Adipose-derived stem cell-derived microvesicle-released miR-210 promoted proliferation, migration and invasion of endothelial cells by regulating RUNX3.
ABSTRACT: The potential mechanism of miRNA released from adipose-derived stem cell (ADSC)-derived micro vesicle (MV) onthe modulation of proliferation, migration and invasion of endothelial cells were explored. In this study, miR-210 level was detected by qT-PCR. Alix, VEGF and RUNX3 expressions were detected by Western blot. The proliferation, migration and invasion of human umbilical vein endothelial cells (HUVECs) were observed by MTT assay and Transwell assay. Luciferase reporter gene assay was conducted to validate the targeting activity of MVs-released miR-210 on RUNX3. We found hypoxia significantly increased the expression of MVs-released miR-210. MVs released from ADSCsin hypoxic group significantly promoted the proliferation, migration and invasion of HUVECs. Overexpression of miR-210 significantly upregulated VEGF expression, and promoted the proliferation, migration and invasion of HUVECs. Besides, RUNX3 was identified as the direct of miR-210 in HUVECs. Overexpression of miR-210 decreased RUNX3 expression and promoted the proliferation, migration and invasion of HUVECs, while overexpression of RUNX3 inhibited these promotion effects. In vivo experiment showed that MVs derived from ADSCs under hypoxia increased miR-210 level and capillary density, and inhibition of miR-210 decreased capillary density. We also found MVs downregulated RUNX3 expression, and inhibition of miR-210 upregulated RUNX3 expression. Therefore, miR-210 released from ADSCs-derived MVs promoted proliferation, migration and invasion of HUVECs by targeting RUNX3, which revealed one of the mechanisms of ADSCs-derived MVs on the promotion of proliferation, migration and invasion of HUVECs. ABBREVIATIONS:ADSC, adipose-derived stem cell; MV, micro vesicle; HUVECs, human umbilical vein endothelial cells; RUNX3, Runtrelatedtranscription factor-3.
Project description:Cell secretion is an important mechanism for stem cell-based therapeutic angiogenesis, along with cell differentiation to vascular endothelial cells or smooth muscle cells. Cell-released microvesicles (MVs) have been recently implicated to play an essential role in intercellular communication. The purpose of this study was to explore the potential effects of stem cell-released MVs in proangiogenic therapy. We observed for the first time that MVs were released from adipose-derived stem cells (ASCs) and were able to increase the migration and tube formation of human umbilical vein endothelial cells (HUVECs). Endothelial differentiation medium (EDM) preconditioning of ASCs upregulated the release of MVs and enhanced the angiogenic effect of the released MVs in vitro. RNA analysis revealed that microRNA was enriched in ASC-released MVs and that the level of microRNA-31 (miR-31) in MVs was notably elevated upon EDM-preconditioning of MV-donor ASCs. Further studies exhibited that miR-31 in MVs contributed to the migration and tube formation of HUVECs, microvessel outgrowth of mouse aortic rings, and vascular formation of mouse Matrigel plugs. Moreover, factor-inhibiting HIF-1, an antiangiogenic gene, was identified as the target of miR-31 in HUVECs. Our findings provide the first evidence that MVs from ASCs, particularly from EDM-preconditioned ASCs, promote angiogenesis and the delivery of miR-31 may contribute the proangiogenic effect.This study provides the evidence that microvesicles (MVs) from adipose-derived stem cells (ASCs), particularly from endothelial differentiation medium (EDM)-preconditioned ASCs, promote angiogenesis. An underlying mechanism of the proangiogenesis may be the delivery of microRNA-31 via MVs from ASCs to vascular endothelial cells in which factor-inhibiting HIF-1 is targeted and suppressed. The study findings reveal the role of MVs in mediating ASC-induced angiogenesis and suggest a potential MV-based angiogenic therapy for ischemic diseases.
Project description:Objective: The study aims to investigate the effects of miR-221-3p in bone marrow mesenchymal stem cell (BMMSC)-derived microvesicles (MVs) on cell cycle, proliferation and invasion of acute myelocytic leukemia (AML). Methods: Bioinformatics was used to predict differentially expressed miRNAs (DEmiRNAs) in AML. The morphology of BMMSC-derived MVs was observed under an electron microscope, and the positional relation of MVs and OCI-AML2 cells was observed by a fluorescence microscope. MTT, Transwell, and flow cytometry assays were used to analyze the effects of MVs on OCI-AML2 cells. The targeted relationship between miR-221-3p and CDKN1C was detected by dual luciferase assay. Results: It was verified that miR-221-3p promoted the proliferation, invasion and migration of OCI-AML2 cells, and induced the cell cycle arrest in G1/S phase as well as inhibited cell apoptosis. Further studies showed that MVs promoted the proliferation, migration and invasion of AML, and induced the cell cycle arrest in G1/S phase through miR-221-3p. It was confirmed that miR-221-3p can directly target CDKN1C to regulate cell cycle, proliferation and invasion of AML. Conclusion: miR-221-3p in BMMSC-derived MVs regulated AML cell cycle, cell proliferation and invasion through targeting CDKN1C. miR-221-3p and CDKN1C were considered to be potential targets and biomarkers for the treatment of AML in clinic.
