCL12对人胰腺癌细胞株PANC-1侵袭能力的影响及其作用机制.方法 取人胰腺癌细胞株PANC-1,用不同浓度的CXCL12处理后,采用Transwell侵袭实验检测不同浓度组中PANC-1细胞侵袭能力的变化;用RT-PCR法检测细胞中MMP-9 mRNA和VEGF mRNA的表达,用ELISA法检测细胞培养液中MMP-9和VEGF蛋白的表达.结果 经CXCL12作用后,人胰腺癌PANC-1细胞侵袭能力明显增强,并具有一定的量效关系;细胞和上清液中MMP9和VEGF的表达亦呈剂量依赖性增强.结论 CXCL12可以通过上调MMP-9和VEGF的表达从而增强人胰腺癌细胞PANC-1的体外侵袭能力.     

lncRNA XIST attenuates hypoxia-induced H9c2 cardiomyocyte injury by targeting the miR-122-5p/FOXP2 axis

ObjectiveTo investigate the effect of lncRNA XIST on apoptosis induced by hypoxia.MethodsWe analyzed the expression levels of lncRNA XIST and miR-122-5p using RT-qPCR in hypoxia-induced cardiomyocytes. The mechanism by which lncRNA XIST affects myocardial ischemia was investigated using the cell transfection, CCK-8, and dual-luciferase reporter assays, as well as by flowcytometry, western blotting, and RNA immunoprecipitation.ResultsHypoxic H9c2 cells demonstrated a decrease in their migration and invasion abilities and XIST expression and an increase in the extent of their apoptosis and expression of microRNA-122-5p. Overexpression of XIST significantly increased the H9c2 cell viability, enhanced cell migration and invasion, and decreased cell apoptosis in a hypoxic environment. The luciferase activity of XIST-WT in H9c2 cells co-transfected with XIST-WT and microRNA-122-5p mimics had decreased. The results of RNA immunoprecipitation showed that XIST interacted directly with miRNA-122-5p. Overexpression of XIST decreased the level of miRNA-122-5p significantly. mi-122-5p mimics increased H9c2 cell apoptosis and downregulated FOXP2 expression. Overexpression of FOXP2 upregulated the expression of the Bcl-2 protein in H9c2 cells transfected with microRNA-122-5p mimics and inhibited the expression of HIF-alpha, Bax, and the cleaved-caspase 9 protein.ConclusionlncRNA XIST could regulate the miR-122-5p/FOXP2 axis to attenuate hypoxia-induced H9c2 cardiomyocyte injury.     

Upregulated microRNA‐126 induces apoptosis of dental pulp stem cell via mediating PTEN‐regulated Akt activation

IntroductionHuman dental pulp stem cells (DPSCs) have potential applications in regenerative medicine. The molecular mechanisms underlying DPSCs viability and apoptosis are not completely understood. Here, we investigated the role of miR‐126 in DPSCs viability and apoptosis.Material and methodsSenescent DPSCs were compared with early passage DPSCs. real‐time PCR and microARRAY were performed to identify the differential expression of miR‐126, and western blot was performed to detect the expression of PTEN. MTT assay was utilized to reveal the proliferative rate of both senescent and early passage DPSCs. Flow cytometry was used to examine the apoptotic rate of DPSCs. Dual‐luciferase reporter assay was carried out to detect the interaction of miR‐126 and PTEN.ResultsSenescent DPSCs showed a high level of apoptosis. Further study showed that miR‐126 is upregulated in senescent DPSCs and its overexpression in early passaged DPSCs induced apoptosis. Phosphatase and tensin homolog gene (PTEN) was identified as a target of miR‐126. PTEN was downregulated in senescent DPSCs, whereas miR‐126 inhibition upregulated PTEN level, and subsequently activated Akt pathway and suppressed the apoptotic phenotype of senescent DPSCs. In addition, PTEN overexpression rescued apoptosis of DPSCs at later stage.ConclusionOur results demonstrate that the miR‐126‐PTEN‐Akt axis plays a key role in the regulation of DPSCs apoptosis and provide a candidate target to improve the functional and therapeutic potential of DPSCs.     

