Recent studies reported miR-497 exhibited inhibitory effects in various cancers. However, whether miR-497 is involved in inhibiting angiogenesis, which is critical for tumor growth and metastasis, is still unknown. The purpose of this study was to investigate the potential role of miR-497 in tumor angiogenesis. In this work, cell proliferation and apoptosis analyses were conducted to explore the potential function of miR-497 in HUVECs by using MTT and TUNEL assays. Western blotting (WB) was employed to validate the downstream targets of miR-497. Furthermore, in order to disclose the role of miR-497 on angiogenesis, VEGFR2-luc transgenic mice were treated with miR-497 mimic and applied to monitor tumor angiogenesis and growth by in vivo bioluminescent imaging (BLI). The results demonstrated that overexpression of miR-497 showed inhibitory effects on VEGFR2 activation and downstream Raf/MEK/ERK signal pathways in vitro and in vivo. Moreover, overexpression of miR-497 effectively induced HUVECs apoptosis by targeting VEGFR2 and downstream PI3K/AKT signaling pathway. Furthermore, miR-497 exhibited anti-angiogenesis and anti-tumor effects in the VEGFR2-luc breast tumor model proven by BLI, WB and immunohistochemistry analysis. In summary, miR-497 inhibits tumor angiogenesis and growth via targeting VEGFR2, indicating miR-497 can be explored as a potential drug candidate for cancer therapy.

MicroRNA-21 (miR-21) is overexpressed in a wide range of cancers and involved in tumor proliferation and metastasis. However, the potential function of miR-21 in regulating tumor angiogenesis has been little disclosed. In this study, we treated the cultured 4T1 murine breast cancer cells and human umbilical vein endothelial cells (HUVECs) with miR-21 mimic, antagomir-21 or negative control (scramble), which were subjected to MTT, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), quantitative Reverse Transcriptase PCR (qRT-PCR) and immunoblotting analysis. In addition, 4T1 cells were implanted beneath the right breast fat pad of the VEGFR2-luc transgenic mice, which were randomly divided into three groups and received saline, antagomir-21 or scramble treatment once respectively after tumor model establishment. Bioluminescent imaging was used to monitor tumor growth and angiogenesis in vivo at 0d, 3d, 5d, 7d, 10d, and 14d after treatment. Mice were killed at the end of study and tumor tissues were collected for use. The results showed that knockdown of miR-21 by antagomir-21 decreased cell proliferation and induced apoptosis via targeting PTEN both in 4T1 cells and HUVECs. We also found the anti-angiogenesis and anti-tumor effects of antagomir-21 in the VEGFR2-luc transgenic mouse model using bioluminescent imaging. Moreover, the Western blotting data revealed that antagomir-21 inhibited tumor angiogenesis through suppressing HIF-1α/VEGF/VEGFR2-associated signaling pathway. In conclusion, the results from current study demonstrate that antagomir-21 can effectively suppress tumor growth and angiogenesis in VEGFR2-luc mouse breast tumor model and bioluminescent imaging can be used as a tool for noninvasively and continuously monitoring tumor angiogenesis in vivo.