Project description:Current evidence indicates that microRNAs are widely down-regulated in various tumors including colorectal carcinoma, liver cancer and lung cancer, and function as tumor suppressors through inhibiting cancer cell growth, invasion and migration. Here, we demonstrated that miR-210-3p level was significantly reduced in the bladder cancer compared to paratumor tissues, and attempt to reveal the regulatory role of miR-210-3p in bladder cancer progression. Exogenous overexpression of miR-210-3p inhibited the proliferation, migration and invasion of bladder cancer cells in vitro. In addition, the nude mouse xenograft model showed that miR-210-3p over-expressing inhibited bladder cancer growth and liver metastasis whereas silencing miR-210-3p caused an opposite outcome, which is mainly regulated by targeting fibroblast growth factor receptor-like 1 (FGFRL1). We also demonstrated that the expression of FGFRL1 in bladder cancer specimens were negatively correlated with miR-210-3p level, and FGFRL1 overexpression rescued the cell proliferation and invasion inhibited by ectopic expression of miR-210-3p. Moreover, knockdown of FGFRL1 was able to mimic the cell growth and metastasis effects induced by miR-210-3p over-expressing in bladder cancer cells. Together, these results indicate that miR-210-3p plays an important role in the regulation of bladder cancer growth and metastasis in vitro and in vivo through targeting FGFRL1.
Project description:Irreversible pulmonary fibrosis induced by paraquat (PQ) poisoning is the major cause of death in patients with PQ poisoning. The epithelial-mesenchymal transition (EMT) is postulated to be one of the main mechanisms of pulmonary fibrosis. Here, we investigated the role of miR-210 in PQ-induced EMT and its relationship with hypoxia-inducible factor-1? (HIF-1?). Western blotting, immunofluorescence, immunoprecipitation and other methods were used in this study. We found that miR-210 expression was significantly increased after PQ poisoning, and it may be regulated by HIF-1?. Overexpression of miR-210 further increased the HIF-1? protein level and promoted EMT. Moreover, miR-210 knock-down reduced the HIF-1? protein level and decreased the degree of EMT. Runt-related transcription factor-3 (RUNX3), a direct target of miR-210, was inhibited by miR-210 in response to PQ poisoning. RUNX3 increased the hydroxylation ability of prolyl hydroxylase domain-containing protein 2 (PHD2), a key enzyme that promotes HIF-1? degradation. PHD2 immunoprecipitated with RUNX3 and its level changed similarly to that of RUNX3. The expression of the HIF-1? protein was significantly reduced when RUNX3 was overexpressed. HIF-1? protein levels were markedly increased when RUNX3 was silenced. Based on these results, a positive feedback loop may exist between miR-210 and HIF-1?. The mechanism may function through miR-210-mediated repression of RUNX3, which further decreases the hydroxylation activity of PHD2, enhances the stability of HIF-1?, and promotes PQ-induced EMT, aggravating the progression of pulmonary fibrosis. This study further elucidates the mechanism of PQ-induced pulmonary fibrosis and may provide a new perspective for the future development of therapies.
Project description:Exosomes are reported to mediate several disease-related microRNAs (miRNAs) to affect the progression of diseases, including atherosclerosis. Here, we aimed to screen the atherosclerosis-associated miRNAs and preliminarily investigate the potential regulatory mechanism of atherosclerosis. First, the lesion model for human umbilical vein endothelial cells (HUVECs) was favorably constructed. Later, through RNA-sequencing and bioinformatics analyses, miR-342-5p was identified in lesion model for HUVECs. MiR-342-5p overexpression or knockdown evidently promoted or inhibited the apoptosis of HUVECs impaired by H2O2. Mechanistically, PPP1R12B was found to have great potential as a target of miR-342-5p in HUVECs impaired by H2O2, supported by RNA-sequencing and a series of bioinformatics analyses. Meanwhile, the effect of miR-342-5p on PPP1R12B expression in HUVECs' lesion model was explored, revealing that miR-342-5p had an inhibitory role in PPP1R12B expression. Additionally, adipose-derived mesenchymal stem cells (ADSCs) in spindle-like shape and their derived exosomes with 30 to 150 nm diameter were characterized. Furthermore, results showed miR-342-5p was evidently decreased in the presence of ADSCs-derived exosomes. These findings indicated ADSCs-derived exosomes restrained the expression of miR-324-5p in lesion model. Collectively, this work demonstrates an atherosclerosis-associated miR-342-5p and reveals a preliminary possible mechanism in which miR-342-5p mediated by ADSCs-derived exosomes protects endothelial cells against atherosclerosis.
