Receptor-Ligand Interaction Mediates Targeting of Endothelial Colony Forming Cell-derived Exosomes to the Kidney after Ischemic Injury.
ABSTRACT: Endothelial colony forming cell (ECFC)-derived exosomes protect mice against ischemic kidney injury, via transfer of microRNA-(miR)-486-5p. Mechanisms mediating exosome recruitment to tissues are unclear. We hypothesized that ECFC exosomes target ischemic kidneys, involving interaction between exosomal CXC chemokine receptor type 4 (CXCR4) and stromal cell-derived factor (SDF)-1?. Ischemia-reperfusion was induced in mice by bilateral renal vascular clamp, with intravenous infusion of exosomes at reperfusion. Optical imaging determined exosome biodistribution, and miR-486-5p was measured by real-time PCR. Human umbilical vein endothelial cells (HUVECs) were cultured to study the CXCR4/SDF-1? interaction. Targeting of administered exosomes to ischemic kidneys was detected 30?min and 4 hrs after reperfusion. Exosomes increased miR-486-5p levels only in kidneys, within proximal tubules, glomeruli, and endothelial cells. Uptake of fluorescently-labeled exosomes into HUVECs, and exosomal transfer of miR-486-5p were enhanced by hypoxia, effects blocked by neutralizing antibody to SDF-1? or by the CXCR4 inhibitor plerixafor. Infusion of ECFC exosomes prevented ischemic kidney injury in vivo, an effect that was not observed when exosomes were pre-incubated with plerixafor. These data indicate that ECFC exosomes selectively target the kidneys after ischemic injury, with rapid cellular transfer of miR486-5p. Targeting of exosomes may involve interaction of CXCR4 with endothelial cell SDF-1?.
Project description:BACKGROUND The therapeutic potential of endothelial colony-forming cells (ECFCs) may be impaired in an ischemic environment. Direct injection of ECFCs is not an effective method of rescuing the ischemic heart, but exosomes derived from these cells may be a promising therapeutic tool. However, exosomes produced under normoxia and hypoxia may not be identical. Therefore, the purpose of this study was to investigate alterations in the anti-fibrotic effects of hypoxia-treated ECFC-derived exosomes and the underlying mechanism involved. MATERIAL AND METHODS ECFCs were isolated from peripheral blood and exosomes were collected from ECFCs treated with normoxia (nor-exo) or hypoxia (hyp-exo). Effects of exosomes on cardiac fibroblast activation were evaluated in vitro. MicroRNAs (miRNAs) inside the exosomes were extracted and compared using next-generation RNA sequencing. Predicted target mRNAs of miR-10b-5p were validated using a dual-luciferase reporter gene assay method. RESULTS Nor-exo significantly ameliorated cardiac fibroblast activation in vitro. These effects were attenuated in the hyp-exo-treated group. miR-10b-5p was enriched in nor-exo but not in hyp-exo. Dual-luciferase reporter gene assay found that both SMAD-specific E3 ubiquitin protein ligase 1 (Smurf1) and histone deacetylase 4 (HDAC4) mRNAs were inhibited by miR-10b-5p. The expression of neutral sphingomyelinase 2 (N-SMase2) was decreased in hypoxia ECFCs, and this result was consistent with the changes in miR-10b-5p in hyp-exo. CONCLUSIONS Due to a reduction of miR-10b-5p, which targets the fibrotic genes Smurf1 and HDAC4, the anti-fibrotic effects of hyp-exo were abolished.
Project description:We have previously demonstrated that a stromal cell-derived factor-1 (SDF-1; CXCL12)/CXCR4 system is involved in the establishment of metastasis in oral cancer. Recently, small non coding RNAs, microRNAs (miRNAs) have been shown to be involved in the metastatic process of several types of cancers. However, the miRNAs that contribute to metastases induced by the SDF-1/CXCR4 system in oral cancer are largely unknown. In this study, we examined the metastasis-related miRNAs induced by the SDF-1/CXCR4 system using B88-SDF-1 oral cancer cells, which exhibit functional CXCR4 and distant metastatic potential in vivo. Through miRNA microarray analysis, we identified the upregulation of miR-518c-5p in B88-SDF-1 cells, and confirmed the induction by real-time PCR analysis. Although an LNA-based miR-518c-5p inhibitor did not affect cell growth of B88-SDF-1 cells, it did significantly inhibit the migration of the cells. Next, we transfected a miR-518c expression vector into parental B88 cells and CAL27 oral cancer cells and isolated stable transfectants, B88-518c and CAL27-518c cells, respectively. The anchorage-dependent and -independent growth of miR-518c transfectants was significantly enhanced compared with the growth of mock cells. Moreover, we detected the enhanced migration of these cells. The LNA-based miR-518c-5p inhibitor significantly impaired the enhanced cell growth and migration of miR-518c transfectants, indicating that these phenomena were mainly dependent on the expression of miR-518c-5p. Next, we examined the function of miR-518c-5p in vivo. miR-518c transfectants or mock transfectants were inoculated into the masseter muscle or the blood vessels of nude mice. Tumor volume, lymph nodes metastasis, and lung metastasis were significantly increased in the mice inoculated with the miR-518c transfectants. These results indicated that miR-518c-5p regulates the growth and metastasis of oral cancer as a downstream target of the SDF-1/CXCR4 system.