It has been demonstrated that mesenchymal stem cells (MSCs) differentiated from human embryonic stem cells (hESCs), name EMSCs, can treat a variety of autoimmune and inflammatory diseases, with similar efficacies to those achieved with MSCs derived from somatic tissues such as bone marrow (BMSCs). The chance increases even higher for EMSCs, than somatic tissue derived MSCs​, to become a cell drug as the former can be produced in large scale from an unlimited hESC line with easier quality control and less biosafety concern. We have further demonstrated that both human ESCs and EMSCs, after aggregation to form spheroids, can tolerate hypoxic and ambient conditions (AC) for over 4 and 10 days, respectively, without loss of their viability and alteration of their functions. Based on these advantages, we decided to test whether EMSC spheroids, made in large quantity and delivered through a long-term distance at AC, can treat osteoarthritis spontaneously developed in rhesus macaques (M. mulatta) monkeys as well as the allogenic MSCs.Methods: Xenogeneic AC-transported EMSC spheroids or allogenic BMSCs were injected into the articular cavity of both knees of the monkeys at 3 animals per group. Another two macaques were injected the same way with saline as controls.Results: Both EMSCs and BMSCs groups showed significant amelioration indicated by the reduction of swelling joint size and amplification of keen flare angle post-treatment, compared to the control group. Examinations via X-ray and MRI also indicated the decrease of inflammation and osteophyma, and recovery of the synovium and cartilage in both treated groups. No sign of allergy or graft versus host disease was observed in the animals.Conclusion: Our results demonstrate that human EMSC spheroids can prevent the osteoarthtitis progression and ameliorate osteoarthritis in the rhesus macaques as well as allogenic BMSCs, and this study shall help advance the clinical application of EMSCs.

Background: Growing evidence shows that microRNAs (miRNAs) are involved in various cardiac processes including cardiac hypertrophy. However, the modulation of miRNA by pharmacological intervention in cardiomyocyte hypertrophy has not been disclosed yet. Methods: We constructed neonatal rat cardiomyocyte hypertrophy induced by angiotensin II stimulation and subjected to cardiomyocyte immunochemistry, qRT-PCR and immunoblotting analysis. In addition, we constructed the mouse cardiac hypertrophy using angomir-22 stimulation and demonstrated the potential antihypertrophic mechnism of atorvastatin. Results: The results showed that a collection of miRNAs were aberrantly expressed in hypertrophic cardiomyocytes induced by angiotensin II stimulation. In addition, overexpression of miR-22 was found in angiotensin II-induced hypertrophic cardiomyocytes, whereas administration of atorvastatin could reverse the upregulation of miRNA-22 nearly back to the control level. Furthermore, up-regulation of miRNA-22 in cardiomyocytes in vitro and in vivo could induce cardiac hypertrophy, which could suppress the protein level of phosphatase and tensin homolog deleted on chromosome ten (PTEN). Concomitantly, the production of ANP, BNP and β-MHC was enhanced and cardiomyocyte size was increased. However, atorvastatin could markedly knockdown miRNA-22 expression and reverse these changes in cardiomyocytes. These results suggest that the contribution of atrovastatin to cardiomyocyte hypertrophy may be involved in downregulation of miRNA-22 expression, which modulates the activity of PTEN in cardiomyocyte hypertrophy. Conclusion: This study demonstrates for the first time the modulation of miRNA-22 can be achieved by pharmacological intervention. The data generated from this study provides a rationale for the development of miRNA-based strategies for antihypertrophic treatment.

Background and objectiveTumor angiogenesis is vital for tumor growth. Recent evidence indicated that bone marrow-derived mesenchymal stem cells (BMSCs) can migrate to tumor sites and exert critical effects on tumor growth through direct and/or indirect interactions with tumor cells. However, the effect of BMSCs on tumor neovascularization has not been fully elucidated. This study aimed to investigate whether fusion cells from glioma stem cells and BMSCs participated in angiogenesis.MethodsSU3-RFP cells were injected into the right caudate nucleus of NC-C57Bl/6 J-GFP nude mice, and the RFP+/GFP+ cells were isolated and named fusion cells. The angiogenic effects of SU3-RFP, BMSCs and fusion cells were compared in vivo and in vitro.ResultsFusion cells showed elevated levels of CD31, CD34 and VE-Cadherin (markers of VEC) as compared to SU3-RFP and BMSCs. The MVD-CD31 in RFP+/GFP+ cell xenograft tumor was significantly greater as compared to that in SU3-RFP xenograft tumor. In addition, the expression of CD133 and stem cell markers Nanog, Oct4 and Sox2 were increased in fusion cells as compared to the parental cells. Fusion cells exhibited enhanced angiogenic effect as compared to parental glioma cells in vivo and in vitro, which may be related to their stem cell properties.ConclusionFusion cells exhibited enhanced angiogenic effect as compared to parental glioma cells in vivo and in vitro, which may be related to their stem cell properties. Hence, cell fusion may contribute to glioma angiogenesis.

