CXCR4 mRNA Research Articles

Effect and Mechanism of Cxcr4 Gene-Modified BMSC-Derived Exosomes on Aplastic Anemia

To explore the improvement effect of CXC chemokine receptor 4 (Cxcr4) gene-modified bone marrow mesenchymal stem cell (BMSC)-derived exosomes on aplastic anemia (AA), and make a preliminary exploration of the mechanism. Mouse BMSCs were isolated and cultured, then infected by recombinant lentivirus carrying Cxcr4 gene. The expression of green fluorescence was observed through fluorescence microscope, the expression of Cxcr4 mRNA was detected by real-time fluorescence quantitative PCR, and the BMSC-derived exosomes modified with Cxcr4 gene were extracted. Mouse models of AA were constructed, and control group, model group (AA), AA+BMSC group, AA+NC-BMSC group, AA+Cxcr4-BMSC group were set up. Except control group and model group, the other three groups of mice were injected 400 μl exosomes from different sources via the tail vein, after 2 weeks, the routine blood indices and the number of bone marrow nucleated cells were detected, the pathological changes of bone marrow were observed by HE staining, and the expression level of Treg cells was detected by flow cytometry. Mouse BMSCs were successfully isolated, and BMSCs with high expression of Cxcr4 and their exosomes were obtained. Compared with the control group, the number of red blood cell (RBC), white blood cell (WBC), and platelet (PLT), the hemoglobin (Hb) content and proportion of Treg cells in the peripheral blood of mice in the model group significantly decreased (P<0.01), as well as the number of bone marrow nucleated cells (P<0.01). The proliferation level of nucleated cells was low, and the medullary cavity was filled with a large number of fat cells. Compared with the model group, the number of RBC, WBC, PLT, the Hb content and proportion of Treg cells in the peripheral blood of mice in the AA+BMSC group, AA+NC-BMSC group, and AA+Cxcr4-BMSC group significantly increased (P<0.01), as well as the number of bone marrow nucleated cells (P<0.01), and pathological changes of bone marrow were improved. In addition, the number of RBC, WBC, PLT, the Hb content and proportion of Treg cells in the peripheral blood of mice in the AA+Cxcr4-BMSC group were significantly higher than those in the AA+BMSC group (P<0.01), as well as the number of bone marrow nucleated cells (P<0.01). Injection of Cxcr4 gene-modified BMSC-derived exosomes has a certain improvement effect on AA mice, and the mechanism may be related to an increase of the proportion of Treg cells.

CXCR7 Promotes Adipogenesis and Insulin Resistance in Human Visceral Adipocytes

Obesity is a multifactorial chronic condition that increases the risk of cardiovascular diseases, type II diabetes, and certain types of cancer. It has been known that some chemokines and their receptors facilitate the development of obesity, leading to other related comorbidities. However, the exact role of atypical chemokine receptor CXCR7 in obesity and obesity‐induced type II diabetes remains unknown. We aimed to study whether CXCR7 promotes adipogenesis and insulin resistance in human visceral adipocytes in vitro. Using RT‐PCR and flow cytometry studies, we found that both CXCR7 mRNA and its protein expressions were significantly upregulated during the differentiation of human visceral adipocytes. Stimulation of CXCR7 by SDF‐1 or LIH383 (a CXCR7‐selective peptide ligand) increased lipid droplets formation during adipocytes differentiation. In addition, we found that SDF‐1 or LIH383 treatment inhibited AKT phosphorylation in a dose‐dependent manner with no effect on ERK1/2 and JNK signaling pathways, which indicates a selective suppression of the AKT signaling axis by CXCR7. Furthermore, activation of CXCR7 by SDF‐1 inhibited insulin signaling to the AKT pathway and insulin‐induced glucose uptake. In contrast, ablation of CXCR7 by CRISPR/Cas9 system restored AKT signaling and enhanced insulin sensitivity in human visceral adipocytes. We concluded that CXCR7 plays a critical role in human visceral adipogenesis and insulin resistance by suppressing the AKT signaling pathway, highlighting CXCR7 as a potential new drug target against obesity and type II diabetes.