Project description:Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. The alveolar subtype (ARMS) is clinically more aggressive, and characterized by an oncogenic fusion protein PAX3-FOXO1 that drives oncogenic cellular properties. Exosomes are small, secreted vesicles that affect paracrine signaling. We show that PAX3-FOXO1 transcript alters exosome content of C2C12 myoblasts, leading to pro-tumorigenic paracrine effects in recipient cells. Microarray analysis revealed alteration in miRNA content of exosomes, affecting cellular networks involved in cell metabolism, growth signaling, and cellular invasion. Overexpression and knockdown studies showed that miR-486-5p is an effector of PAX3-FOXO1, and mediates its paracrine effects in exosomes, including promoting recipient cell migration, invasion, and colony formation. Analysis of human RMS cells showed miR-486-5p is enriched in both cells and exosomes, and to a higher extent in ARMS subtypes. Analysis of human serum samples showed that miR-486-5p is enriched in exosomes of patients with RMS, and follow-up after chemotherapy showed decrease to control values. Our findings identify a novel role of both PAX3-FOXO1 and its downstream effector miR-486-5p in exosome-mediated oncogenic paracrine effects of RMS, and suggest its possible use as a biomarker.
Project description:Objective:This study aimed to describe the mechanism of exosome-derived miR-486-5p underlying the cell cycle and progression in lung adenocarcinoma (LUAD). Methods:Bioinformatics methods were applied for identifying the differentially expressed genes (DEGs) in the GEO-LUAD dataset, predicting where the potential target miRNA was expressed and exploring the corresponding downstream target mRNA. qRT-PCR was conducted to detect the levels of the target genes in cancer cells. Thereafter, a series of in vitro experiments were performed for cell activities evaluation, including CCK-8, EdU, colony formation assay and transwell. Besides, Western blot was applied to determine the protein levels of the migration and invasion-related factors (NEK2, E-cadherin, N-cadherin, Vimentin, MMP-2, and MMP-9). Dual-luciferase reporter gene assay was employed for validating the targeted relationship between the target genes. Furthermore, nude mouse transplantation tumor experiment was conducted to further validate the role of the target miRNA in tumor development, and immunohistochemistry was used for Ki67 detection and TUNEL was applied for cell apoptosis assay. Results:miR-486-5p was observed to be enriched in serum exosomes, and seen to be significantly down-regulated in cancer tissues as well as in cancer serum exosomes. It was proven that exosomes could release miR-486-5p, thus regulating LUAD progression and affecting cell cycle. Moreover, NEK2 was identified as a target of miR-486-5p both in vivo and in vitro. Enrichment analysis revealed that NEK2 was mainly activated in cell cycle and mitosis-related pathways. Meanwhile, NEK2 was found to present significant difference in different TNM stages. Furthermore, rescue experiments indicated that the inhibitory effect of miR-486-5p overexpression on LUAD progression could be abrogated when miR-486-5p and NEK2 were simultaneously up-regulated. Conclusion:Exosome-derived miR-486-5p is responsible for cell cycle arrest as well as the inhibition of cell proliferation and metastasis in LUAD via targeting NEK2.