肿瘤微环境促进子宫内膜癌细胞分泌雌激素

目的:筛选子宫内膜癌微环境中促进子宫内膜癌细胞雌激素分泌的细胞因子,并探讨相关机制。方法:提取子宫内膜癌间质细胞,用双重免疫荧光法进行鉴定。检测子宫内膜癌细胞(RL95-2、HEC-1A和HEC-1B)与间质细胞共培养和单独培养模式下,培养液上清雌激素浓度和肿瘤细胞芳香化酶的表达情况。用细胞因子芯片筛选2种模式下差异性表达的细胞因子,并用real-time PCR进行验证。结果:成功提取肿瘤间质细胞(波形蛋白为阳性)。共培养组上清雌激素浓度和肿瘤细胞中芳香化酶mRNA表达量均高于单独培养组(P<0.01)。共培养组上清中生长分化因子-15(GDF-15)的表达高于单独培养组,10 ng/mL的GDF-15可显著上调子宫内膜癌细胞中芳香化酶的表达(P<0.01)。结论:子宫内膜癌细胞与间质细胞共培养情况下,子宫内膜癌细胞中芳香化酶的表达,进而促进间质细胞雌激素浓度的合成,其中子宫内膜癌细胞中GDF-15可能发挥重要作用。     

Expression of recombinant Schistosoma japonicum protein rSjE16 and its effects on LX-2 cells in vitro

ObjectiveThe activation of hepatic stellate cells (HSCs) is a key event in schistosome-induced liver fibrosis. Previous studies have shown that soluble egg antigens and the recombinant P40 protein from Schistosoma japonicum eggs inhibit HSC activation. In the present study, we observed the direct effect of the S. japonicum recombinant (r)SjE16 protein on HSCs.MethodsThe sequence of SjE16 was analyzed by bioinformatics. Then western blotting, quantitative PCR, and MTT assays were performed to observe the effects of rSjE16 on HSCs.ResultsThe SjE16 protein has no signal peptide or transmembrane region. rSjE16 significantly inhibited expression levels of α-smooth muscle actin and collagen I protein in LX-2 cells. rSjE16 also significantly increased the expression levels of interleukin (IL)-6 and IL-8, and enhanced the expression of matrix metalloproteinase (MMP)-2, MMP-9, and peroxisome proliferator-activated receptor-γ in LX-2 cells. LX-2 cell viability was not inhibited by rSjE16.ConclusionrSjE16 may be involved in the progression of HSC activation via a complex molecular mechanism, which requires further study to fully understand.     

MiR-125a regulates ovarian cancer proliferation and invasion by repressing GALNT14 expression

Increasing evidence has suggested that dysregulation of microRNAs (miRNAs) could contribute to tumor progression. The miR-125a was downregulated in several types of cancer, however, the molecular mechanism of miR-125a in the ovarian cancer remains unclear. The aim of the paper was to reveal the mechanism of miR-125a regulating cell proliferation and metastasis in ovarian cancer. In this study, western blotting, immunohistochemistry and serum-ELISA assay revealed that polypeptide N-acetylgalactosaminyl transferase 14 (GALNT14) expression was upregulated and correlated with the cancer stage in ovarian cancer. The expression levels of miR-125a were downregulated and negatively related to GALNT14 expression in clinical ovarian cancer tissues. Moreover, luciferase reporter assay identified polypeptide N-acetylgalactosaminyl transferase 14 (GALNT14) as a direct target of miR-125a, and overexpression of miR-125a markedly reduced the expression of GALNT14 in ovarian cancer. Functional characterization of miR-125a was accomplished by reconstitution of miR-125a and silencing GALNT14 expression in ovarian cancer cells to determine changes in proliferation and invasion. The MTT assay and transwell assay revealed that miR-125a transfectant significantly inhibits cell proliferation and invasion, by repressing GALNT14 expression. Furthermore, the gelatin zymography assay miR-125a mimics and GALNT14 siRNA suppressed the activity of MMP2 and MMP9. Taken together, our findings show that miR-125a functions as tumor suppressor in ovarian cancer by targeting GALNT14, and miR-125a may therefore serve as a biomarker for diagnosis and therapeutics in ovarian cancer.     