A targeting delivery system for effective genome editing in leukemia cells to reverse malignancy

Therapy resistance associated with relapse is a main cause of death in acute myeloid leukemia (AML). To address this issue, a dual-targeting CRISPR-Cas9 genome editing nanosystem was constructed for CXCR4 knockout to reverse the malignancy of leukemia cells. The surface of the dual-targeting nanosystem is composed of MUC1 specific aptamer incorporated alginate (MUC1 aptamer-alginate) and T22-NLS peptide with T22 sequence targeting CXCR4; the core of the nanosystem consists of protamine complexed with CRISPR-Cas9 plasmid. The in vitro study shows that the nanosystem mediated genome editing induces cell apoptosis, cell cycle arrest, as well as inhibited cell migration and adhesion in edited THP-1 cells after CXCR4 knockout. Further, the unprocessed peripheral blood from acute myeloid leukemia (AML) patients was directly used to carry out ex vivo study. The results show the genome editing nanosystem can effectively knock out CXCR4 in leukemia cells, leading to attenuated CXCR4 protein as studied by antibody labeling and reduced CXCR4 mRNA as probed by a molecular beacon delivery system. In addition to developing a promising delivery vector for gene therapy on AML, this study also provides an effective strategy to evaluate the therapeutic efficiency of particular treatments by peripheral blood-based ex vivo studies.

Elevated Plasma Levels of CXCL13 Chemokine in Saudi Patients With Asthma Exacerbation.

Background: Bronchial asthma is a lung disorder characterized by chronic allergic inflammation of the airways, and several of the immune and non-immune cells contribute to asthma's pathogenicity. B-cell activation plays an essential role in developing allergic inflammation in the lungs. CXCL13 is a potent B-cell chemoattractant chemokine, which has a crucial role in the recruitment and trafficking of B cells after interaction with its receptor CXCR5. This study is aimed to evaluate plasma levels of CXCL13 and its receptor CXCR5 in Saudi patients with asthma exacerbation relative to healthy controls.Methods: A total of 23 patients with asthma exacerbation and 20 healthy controls participated in this study. Total immunoglobulin E (IgE) and CXCL13 protein levels were measured in the plasma of patients with asthma exacerbations and healthy controls by specific enzyme-linked immunosorbent assay (ELISA). Gene expression mRNA for CXCR5 was measured using real-time polymerase chain reaction (RT-PCR).Results: Total IgE protein concentrations were elevated significantly in asthma exacerbation patients than that in healthy controls. CXCL13 protein levels were increased significantly in the asthma group relative to healthy controls. In addition, CXCR5 mRNA levels were elevated significantly in the asthma group than in the healthy controls.Conclusions: Measurement of CXCL13 and CXCR5 may be used as an additional biomarker of asthma exacerbation, and targeting CXCL13 or its receptor may be used as new treatment options in asthma.

Circular RNA circFGFR1 Functions as an Oncogene in Glioblastoma Cells through Sponging to hsa-miR-224-5p.

Recently, increased studies have shown the important regulatory role of circular RNA (circRNA) in cancer progression and development, including glioblastoma (GBM). However, the function of circRNAs in glioblastoma is still largely unclear. Here, we state that circFGFR1 is elevated in glioma cells, resulting in aggravated glioma aggravated malignancy. The upregulation of circFGFR1 also promotes glioma growth in mouse xenograft models. Furthermore, CXCR4 level in glioma cells is positively correlated with circFGFR1 level, and higher CXCR4 expression is found in circFGFR1 overexpression groups. The effect of circFGFR1 on glioma malignancy is abolished in CXCR4 knockout cells. Then, RIP, RNA pull-down, and luciferase reporter assay results showed that hsa-miR-224-5p directly binds to circFGFR1 and CXCR4 mRNA. The CXCR4 3′-untranslated region (UTR) activated luciferase activity was reduced with hsa-miR-224-5p transfection, while it is reversed when cotransfected with circFGFR1, indicating that circFGFR1 acts as a hsa-miR-244-5p sponge to increase CXCR4 expression. The hsa-miR-224-5p expression is negatively corrected with the glioma malignancy through inhibiting CXCR4 level. Besides, the circFGFR1-induced regulation in glioma malignancy is also abrogated in hsa-miR-224-5p knockout cells. Taken together, our findings suggest that circFGFR1 plays a critical role in the tumorigenic behaviors in glioma cells by upregulating CXCR4 expression via sponging to hsa-miR-224-5p. These findings provide a new perspective on circRNAs during GBM development.

High Expression of CXCL10/CXCR3 in Ventilator-Induced Lung Injury Caused by High Mechanical Power.