Project description:In colorectal cancer, increased expression of the CXC chemokine receptor 4 (CXCR4) has been shown to provoke metastatic disease due to the interaction with its ligand stromal cell-derived factor 1 (SDF-1). Recently, a second SDF-1 receptor, CXCR7, was found to enhance tumor growth in solid tumors. Albeit signaling cascades via SDF-1/CXCR4 have been intensively studied, the significance of the SDF-1/CXCR7-induced intracellular communication triggering malignancy is still only marginally understood. In tumor tissue of 52 colorectal cancer (CRC) patients, we observed that expression of CXCR7 and CXCR4 increased with tumor stage, tumor size, and lymph node infiltration. Asking whether activation of CXCR4 or CXCR7 might result in a similar expression pattern, we performed microarray expression analyses using lentivirally CXCR4- and/or CXCR7-overexpressing SW480 colon cancer cell lines with and without stimulation by SDF-1α. Gene regulation via SDF-1α/CXCR4 and SDF-1α/CXCR7 was completely different and partly antidromic. Expressions of the differentially expressed genes AKR1C3, AXL, EGFR, IGFBP7, IL24, TNNC1, TRIP6 were confirmed by qPCR. Differentially regulated genes were assigned by GO to migration and lipid metabolic processes. Furthermore, using the in silico gene set enrichment analysis we showed for the first time that expressions of miR-217 and miR-218 were increased in CXCR4 and reduced in CXCR7 cells after stimulation with SDF-1α. As expected, their putative target mRNAs were inversely expressed. Functional assays exerted that exposure to SDF-1α resulted in strongly amplified invasiveness and chemosensitivity of CXCR4-expressing cells. CXCR7 overexpression led to reduced invasiveness which could only be marginally increased by SDF-1α. The CXCR4 antagonist plerixafor significantly reduced invasiveness of CXCR4-overexpressing cells only. Similarly, compared to control cells, CXCR4 cells showed increased sensitivity against 5-FU, while CXCR7 cells were more chemoresistant. These opposing results for CXCR4- or CXCR7-overexpressing colon carcinoma cells demand an unexpected attention in the clinical application of chemokine receptor antagonists like Plerixafor. 24 samples
Project description:As one of the most common cancers worldwide, colorectal cancer (CRC) causes a large number of mortality annually. Aberrant expression of microRNAs (miRNAs) is significantly associated with the initiation and development of CRC. Further investigations regarding the regulatory mechanism of miRNAs is warranted. In this study, we discovered that miR-486-5p was remarkably downregulated in CRC, which partially results from higher DNA methylation in the promoter region detected by using methylation-specific PCR, bisulfite sequencing PCR, and DNA demethylation treatment. Besides, decreased miR-486-5p was obviously associated with advanced TNM stage, larger tumor size, lymphatic metastasis, and poor prognosis in CRC. Upregulated miR-486-5p inhibited the proliferation and migration of CRC through targeting PLAGL2 expression and subsequent repressing IGF/?-catenin signal pathways both in vitro and in vivo. Notably, plasma miR-486-5p expression was significantly upregulated in CRC patients and we identified plasma miR-486-5p as a novel diagnostic biomarker of CRC using receiver operating characteristic (ROC) curve analysis. Moreover, exploration in GEO dataset revealed that circulating miR-486-5p is tumor derived through being packaged into secretory exosomes. Taken together, our data demonstrated that miR-486-5p promotes colorectal cancer proliferation and migration through activation of PLAGL2/IGF2/?-catenin signal pathway, which is a promising therapeutic target of CRC treatment.
Project description:Plasma exosomal microRNAs (miRNAs) are considered as valid circulating biomarkers for cancer diagnosis and prognosis. Quantitative real-time polymerase chain reaction (qRT-PCR), the most commonly used technique to assess circulating miRNA levels, requires a normalization step involving uniformly expressed endogenous miRNAs. However, there is still no consensus on reference miRNAs for plasma exosomal miRNA abundance normalization. In this study, we identified a panel of miRNAs with stable abundance by analyzing public plasma exosome RNA-seq data and selected miR-486-5p, miR-26a-5p, miR-423-5p and miR191-5p as candidate normalizers. Next, we tested the abundance variation of these miRNAs by qRT-PCR in plasma exosomes of healthy donors and pediatric patients with anaplastic large cell lymphoma, Burkitt lymphoma, Hodgkin lymphoma and mature B-cell acute lymphoblastic leukemia. MiR-486-5p and miR-26a-5p showed the most stable levels, both between healthy controls and patients and among the malignancies analyzed. In light of previous reports on miRNA stability in different exosome isolation methods, our data indicated that miR-26a-5p is a bona fide reference miRNA for qRT-PCR normalization to evaluate miRNA abundance from circulating plasma exosomes in studies of hematological malignancies.