High expression of DHX9 promotes the growth and metastasis of hepatocellular carcinoma

BackgroundDHX9, an NTP‐dependent RNA helicase, is closely associated with the proliferation and metastasis of some tumor cells and the prognosis of patients, but its role in hepatocellular carcinoma (HCC) is not well‐known. This study was performed to explore the expression and role of DHX9 in HCC.MethodsThe expression of DHX9 in HCC tissues and cell lines was detected by TCGA database, qPCR, western blotting, and immunohistochemistry. The relationship between the DHX9 expression level and the prognosis of patients with HCC was accessed. Then, the function of DHX9 knockdown in HCC cells was examined by CCK‐8, scratch, Transwell, and apoptosis assays. Epithelial‐mesenchymal transition (EMT) was detected by western blotting.ResultsDHX9 was highly expressed in HCC tissues by analyzing both TCGA database and clinical samples. High DHX9 expression level was associated with TNM stage, vascular invasion and metastasis of HCC patients, and was an independent adverse prognostic factor. DHX9 knockdown significantly inhibited cell proliferation, migration, invasion and EMT and increased cell apoptosis in HCC cells.ConclusionOur findings suggest that DHX9 participates in the progression of HCC as an oncogene and may be a potential target for the clinical diagnosis and therapy of HCC.     

CXCL5 limits macrophage foam cell formation in atherosclerosis

The ELR⁺-CXCL chemokines have been described typically as potent chemoattractants and activators of neutrophils during the acute phase of inflammation. Their role in atherosclerosis, a chronic inflammatory vascular disease, has been largely unexplored. Using a mouse model of atherosclerosis, we found that CXCL5 expression was upregulated during disease progression, both locally and systemically, but was not associated with neutrophil infiltration. Unexpectedly, inhibition of CXCL5 was not beneficial but rather induced a significant macrophage foam cell accumulation in murine atherosclerotic plaques. Additionally, we demonstrated that CXCL5 modulated macrophage activation, increased expression of the cholesterol efflux regulatory protein ABCA1, and enhanced cholesterol efflux activity in macrophages. These findings reveal a protective role for CXCL5, in the context of atherosclerosis, centered on the regulation of macrophage foam cell formation.     

子宫内膜癌组织中趋化因子CXCL12及其受体CXCR4表达水平研究

目的 观察子宫内膜癌组织中趋化因子CXCL12及其受体CXCR4表达水平,探讨其与子宫内膜癌患者临床病理特征相关性。方法 收集2012年1月～2014年12月间入院接受手术的子宫内膜癌患者52例病理标本,年龄55.6±19.2岁,以正常子宫内膜26例为对照组,年龄52.3±16.5岁。采用免疫组化技术检测趋化因子CXCL12及其受体CXCR4在子宫内膜癌组织中的表达情况,并分析它们与FIGO分期、细胞分化程度、淋巴结转移和病理类型等临床病理特征的关系。结果 CXCL12和CXCR4在子宫内膜癌组织中阳性表达率分别为76.92%和69.23%,而在正常子宫内膜组织中则分别为34.62%和30.77%,差异具有统计学意义(χ²=11.826,P＜0.01)。CXCR4的表达与子宫内膜癌细胞分化程度存在差异,有统计学意义(均P＜0.05),且与分化高低呈正相关(r=0.386,P＜0.05)。在子宫内膜癌组织中,除CXCR4表达与细胞分化程度呈正相关外,CXCL12和CXCR4表达与临床分期、淋巴结转移和病理类型无相关性(P＞0.05)。结论 CXCL12和CXCR4在子宫内膜癌组织中表达明显升高,这可能在子宫内膜癌发生发展过程中发挥重要生物学作用。     