Background The energy delivered by a ventilator to the respiratory system in one minute is defined as mechanical power (MP). However, the effect of ventilator-induced lung injury (VILI) in patients suffering from acute respiratory distress syndrome (ARDS) is still unknown. Our previous studies revealed that CXCL10 may be a potential biomarker of lung injury in ARDS. Therefore, the aim of this study was to compare the lung injury of rats and patients under different MP conditions to explore the involvement of CXCL10 and its receptor CXCR3 in VILI. Methods Patients were divided into the high mechanical power group (HMPp group) and low mechanical power group (LMPp group), while rats were assigned to the high mechanical power group (HMPr group), medium mechanical power group (MMPr group), and low mechanical power group (LMPr group). CXCL10 and CXCR3 plasma content in ARDS patients and rats under ventilation at different MP was measured, as well as their protein and mRNA expression in rat lungs. Results CXCL10 and CXCR3 content in the plasma of ARDS patients in the HMPp was significantly higher than that in the LMPp. The increase of MP during mechanical ventilation in the rats gradually increased lung damage, and CXCL10 and CXCR3 levels in rat plasma gradually increased with the increase of MP. CXCL10 and CXCR3 protein and mRNA expression in the HMPr group and MMPr group was significantly higher than that in the LMPr group (P < 0.05). More mast cells were present in the trachea, bronchus, blood vessels, and lymphatic system in the rat lungs of the HMPr group, and the number of mast cells in the HMPr group (13.32 ± 3.27) was significantly higher than that in the LMPr group (3.25 ± 0.29) (P < 0.05). Conclusion The higher the MP, the more severe the lung injury, and the higher the CXCL10/CXCR3 expression. Therefore, CXCL10/CXCR3 might participate in VILI by mediating mast cell chemotaxis.

Regulation of CXCR4 Expression by Taurine in Macrophage-Like Cells.

Taurine (2-aminoethanesulfonic acid) is a free β-amino acid found at high concentrations in many mammalian tissues. Taurine plays a role in several essential biological processes, including anti-oxidation, anti-inflammation, and osmoregulation. However, its regulatory mechanisms, especially at the genetic and molecular levels, have not been elucidated. Here, we targeted immune-related genes and investigated the effects of taurine on immune-related gene expression in macrophage-like cells. J774.1 cell line was used, and the effect of taurine on mRNA expression of immune-related genes such as cytokines, their receptors, and toll-like receptors was examined. Among these, taurine significantly increased the mRNA levels of C-X-C chemokine receptor 2 (CXCR2), chemokine receptor. Furthermore, we found that the taurine-induced increase in CXCR4 mRNA levels was higher than that in CXCR2 mRNA levels. Taurine increased both mRNA and protein expression levels of CXCR4. Additionally, we examined the effects of taurine analogs, including hypotaurine, β-alanine, and γ-aminobutyric acid (GABA). While GABA increased the mRNA expression of CXCR4, hypotaurine slightly increased this expression, and β-alanine had no effect, although these taurine analogs are the substrates of taurine transporter. These findings demonstrate that taurine specifically affects CXCR4 mRNA expression in macrophage-like cells.

Implication of microglia in ketamine-induced long-term cognitive impairment in murine pups.

Ketamine, a non-competitive N-methyl-D-aspartate receptor (NMDAR) antagonist, is widely applicable to anesthesia, analgesia, and sedation. However, the function and mechanisms of ketamine in the long-term learning and memory function of neonatal mice are unclear. The present study aims to investigate whether long-term learning and memory function will be affected by multiple ketamine exposures in the early development period. The mRNA and protein levels were measured by RT-qPCR and western blot, respectively. The Morris Water Maze test was performed to assess spatial learning and memory. We identified that neonatal exposure to ketamine downsized the positive neurons for microtubule-associated protein doublecortin (DCX) and Ki67 in hippocampal dentate gyrus at the juvenile and late adolescence stages. Double-labeling tests demonstrated that the counts of Iba1+ cells and Ki67+ cells were pronouncedly diminished with exposure to ketamine. Further, qPCR assays to screen the key factors predisposing the populations and maturation of microglia exhibited remarkable decline of CX3CR1 mRNA levels in ketamine group versus the control group. The close relation of microglia to synaptic plasticity was depicted by the significantly downregulated synaptic plasticity-related proteins NR2B and PSD-95 subsequent to multiple exposures to ketamine. Finally, we found that both the protein and mRNA levels of BDNF were markedly decreased in ketamine group versus the control group. We found that multiple exposures to ketamine in neonatal mice lead to spatial learning and memory dysfunction. The alterations of microglial development and function are the possible mechanisms of long-term learning and memory impairment.