Project description:In spite of advances in the treatment of pediatric acute lymphoblastic leukemia (ALL), a significant number of children with ALL are not cured of their disease. We and others have shown that signaling from the bone marrow microenvironment confers therapeutic resistance, and that the interaction between CXCR4 and stromal cell-derived factor-1 (SDF-1 or CXCL12) is a key mediator of this effect. We demonstrate that ALL cells that upregulate surface CXCR4 in response to chemotherapy treatment are protected from chemotherapy-induced apoptosis when co-cultured with bone marrow stroma. Treatment with the CXCR4 inhibitor plerixafor diminishes stromal protection and confers chemosensitivity. Using xenograft models of high-risk pediatric ALL, plerixafor plus chemotherapy induces significantly decreased leukemic burden, compared to chemotherapy alone. Further, treatment with plerixafor and chemotherapy influences surface expression of CXCR4, VLA-4, and CXCR7 in surviving ALL blasts. Finally, prolonged exposure of ALL blasts to plerixafor leads to a persistent increase in surface CXCR4 expression, along with modulation of surface expression of additional adhesion molecules, and enhanced SDF-1?-induced chemotaxis, findings that may have implications for therapeutic resistance. Our results suggest that while CXCR4 inhibition may prove useful in ALL, further study is needed to understand the full effects of targeting the leukemic microenvironment.
Project description:High-dose chemotherapy and autologous transplantation of hematopoietic cells is a crucial treatment option for hematologic malignancy patients. Current mobilization regimes often do not provide adequate numbers of CD34(+) cells. The chemokine receptor CXCR4 and ligand SDF-1 are integrally involved in homing and mobilization of hematopoietic progenitor cells. Disruption of the CXCR4/SDF-1 axis by the CXCR4 antagonist, plerixafor, has been demonstrated in Phase II and Phase III trials to improve mobilization when used in conjunction with granulocyte colony-stimulating factor (G-CSF). This approach is safe with few adverse events and produces significantly greater numbers of CD34(+) cells when compared to G-CSF alone. New plerixafor initiatives include use in volunteer donors for allogeneic hematopoietic cell transplant and in other disease targets.
Project description:Functional impairment of endothelial colony-forming cells (ECFCs), a specific cell lineage of endothelial progenitor cells (EPCs) is highly associated with the severity of coronary artery disease (CAD), the most common type of cardiovascular disease (CVD). Emerging evidence show that circulating microRNAs (miRNAs) in CAD patients' body fluid hold a great potential as biomarkers. However, our knowledge of the role of circulating miRNA in regulating the function of ECFCs and the progression of CAD is still in its infancy. We showed that when ECFCs from healthy volunteers were incubated with conditioned medium or purified exosomes of cultured CAD ECFCs, the secretory factors from CAD ECFCs dysregulated migration and tube formation ability of healthy ECFCs. It is known that exosomes influence the physiology of recipient cells by introducing RNAs including miRNAs. By using small RNA sequencing (smRNA-seq), we deciphered the circulating miRNome in the plasma of healthy individual and CAD patients, and found that the plasma miRNA spectrum from CAD patients was significantly different from that of healthy control. Interestingly, smRNA-seq of both healthy and CAD ECFCs showed that twelve miRNAs that had a higher expression in the plasma of CAD patients also showed higher expression in CAD ECFCs when compared with healthy control. This result suggests that these miRNAs may be involved in the regulation of ECFC functions. For identification of potential mRNA targets of the differentially expressed miRNA in CAD patients, cDNA microarray analysis was performed to identify the angiogenesis-related genes that were down-regulated in CAD ECFCs and Pearson's correlation were used to identify miRNAs that were negatively correlated with the identified angiogenesis-related genes. RT-qPCR analysis of the five miRNAs that negatively correlated with the down-regulated angiogenesis-related genes in plasma and ECFC of CAD patients showed miR-146a-5p and miR-146b-5p up-regulation compared to healthy control. Knockdown of miR-146a-5p or miR-146b-5p in CAD ECFCs enhanced migration and tube formation activity in diseased ECFCs. Contrarily, overexpression of miR-146a-5p or miR-146b-5p in healthy ECFC repressed migration and tube formation in ECFCs. TargetScan analysis showed that miR-146a-5p and miR-146b-5p target many of the angiogenesis-related genes that were down-regulated in CAD ECFCs. Knockdown of miR-146a-5p or miR-146b-5p restores CAV1 and RHOJ levels in CAD ECFCs. Reporter assays confirmed the direct binding and repression of miR-146a-5p and miR-146b-5p to the 3'-UTR of mRNA of RHOJ, a positive regulator of angiogenic potential in endothelial cells. Consistently, RHOJ knockdown inhibited the migration and tube formation ability in ECFCs. Collectively, we discovered the dysregulation of miR-146a-5p/RHOJ and miR-146b-5p/RHOJ axis in the plasma and ECFCs of CAD patients that could be used as biomarkers or therapeutic targets for CAD and other angiogenesis-related diseases.