CCL5 secreted by tumor associated macrophages may be a new target in treatment of gastric cancer

AimTo investigate the role of CCL5 secreted by tumor associated macrophages (TAMs) in gastric cancer, and to explore how CCL5/CCR5 axis modulates phenotypes of gastric cancer cells.MethodsExpression of CCL5 and TAM surface marker CD68 in gastric cancer tissues was examined using SP immunohistochemistry. Serum CCL5 levels of patients were assessed using ELISA. Cross-analyses of CCL5 and CD68 expression with clinicopathological data were done. Correlation between CCL5 and CD68 in gastric cancer tissues was also studied. In vitro functional characterization of CCL5 in gastric cancer was done in co-culture of AGS and THP-1 derived macrophages using MTS assay, plate clone formation assay, and transwell experiment. Expression of chemokines and its receptors were detected by RT-PCR, while Stat3 phosphorylation and downstream target proteins were studied using western blot.ResultsCCL5 and CD68 were both highly expressed in tissues gastric cancer, of which the expressions were positively correlated with each other, and of clinical importance, were associated with the depth of invasion, lymph node metastasis, TNM staging and tumor differentiation. Serum CCL5 was also elevated in patients with gastric cancer comparing to healthy volunteers. Co-culture of AGS cells with THP-1 derived macrophages increased cell proliferation, clone forming ability as well as migration of AGS cells. Migration of AGS cells across transwell membrane was also enhanced by increasing exogenous CCL5. Meanwhile, mRNA expression of CCL5, MMP2, MMP9, and CCR5 was also highly expressed in the cells. Stat3 signaling as reflected by its phosphorylation was also increased in AGS cells upon co-culture with THP-1 derived macrophages.ConclusionCCL5 secreted by TAMs may promote the proliferation, invasion and metastasis of gastric cancer cells, in which Stat3 signaling pathway is likely to play an important role. The correlation of CCL5 with clinicopathological parameters suggested CCL5 holds promise as important molecular marker of gastric cancer staging and disease progression.     

Soluble C-X-C chemokine ligand 16 levels are increased in gout patients

ObjectivesSoluble C-X-C chemokine ligand 16 (CXCL16) was shown to recruit polymorphonuclear cells into synovial tissue in gout patients. The aim of this study was to explore the pathophysiological characteristics of CXCL16 in gout patients with or without chronic kidney disease (CKD).Design and methods42 gout patients, 22 CKD and 20 healthy subjects were enrolled. Plasma CXCL16 and other biochemical parameters were tested.ResultsPlasma CXCL16 levels in gout subjects with CKD were significantly increased compared with healthy, CKD and gout subjects without CKD. Soluble CXCL16 levels in gout subjects were closely correlated with renal function and lipid profiles, and independently associated with 24 h proteinuria, creatinine clearance rate and C-reactive protein.ConclusionOur data indicated that plasma CXCL16 levels are significantly increased in gout patients with and without CKD, and are independently associated with renal function. Elucidating the pathophysiologcial role of CXCL16 in gout patients requires further study.     

Circular RNA circ_0010235 sponges miR-338-3p to play oncogenic role in proliferation,migration and invasion of non-small-cell lung cancer cells through modulating KIF2A