Secreted Protein Acidic and Rich in Cysteine (SPARC) and Chemokine’s mRNA expression in Endometrial Cancer versus Normal Endometrium: A Possible Pattern of Haematogenous and Lymphovascular Metastases

Objectives: Secreted Protein Acidic and Rich in Cysteine (SPARC) is a multifunctional glycoprotein, participating in tissue remodeling, morphogenesis and bone mineralization. Furthermore, SPARC controls important mechanisms involved in cancer progression, including angiogenesis regulation. However, in some studies SPARC was found to show tumor suppression while in other a protumorigenic and prometastatic action. In tumor microenvironment some chemokines and their receptors, thanks to their ability to modulate cancer cells migration and proliferation, are involved in the angiogenetic and metastatic process. In this study we compared, in human endometrial cancer tissue (EC) vs normal endometrium counterpart (NE), SPARC with CXCL12, CXCL11, CXCL8, and CXCR7 mRNA expression. Material and Methods: Fresh specimens from 15 patients with EC and corresponding NE were stored at –80°. One mcg of mRNA was reverse-transcribed in cDNA. A Real-Time PCR determined relative cDNA levels of targeted gene mRNA. Results: In EC vs NE, we observed down-regulation of SPARC mRNA in 91% (P&lt;.05), down regulation of CXCL12 mRNA in 91% (P&lt;.001), and down-regulation of CXCR7 mRNA in 91% (P&lt;.001). In EC, SPARC mRNA down-regulation was directly related in 100% of samples to CXCL12 and CXCR7 (P&lt;.001) and in 73% of samples to VEGF (P&lt;.03). Conclusion: In endometrial cancer, under expression of SPARC is directly related to CXCL12 and CXCR7 and this result might be consistent with a SPARC function on tumor progression and invasion mediated by CXCL12 and CXCR7 on blood and lymphatic spread, respectively.

Interleukin-1 receptor antagonist inhibits metastatic potential by down-regulating CXCL12/CXCR4 signaling axis in colorectal cancer

BackgroundThe aim of this study was to investigate the co-operative role of CXCR4/CXCL12 axis and IL-1Ra in metastatic processes mechanism by interactions between colorectal cancer cells and stromal cells in their microenvironment.MethodsExpression of IL-1α, interleukin-1 receptor type I (IL-1 RI), CXCL12 and CXCR4 mRNA and proteins were determined by RT-PCR and Western blot. The effect of secreted level of CXCL12 by IL-1Ra on fibroblasts was measured by ELISA. CXCL12 regulate metastatic potential of colorectal cancer was evaluated by proliferation, invasion and angiogenesis assays, respectively, in which invasion and angiogenesis assays used an in vitro system consisting of co-cultured colorectal cells and stromal cells.ResultsIL-1α was expressed in high liver metastatic colorectal cancer cell lines (HT-29 and WiDr). The colorectal cancer cell-derived IL-1α and rIL-1α significantly promoted CXCL12 expression by fibroblasts, and this enhancing effect can be significantly inhibited by IL-1Ra (P < 0.01). CXCL12 not only enhanced the migration and proliferation of human umbilical vein endothelial cells, but also significantly enhanced angiogenesis (P < 0.01). Furthermore, the high liver-metastatic colorectal cancer cell line (HT-29), which secretes IL-1α, significantly enhanced angiogenesis compared to the low liver-metastatic cell line (CaCo-2), which does not produce IL-1α (P < 0.01). On the contrary, IL-1Ra can significantly inhibit migration, proliferation and angiogenesis (P < 0.01).ConclusionAutocrine IL-1α and paracrine CXCL12 co-enhances the metastatic potential of colorectal cancer cells; IL-1Ra can inhibit the metastatic potential of colorectal cancer cells via decrease IL-1α/CXCR4/CXCL12 signaling pathways.Graphical 7Qx8An2hePDujdT1wSZV-LVideo

Cellular detection of the chemokine receptor CXCR4 in bovine mammary glands and its distribution and regulation on bovine leukocytes