Project description:MicroRNAs (miRNAs) influence stem cell functions, including mobilization, proliferation, and differentiation. miR-150 is abundantly expressed in monocytes. Knockdown of miR-150 promotes bone marrow stem cell migration. The role of miR-150 in adipose-derived stem cells (ADSCs) is unclear. In this study, the effects of miR-150 on adipogenic differentiation and proliferation of ADSCs were investigated. ADSCs were isolated from the inguinal adipose tissue of wild-type (WT) and miR-150 knockout (KO) mice and were induced for adipogenic differentiation. The miR-150 level was detected by real-time PCR. ADSCs were transfected by miR-150 or small-interfering RNA (siRNA) of Notch3. MTT assay and colony formation assay were performed in miR-150 knockdown and control ADSCs. Real-time PCR showed that miR-150 was expressed in ADSCs. miR-150 knockdown significantly decreased the capacity of adipogenic differentiation of ADSCs, as compared with their counterparts from WT mice. It is intriguing that the overexpression of miR-150 significantly increased C/EBP? and PPAR-? expression and lipid formation in ADSCs with adipogenic induction. Overexpression of miR-150 significantly decreased Notch3 expression in ADSCs compared with the control groups. Furthermore, Notch3 inhibition promoted the adipogenic differentiation in ADSCs. miR-150 also suppressed proliferation potential and the expression of Nanog in ADSCs. In summary, this study demonstrates, for the first time, that miR-150 promotes adipogenic differentiation and inhibits proliferation of ADSCs. miR-150 regulates adipogenic differentiation of ADSCs, likely mediated by the downregulation of Notch3.
Project description:We developed anticancer drug-conjugated biodegradable polymer-nanoparticle-loaded adipose-derived stem cells (AdSCs) as a tool for biodrug delivery systems for cancer therapy. Pirarubicin was conjugated in polylactic/glycolic acid (PLGA) followed by formation of nanoparticles (NPs), which were loaded with human AdSCs and cocultured. The pirarubicin-conjugated PLGA NP-loaded AdSCs (PirNP-AdSCs) were overall viable within 48?h and exhibited significantly enhanced migration activity. We confirmed that pirarubicin was gradually released into the culture medium from PirNP-AdSCs, and the conditioned medium significantly inhibited the proliferation activity and induced the apoptosis of human pancreatic cancer cells (KP1N). PirNP-AdSCs also significantly induced tumor cell apoptosis in an ex vivo culture system with KP1N-derived tumors, and there was increased invasion/migration of PirNP-AdSCs inside the tumor. Finally, we compared the therapeutic efficacy of the PirNP-AdSCs on KP1N-derived tumor growth with that of treatments of AdSCs alone, PirNPs alone or normal saline (control) in immunodeficient mice. Subcutaneous local administration of PirNP-AdSCs significantly inhibited tumor growth, inducing the apoptosis of tumor cells and vasculature compared with the other groups. The present therapeutic strategy might give rise to a novel cancer therapy minimizing the adverse side effects of anticancer drugs in patients who suffer from cancer.
Project description:DICER is a key rate-limiting enzyme in the canonical miRNAs biogenesis pathway, and DICER and DICER-dependent miRNAs have been proved to play essential roles in many physiological and pathological processes. However, whether DICER is involved in placentation has not been studied. Successful spiral artery remodelling is one of the key milestones during placentation, which depends mostly on the invasion of trophoblasts and the crosstalk between trophoblasts and endothelial cells. In the present study, we show that DICER knockdown impairs the invasion ability of both primary extravillous trophoblasts (EVT) and HTR8/SVneo (HTR8) cell lines. The decreased invasion of HTR8 cells upon DICER knockdown (sh-Dicer) was partly due to the up-regulation of miR-16-2-3p, which led to a reduced expression level of the collagen type 1 alpha 2 chain (COL1A2) protein. Moreover, microvesicles (MVs) can be secreted by HTR8 cells and promote the tube formation ability of human umbilical cord vein endothelial cells (HUVECs). However, conditioned medium and MVs derived from sh-Dicer HTR8 cells have an anti-angiogenic effect, due to reduced angiogenic factors and increased anti-angiogenic miRNAs (including let-7d, miR-1-6-2 and miR-15b), respectively. In addition, reduced protein expression of DICER is found in PE placenta by immunoblotting and immunohistochemistry. In summary, our study uncovered a novel DICER-miR-16-2-COL1A2 mediated pathway involved in the invasion ability of EVT, and DICER-containing MVs mediate the pro-angiogenic effect of trophoblast-derived conditioned medium on angiogenesis, implying the involvement of DICER in the pathogenesis of PE.