BackgroundCircular RNA microarray analysis showed hsa_circ_0010235 (circ_0010235) was highly upregulated in non-small-cell lung cancer (NSCLC) patients; however, its role in carcinogenesis and development of NSCLC cells was unrevealed. Here, we intended to investigate role and mechanism of circ_0010235 in NSCLC proliferation, migration and invasion.Methods and resultsExpression of circ_0010235, microRNA (miR)-338-3p and kinesin family member 2A (KIF2A) was detected by quantitative real-time PCR, western blotting and immunohistochemistry (IHC). Cell progression was measured by cell-counting kit-8 assay, 5-ethynyl-2-deoxyuridine (EdU) assay, flow cytometry, transwell assay, western blotting, IHC and xenograft experiment. The relationship among circ_0010235, miR-338-3p and KIF2A was determined by dual-luciferase reporter assay, RNA immunoprecipitation and Pearson’s correlation analysis. Expression of circ_0010235 was increased in human NSCLC tissues and cells, accompanied with miR-338-3p downregulation and KIF2A upregulation. Essentially, circ_0010235 could sponge miR-338-3p via target binding, and miR-338-3p downstream targeted KIF2A. Functionally, exhaustion of circ_0010235 induced apoptosis rate of NSCLC cells and curbed cell viability, EdU incorporation, migration rate and invasion rate, accompanied with higher E-cadherin and lower N-cadherin expression. Additionally, re-expression of miR-338-3p prompted above similar effects in NSCLC cells in vitro. Contrarily, miR-338-3p blockage partially counteract the effects of circ_0010235 exhaustion; plus, restoration of KIF2A could attenuate miR-338-3p role, as well. Notably, interfering circ_0010235 delayed tumour growth of NSCLC cells by promoting miR-338-3p and E-cadherin expression, and depressing KIF2A, ki-67 and N-cadherin expression.Conclusionscirc_0010235 could be a novel identified oncogenic circRNA in NSCLC, and targeting miR-338-3p/KIF2A axis was one regulatory mechanism underlying circ_0010235.

KEY MESSAGE

• Circ_0010235 was an upregulated circRNA in NSCLC patients and cells.
• Interfering circ_0010235 restrained NSCLC cell proliferation and metastasis in vitro and in vivo.
• miR-338-3p per se suppressed NSCLC in vitro and its downregulation diminished the tumour-suppressive role of circ_0010235 blockage in NSCLC cells.
• miR-338-3p could downstream target KIF2A and be sponged by circ_0010235.

     

CXCL4L1 inhibits angiogenesis and induces undirected endothelial cell migration without affecting endothelial cell proliferation and monocyte recruitment

Summary. Background and Objectives: The non‐allelic variant of CXCL4/PF4, CXCL4L1/PF4alt, differs from CXCL4 in three amino acids of the C‐terminal α‐helix and has been characterized as a potent anti‐angiogenic regulator. Although CXCL4 structurally belongs to the chemokine family, it does not behave like a ‘classical’ chemokine, lacking significant chemotactic properties. Specific hallmarks are its angiostatic, anti‐proliferative activities, and proinflammatory functions, which can be conferred by heteromer‐formation with CCL5/RANTES enhancing monocyte recruitment. Methods and Results: Here we show that tube formation of endothelial cells was inhibited by CXCL4L1 and CXCL4, while only CXCL4L1 triggered chemokinesis of endothelial cells. The chemotactic response towards VEGF and bFGF was attenuated by both variants and CXCL4L1‐induced chemokinesis was blocked by bFGF or VEGF. Endothelial cell proliferation was inhibited by CXCL4 (IC₅₀ 6.9 μg mL^?1) but not by CXCL4L1, while both chemokines bound directly to VEGF and bFGF. Moreover, CXCL4 enhanced CCL5‐induced monocyte arrest in flow adhesion experiments and monocyte recruitment into the mouse peritoneal cavity in vivo, whereas CXCL4L1 had no effect. CXCL4L1 revealed lower affinity to CCL5 than CXCL4, as quantified by isothermal fluorescence titration. As evidenced by the reduction of the activated partial thromboplastin time, CXCL4L1 showed a tendency towards less heparin‐neutralizing activity than CXCL4 (IC₅₀ 2.45 vs 0.98 μg mL^?1). Conclusions: CXCL4L1 may act angiostatically by causing random endothelial cell locomotion, disturbing directed migration towards angiogenic chemokines, serving as a homeostatic chemokine with a moderate structural distinction yet different functional profile from CXCL4.