Mastitis has a high incidence in dairy cows. Experimental infection with Escherichia coli increased the number of leukocytes in milk and the gene expression of the chemokine receptor CXCR4 in mammary gland tissues. A link between CXCR4 expression and lipopolysaccharide sensing was demonstrated in other species using in vitro models. The receptor that binds the chemokine stomal cell-derived factor 1 might be associated with the inflammatory response in bovine mammary glands. However, studies in cows are rare, and data on the localization of CXCR4 in bovine mammary glands and its distribution in bovine leukocytes are lacking. Fatty acids (FA) affect the inflammatory response. In human peripheral blood monocytes, exposure to conjugated linoleic acids (CLA) decreases the expression of CXCR4, leading to a decreased inflammatory response in these cells. In this study, we analyzed the expression of CXCR4 in the mammary glands of dairy cows by immunohistochemistry (n = 5) and laser capture microdissection followed by qualitative PCR (n = 3). We characterized the surface expression of CXCR4 on bovine leukocytes, including monocyte subpopulations, first by flow cytometry (n = 5) and then confirmed these results by Western blotting (n = 3). Rumen fistulated dairy cows (n = 4; 126 ± 4 d in milk) were fitted with abomasal infusion tubes, arranged in a 4 × 4 Latin square design, and supplemented for 6 wk twice daily with rising doses of FA followed by a 3-wk washout period. Then, CXCR4 expression on leukocytes was analyzed. The cows received a corn-based diet and were supplemented with coconut oil delivering medium-chain FA (38 g/d), linseed-safflower oil mix delivering n-3 FA (EFA, 39 g of linseed oil and 2 g of safflower oil per day), Lutalin (cis-9,trans-11 and trans-10,cis-12 CLA, 5 g/d; BASF), and EFA + CLA. In the bovine mammary gland, the epithelial cells of the lactiferous duct, but not alveolar epithelial cells, showed clear CXCR4 protein and mRNA signals. Among the leukocyte subsets, monocytes displayed the highest percentage of CXCR4-positive cells (87%), whereas circulating neutrophils showed almost no CXCR4 surface expression (3%) but stored the receptor intracellularly. The percentage of CXCR4-positive leukocytes was not affected by the different FA supplements, but FA supplementation reduced the receptor abundance per cell (40% on average). In conclusion, CXCR4 was clearly detected in the lactiferous duct cells of the mammary gland but not in the alveolar epithelial cells. Compared with other leukocytes, bovine monocytes showed the highest signal intensity of CXCR4 on their surface, whereas granulocytes stored CXCR4 intracellularly. Supplementation with all the FA reduced the surface expression of CXCR4 per leukocyte and could therefore potentially affect the inflammatory status associated with the surface expression of CXCR4. The importance of our observations should be verified in cows with mastitis in the future.

Co-culture of mesenchymal stem cell spheres with hematopoietic stem cells under hypoxia: a cost-effective method to maintain self-renewal and homing marker expression.

Hematopoietic stem cell (HSC) transplantation is considered a possible treatment option capable of curing various diseases. The aim of this study was the co-culturing of mesenchymal stem cell (MSC) spheres with HSCs under hypoxic condition to enhance the proliferation, self-renewal, stemness, and homing capacities of HSCs. HSCs were expanded after being subjected to different conditions including cytokines without feeder (Cyto), co-culturing with adherent MSCs (MSC), co-culturing with adherent MSCs + hypoxia (MSC + Hyp), co-culturing with MSCs spheres (Sph-MSC), co-culturing with MSCs spheres + hypoxia (Sph-MSC + Hyp), co-culturing with MSC spheres + cytokines (Sph-MSC + Cyto). After 10days, total nucleated cell (TNC) and CD34+/CD38- cell counts, colony-forming unit assay (CFU), long-term culture initiating cell (LTC-IC), the expression of endothelial protein C receptor (EPCR), nucleostemin (NS), nuclear factor I/X (Nfix) CXCR4, and VLA-4 were evaluated. The TNC, CD34+/CD38- cell count, CFU, and LTC-IC were higher in the Sph-MSC + Hyp and Sph-MSC + Cyto groups as compared with those of the MSC + Hyp group (P < 0.001). The expanded HSCs co-cultured with MSC spheres in combination with hypoxia expressed more EPCR, CXCR4, VLA-4, NS, and Nfix mRNA. The protein expression was also more up-regulated in the Sph-MSC + Cyto and Sph-MSC + Hyp groups. Co-culturing HSCs with MSC spheres under hypoxic condition not only leads to higher cellular yield but also increases the expression of self-renewal and homing genes. Therefore, we suggest this approach as a simple and non-expensive strategy that might improve the transplantation efficiency of HSCs.

MicroRNA-33b replacement effect on growth and migration inhibition in ovarian cancer cells.