Project description:Hypoxia enhances the proliferation and migration of adipose-derived stem cells (ASCs) via the generation of reactive oxygen species (ROS). Therefore, this study primarily investigated whether or not ROS generation could regulate microRNA-210 (miR-210) expression, and increase proliferation/migration of ASCs. In addition, we tried to identify the signaling pathways involved in miR-210 upregulation and the direct target genes of miR-210 that mediate these functions. Various sources of ROS generation such as hypoxia, antimycin, rotenone, and platelet-derived growth factor (PDGF)-BB upregulated miR-210 expression, and increased the proliferation/migration of ASCs. There is a positive feed-forward loop between ROS generation and miR-210, and miR-210 itself increases ROS generation by downregulation of iron-sulfur cluster scaffold homolog 2 (ISCU2). Although hypoxia-inducible factor-1? was not involved in miR-210 expression, pharmacological or small interfering RNA (siRNA)-driven inhibition of Akt and ERK1/2 molecules reduced miR-210 expression. Transfection of siRNAs of NF-?B and Elk1 also reduced miR-210 expression, indicating that these signaling pathways mediate miR-210 upregulation. Protein tyrosine phosphatase, non-receptor type 2 (PTPN2) was selected for miR-210 target gene, and it was downregulated by ROS generators or miR-210 mimic treatment. PTPN2 was first proven to be a direct miR-210 target in luciferase activity assay, and pharmacological inhibition or overexpression of PTPN2 regulated the proliferation and migration of ASC. In conclusion, ROS generation from diverse sources induces miR-210 expression in ASCs via PDGFR-?, Akt and ERK pathways. Transcription of miR-210 expression is regulated by NF-?B and Elk1, and miR-210 increases the proliferation and migration of ASCs via ISCU2 and PTPN2 downregulation.
Project description:Background:Podocyte migration is actively involved in the process of podocyte loss and proteinuria production, which is closely associated with the development of diabetic nephropathy (DN). Exosomes from adipose-derived stem cells (ADSCs-Exos) effectively inhibit podocyte apoptosis in the treatment of DN. However, how ADSCs-Exos affect the migration of podocytes is obscure. This study is aimed at exploring the regulatory role of ADSCs-Exos on cell migration and the underlying mechanism. Methods:ADSCs-Exo was authenticated by transmission electron microscopy (TEM), western blotting, and flow cytometry. Cell viability and migration ability of podocytes were measured by CCK8 and Transwell assays, respectively. Relative expressions of miRNAs and mRNAs were determined by qRT-PCR. The transmitting between PKH26-labeled exosome and podocytes was evaluated by IF assay. Dual luciferase reporter assay was employed to detect the relationship between miR-215-5p and ZEB2. Results:The exposure to serum from DN patient (hDN-serum) significantly inhibited cell viability of podocytes, but ADSCs-Exo addition notably blunts cytotoxicity induced by the transient stimulus of hDN-serum. Besides, ADSCs-Exo administration powerfully impeded high glucose- (HG-) induced migration and injury of podocyte. With the podocyte dysfunction, several miRNAs presented a significant decline under the treatment of HG including miR-251-5p, miR-879-5p, miR-3066-5p, and miR-7a-5p, all of which were rescued by the addition of ADSCs-Exo. However, only miR-251-5p was a key determinant in the process of ADSCs-Exo-mediated protective role on podocyte damage. The miR-251-5p inhibitor counteracted the improvement from the ADSCs-Exo preparation on HG-induced proliferation inhibition and migration promotion. Additionally, miR-215-5p mimics alone remarkably reversed HG-induced EMT process of podocyte. Mechanistically, we confirmed that ADSCs-Exos mediated the shuttling of miR-215-5p to podocyte, thereby protecting against HG-induced metastasis, possibly through inhibiting the transcription of ZEB2. Conclusion:ADSCs-Exo has the protective effect on HG-evoked EMT progression of podocytes thru a mechanism involving ZEB2. Potentially, the ADSCs-Exo preparation is a useful therapeutic strategy for improving podocyte dysfunction and DN symptoms clinically.