Abstract. The ability of tumor cells to autonomously generate tumor vessels has received considerable attention in recent years. However, the degree of autonomy is relative. Meanwhile, the effect of bone marrow-derived mesenchymal stem cells (BMSCs) on tumor neovascularization has not been fully elucidated. The present study aimed to illuminate whether cell fusion between glioma stem cells and BMSC is involved in glioma neovascularization. BMSCs were isolated from transgenic nude mice, of which all nucleated cells express green fluorescent protein (GFP). The immunophenotype and multilineage differentiation potential of BMSC were confirmed. SU3 glioma stem/progenitor cells were transfected with red fluorescent protein (SU3-RFP cells). In a co-culture system of BMSC-GFP and SU3-RFP, RFP + /GFP + cells were detected and isolated by dual colors using FACS. The angiogenic effect of RFP + /GFP + cells was determined in vivo and in vitro. Flow cytometry analysis showed that BMSC expressed high levels of CD105, C44, and very low levels of CD45 and CD11b. When co-cultured with SU3-RFP, 73.8% of cells co-expressing RFP and GFP were identified as fused cells in the 5th generation. The fused cells exhibited tube formation ability in vitro and could give rise to a solid tumor and form tumor blood vessels in vivo. In the dual-color orthotopic model of transplantable xenograft glioma, yellow vessel-like structures that expressed CD105, RFP and GFP were identified as de novo-formed vessels derived from the fused cells. The yellow vessels observed in the tumor-bearing mice directly arose from the fusion of BMSCs and SU3-RFP cells. Thus, cell fusion is one of the driving factors for tumor neovascularization.

This study elucidates the feasibility of using our constructed miRNA reporter imaging system to monitor the location and magnitude of expression levels of miR-22 in cardiac hypertrophy in vitro and in vivo.

Osteosarcoma (OS) is the most common primary bone malignancy. Our previous study revealed an association between the level of epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (EFEMP1) and the invasion, metastasis, and poor prognosis of OS. However, the exact correlation between the serum EFEMP1 level and OS diagnosis and progression was unclear. This study is aimed at determining the value of the serum EFEMP1 level in the diagnosis and prognosis of OS. Fifty-one consecutive OS patients were prospectively registered in this study. The serum EFEMP1 levels were measured using ELISA at diagnosis, after neoadjuvant chemotherapy, and before and after surgical treatment. Sixty-nine healthy subjects in the control group, nine patients with chondrosarcoma, and 12 patients with giant cell tumor of the bone were also enrolled in this study. Surgical orthotopic implantation was used to generate a mouse OS model, and the correlation between the circulating EFEMP1 levels and tumor progression was examined. Then, OS patients had significantly higher mean serum EFEMP1 levels (7.61 ng/ml) than the control subjects (1.47 ng/ml). The serum EFEMP1 levels were correlated with the Enneking staging system (r=0.32, P=0.021) and lung metastasis (r=0.50, P<0.001). There was also a correlation between the serum EFEMP1 level and EFEMP1 expression in the respective OS samples (r=0.49, P<0.001). Additionally, patients with either chondrosarcoma or giant cell tumor of the bone had significantly higher serum EFEMP1 levels than OS patients. Surgical and chemotherapeutic treatment led to an increase in the serum EFEMP1 levels. Then, the destruction of bone tissues might be one of the factors about the EFEMP1 levels. In the mouse OS model, the serum EFEMP1 level was correlated with tumor progression. Our results suggested that serum EFEMP1 levels might be used to distinguish OS patients from healthy controls and as an indicator for OS lung metastasis. Serum EFEMP1 levels could serve as a new and assisted biomarker for the auxiliary diagnosis and prognosis of OS.