Ovarian cancer is a devastating gynecological disease which is considered the major cause of cancer fatality around the world. The down-regulation of microRNA-33b (miR-33b) was reported in some malignancies. Hence, we transfected the miR-33b mimic into SKOV3 cells and evaluated the impacts of this interference on the growth and migration repression of these tumor cells as well as on targeted genes expression. In our study, transfecting the miR-33b mimic and inhibitor, negative control (NC), and NC inhibitor were established using Lipofectamine 2000. The cytotoxic effects of miR-33b were evaluated by MTT. To assess the miR-33b effects on cell migration, a scratching test was applied. The expression levels of miR-33b, ADAMTS, C-Myc, MMP9, K-Ras, and CXCR4 were evaluated using qRT-PCR. These findings indicate that transfection of miR-143mimic had no marked effects on the SKOV3 cell line. As expected, miR-33b relative expression levels were as follows: miR-33b mimic >NC and NC inhibitor >miR-33b inhibitor (p<0.01). Moreover, transfected miR-33b mimic could suppress SKOV3 cells' proliferation, whereas transfected miR-33b inhibitor could promote cell proliferation (p<0.01). MiR-33b overexpression significantly down-regulated the MMP9, CXCR-4, c-Myc, ADAMTS, and K-Ras mRNA levels (p<0.05). As expected, these results confirm the tumor-suppressive effect of miR-33b in the SKOV3 ovarian cancer cell line by reducing cell survival, proliferation, and migration.

Expansion of cord blood stem cells in fibronectin-coated microfluidic bioreactor

BackgroundHematopoietic stem/progenitor cell transplantation is the main treatment option for hematological malignancies and disorders. One strategy to solve the problem of low stem cell doses used in transplantation is pre-transplant expansion. We hypothesized that using fibronectin-coated microfluidic channels would expand HSPCs and keep self-renewal potential in a three-dimensional environment, compared to the conventional method. We also compared stem cell homing factors expression in microfluidic to conventional cultures. Materials and methodsA microfluidic device was created and characterized by scanning electron microscopy. The CD133+ cells were collected from cord blood and purified. They were subsequently cultured in 24-well plates and microfluidic bioreactor systems using the StemSpan serum-free medium. Eventually, we analyzed cell surface expression levels of the CXCR4 molecule and CXCR4 mRNA expression in CD133+ cells cultured in different systems. ResultsThe expansion results showed significant improvement in CD133+ cell expansion in the microfluidic system than the conventional method. The median expression of the CXCR4 in the expanded cell was lower in the conventional system than in the microfluidic system. The CXCR4 gene expression up-regulated in the microfluidic system. ConclusionUtilizing microfluidic systems to expand desired cells effectively is the next step in cell culture. Comparative gene expression profiling provides a glimpse of the effects of culture microenvironments on the genetic program of HSCs grown in different systems.

Acute ischemic stroke triggers a cellular senescence-associated secretory phenotype

Senescent cells are capable of expressing a myriad of inflammatory cytokines and this pro-inflammatory phenomenon is known as senescence-associated secretory phenotype (SASP). The contribution of this phenomenon in brain ischemia was scarce. A mouse model of transient focal cerebral ischemia by compressing the distal middle cerebral artery (tMCAo) for 60 min was used. SASP, pro-inflammatory cytokines and cell cycle mRNAs levels were quantified at 30-min and 72 h post-surgery. Immunohistochemistry in paraffin embedded human brain slides and mouse brain tissue was performed. Our results showed an increase of both p16 and p21 mRNA restricted to the infarct area in the tMCAo brain. Moreover, there was an induction of Il6, Tnfa, Cxc11, and its receptor Cxcr2 mRNA pro-inflammatory cytokines with a high positive correlation with p16/p21 mRNA levels. The p16 was mainly shown in cytoplasm of neurons and cytoplasm/membrane of microglial cells. The p21 was observed in membrane of neurons and also it showed a mixed cytoplasmic and membranous pattern in the microglial cells. In a human stroke patient, an increase of P16 in the perimeter of the MCA infarct area was observed. These suggest a role of SASP in tMCAo mouse model and in human brain tissue. SASP potentially has a physiological role in acute ischemic stroke and neurological function loss.

Pretreatment with licochalcone a enhances therapeutic activity of rat bone marrow mesenchymal stem cells in animal models of colitis.

Objective(s):Colitis has a high prevalence rate, limited treatment options, and needs to be solved urgently. Application of Licochacone A (LA) or rBMMSCs alone in the treatment of colitis has a certain but limited effect. This study aims to develop an LA-based strategy to improve mesenchymal stem cells’ (MSCs’) therapeutic capacity in mice DSS-induced colitis by increasing the number of MSCs migrating to the inflammation site.Materials and Methods: In vivo, we injected MSCs pretreated with LA, MSCs alone, or PBS into the tail vein of colitis mice, and assessed the colon length, disease activity index (DAI) score, body weight, HAI score, and tracked the location of MSCs at day 10. In vitro, we knocked down the CXCR4 gene by siRNA and then treated it with LA, then tested the mRNA level of CXCR4 and the migration ability of group CXCR4, CXCR4+LA, LA, and control to verify the relationship between this effect and the SDF-1-CXCR4 signaling pathway.Results:The mice that received LA- pretreated MSCs had ameliorated body weight loss, preserved colon morphology, and decreased DAI and histological activity index (HAI) compared with the MSCs group. Besides, the number of MSCs migrating to the inflammation site significantly increased in group LA+MSCs, and expression of CXCR4 significantly increased too. Furthermore, we found that LA could partly revise the decrease of the migration of MSCs and the expression of CXCR4 mRNA caused by CXCR4-siRNA.Conclusion:LA may improve the migration ability of MSCs through increasing CXCR4 expression therapy enhancing their therapeutic activity.

Lipopolysaccharide-induced maternal immune activation modulates microglial CX3CR1 protein expression and morphological phenotype in the hippocampus and dentate gyrus, resulting in cognitive inflexibility during late adolescence

Inflammation during pregnancy can disturb brain development and lead to disorders in the progeny, including autism spectrum disorder and schizophrenia. However, the mechanism by which a prenatal, short-lived increase of cytokines results in adverse neurodevelopmental outcomes remains largely unknown. Microglia—the brain’s resident immune-cells—stand as fundamental cellular mediators, being highly sensitive and responsive to immune signals, which also play key roles during normal development.The fractalkine signaling axis is a neuron-microglia communication mechanism used to regulate neurogenesis and network formation. Previously, we showed hippocampal reduction of fractalkine receptor (Cx3cr1) mRNA at postnatal day (P) 15 in male offspring exposed to maternal immune activation induced with lipopolysaccharide (LPS) during late gestation, which was concomitant to an increased dendritic spine density in the dentate gyrus, a neurogenic niche. The current study sought to evaluate the origin and impact of this reduced hippocampal Cx3cr1 mRNA expression on microglia and cognition. We found that microglial total cell number and density are not affected in the dorsal hippocampus and dentate gyrus, respectively, but that the microglial CX3CR1 protein is decreased in the hippocampus of LPS-male offspring at P15. Further characterization of microglial morphology in the dentate gyrus identified a more ameboid phenotype in LPS-exposed offspring, predominantly in males, at P15. We thus explored maternal plasma and fetal brain cytokines to understand the mechanism behind microglial priming, showing a robust immune activation in the mother at 2 and 4 hrs after LPS administration, while only IL-10 tended towards upregulation at 2 hrs after LPS in fetal brains. To evaluate the functional long-term consequences, we assessed learning and cognitive flexibility behavior during late adolescence, finding that LPS affects only the latter with a male predominance on perseveration. A CX3CR1 gene variant in humans that results in disrupted fractalkine signaling has been recently associated with an increased risk for neurodevelopmental disorders. We show that an acute immune insult during late gestation can alter fractalkine signaling by reducing the microglial CX3CR1 protein expression, highlighting neuron-microglial fractalkine signaling as a relevant target underlying the outcomes of environmental risk factors on neurodevelopmental disorders.

MTT assay based inhibition analysis of A2780 cells proliferation using Mollugo nudicaulis Lam. extract with CXCR4 and HER2 expression.

It is of interest to document the inhibition of A2780 cell proliferation using Mollugo nudicaulis Lam.(M.nudicaulis) extract by MTT assay and by monitoring the CXCR4 and HER2 expression through RT-PCR analysis. Results shown that the n-hexane extract of M.nudicaulis have anticancer activity IC50 values of 32.46±0.92 µg/mL on A2780 cell lines. It is further found that the CXCR4 and HER2 mRNA and protein expression were significantly reduced in M.nudicaulis treated A2780 cell lines. Thus, the n-hexane extract of M.nudicaulis is a natural source of bioactive compounds as potential anticancer agents.

MiR-100 Suppresses the Proliferation, Invasion, and Migration of Hepatocellular Carcinoma Cells via Targeting CXCR7.

This study is to elucidate the functions of miR-100 in hepatocellular carcinoma progression and to explore the underlying mechanisms. Expression levels of miR-100 in normal-cancer hepatocellular carcinoma tissues were measured using quantitative real-time PCR (qRT-PCR). The invasive and proliferative abilities of hepatocellular carcinoma cell lines transfected with mimic-NC or mimic-miR-100 were measured using transwell, CCK-8, and colony formation assays. The binding sites between CXCR7 and miR-100 were determined using luciferase reporter assays. The correlation of CXCR7 and miR-100 in hepatocellular carcinoma progression was further confirmed by cotransfection assays. Our results showed that miR-100 was significantly lower expressed in hepatocellular carcinoma tissues and negatively associated with CXCR7 expression. Cell functional assays' results found that upregulation of miR-100 inhibited the proliferative, invasive, and migrative abilities in hepatocellular carcinoma cells. Luciferase reporter assay suggested that CXCR7 mRNA and miR-100 bound one another. Increasing CXCR7 expression reversed the inhibitive effects of upregulated miR-100 in hepatocellular carcinoma cells. Further study showed that miR-100/CXCR7 played a role in hepatocellular carcinoma progression by regulating metalloproteinase-2 (MMP2) and vascular endothelial growth factor (VEGF). Conclusively, miR-100 exerts antitumor effects on hepatocellular carcinoma. Overexpression of miR-100 attenuates the invasive and proliferative abilities of hepatocellular carcinoma cells by targeting CXCR7.

ASXL1 Mutation Change the Migration Ability in JAK2V617F Positive Cell Lines through SDF-1/CXCR4 Axis

ASXL1 plays a vital role in regulation of the histone methylation and acts as a tumor suppressor. Majority of ASXL1 mutations are frame-shift and nonsense mutations. The most frequent mutation is C.1934dupG (P.Gly646TrpfsX12) with a loss of PHD domain and truncated protein formed. Reduced normal ASXL1 protein expression promotes myeloid transformation. ASXL1 mutations change the transcription of certain oncogenes and result in dysfunction of tumor stem cells.We found that ASXL1 mutations were identified in 29.1% (32/110) patients with primary myelofibrosis (PMF). Moreover, JAK2V617F mutated patients with ASXL1 mutations were more likely to present splenomegaly (median LCM: 10 vs. 5 cm, P=0.023), while in JAK2V617F negative patients, the association was gone. There is evidence that SDF-1α/CXCR4 axis plays an important role in the adhesion of leukemic cells and bone marrow stromal cells. Abnormal CXCR4 expression may contribute to constitutive migration of CD34+ cells in PMF. Accordingly, we supposed that on the basis of JAK2V617F mutation, mutated ASXL1 acts on the SDF-1α/CXCR4 axis leading to the stem cell mobilization and splenomagly.To further investigate the molecular mechanism of ASXL1 mutation in patients with JAK2V617F, we performed short hairpin RNA (shRNA)-mediated stable knockdown of ASXL1 in JAK2V617F mutated UKE1 and SET2 cell lines and JAK2V617F negative HL-60 cell line. In JAK2V617F mutated UKE1 and SET2 cells, ASXL1 knockdown accompanied with the significant elevation of CXCR4 mRNA and protein levels (Figure 1A); while in HL-60 cell, ASXL1 knockdown accompanied with the significant decreased expression of CXCR4 (Figure 1A). Next, we tested the effect of ASXL1 expression on cell migration ability by using transwell migration assay. Our observation showed that ASXL1 knockdown cells showed obvious promotion in migration in JAK2V617F mutated UKE1 and SET2 cells (P=0.028 and P<0.001 ), and CXCR4 antagonist AMD3100 could disrupt this effect (Figure 1B), and down regulation of ASXL1 expression accompanied with declined migration ability in HL-60 cells (P=0.0025, Figure 1B). Flow cytometry also confirmed the increased membrane CXCR4 expression in JAK2V617F mutated UKE1 and SET2 cells. In order to further explore the mechanism of ASXL1 effect on cell migration, we performed mRNA expression microarrary and the results showed that the function of cell adhesion ability, cell-matrix adhesion and leukocyte migration ability were up regulated in ASXL1 knockdown SET2 cells (Figure 1C).In summary, our data demonstrated that ASXL1 knockdown in JAK2V617F mutated UKE1 and SET2 cells resulted in increased expression of CXCR4 and enhanced cell migration abilities, which may indicated that ASXL1 mutation may act on SDF-1/CXCR4 axis. We speculate that under the JAK2V617F mutated background, ASXL1 knockdown may promote the migration ability of tumor stem cells and formation of tumor microenvironment through acting on SDF-1/CXCR4 axis and result in splenomegaly and extramedullary hematopoiesis in PMF patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.