MicroRNA let-7f alleviates vascular endothelial cell dysfunction via targeting HMGA2 under oxygen-glucose deprivation and reoxygenation.

Stroke is a fatal disease with high disability and mortality and there is no credible treatment for stroke at present. Studies on stroke are extensively developed to explore the underlying mechanisms of ischemic and reperfusion injuries. Herein, we investigated the functions of microRNA let-7f (also termed let-7f-5p) in vascular endothelial cell dysfunction. The bEnd.3 cells were stimulated with oxygen-glucose deprivation and reoxygenation (OGD/R) to mimic cell injury in vitro. CCK-8 assays, flow cytometry and western blot analyses were conducted to examine the viability and apoptosis of bEnd.3 cells. Reverse transcription quantitative polymerase chain reaction analyses were employed to measure RNA expression. Endothelial cell permeability in vitro assay was employed to assess endothelial permeability of bEnd.3 cells, and expression levels of proteins associated with cell apoptosis or blood-brain barrier (BBB) were detected by western blot analyses. Luciferase reporter assay was conducted to explore the combination between let-7f and HMGA2. We found that OGD/R induced injuries on endothelial cells (bEnd.3) by decreasing cell viability and promoting cell apoptosis. Let-7f exhibited low expression in bEnd.3 cells under OGD/R. Let-7f overexpression increased the viability of bEnd.3 cells and inhibited cell apoptosis. In addition, the endothelial permeability of bEnd.3 cells was increased by OGD/R and reversed by let-7f overexpression. The levels of tight junction proteins (ZO-1 and occludin) were downregulated by OGD/R and then reversed by let-7f overexpression. Mechanistically, HMGA2 is a target gene of let-7f and its expression was negatively regulated by let-7f. Rescue assays revealed that HMGA2 overexpression reversed the effects of let-7f overexpression on cell viability, cell apoptosis, endothelial permeability, and BBB function. In conclusion, let-7f alleviates vascular endothelial cell dysfunction by downregulating HMGA2 expression under OGD/R.

miR-152-3p aggravates vascular endothelial cell dysfunction by targeting DEAD-box helicase 6 (DDX6) under hypoxia.

Stroke is a main cause of disability and death worldwide, and ischemic stroke accounts for most stroke cases. Recently, microRNAs (miRNAs) have been verified to play critical roles in the development of stroke. Herein, we explored effects of miR-152-3p on vascular endothelial cell functions under hypoxia. Human umbilical vein endothelial cells (HUVECs) were treated with hypoxia to mimic cell injury in vitro. Reverse transcription quantitative polymerase chain reaction revealed that miR-152-3p exhibited high expression in HUVECs treated with hypoxia. The inhibition of miR-152-3p reversed hypoxia-induced decrease in cell viability and the increase in angiogenesis, according to the results of cell counting kit-8 assays and tube formation assays. miR-152-3p inhibition reversed the increase in endothelial cell permeability mediated by hypoxia, as shown by endothelial cell permeability in vitro assays. In addition, the increase in protein levels of angiogenetic markers and the decrease in levels of tight junction proteins induced by hypoxia were reversed by miR-152-3p inhibition. Mechanistically, miR-152-3p directly targets 3'-untranslated region of DEAD-box helicase 6 (DDX6), which was confirmed by luciferase reporter assays. DDX6 is lowly expressed in HUVECs under hypoxic condition, and mRNA expression and protein level of DDX6 were upregulated in HUVECs due to miR-152-3p inhibition. Rescue assays showed that DDX6 knockdown reversed effects of miR-152-3p on cell viability, angiogenesis and endothelial permeability. The results demonstrated that miR-152-3p aggravates vascular endothelial cell dysfunction by targeting DDX6 under hypoxia.

Bufalin Inhibits Cellular Proliferation and Cancer Stem Cell-Like Phenotypes via Upregulation of MiR-203 in Glioma.

BACKGROUND/AIMS: Prior studies have shown that bufalin inhibits cellular proliferation and induces apoptosis in various human cancers. MicroRNA-203 (miR-203) has been shown to function as an important regulator of tumor progression at various stages. In this study, we investigated the effect of miR-203 expression and bufalin treatment on glioma cell proliferation and stem cell-like phenotypes. METHODS: We used cell viability assay, colony formation assay, cell apoptosis assay and neurosphere formation assay to dectect the treatment effect of bufalin on U251 and U87 cells. Cells were transfected with the miR-203 mimic without bufalin treatment or cells were transfected with anti-miR-203 under bufalin treatment, the above expreiments were repeated. RT-PCR was employed to quantify miR-203 expression. Western blot was performed to detect the stem cell-like (CSC) markers, OCT4 and SOX2. Luciferase activity assay was used to determine whether the SPARC is the target of miR-203. RESULTS: Bufalin treatment inhibited cell proliferation, colony formation, and CSC phenotypes and increased cell apoptosis and expression of miR-203. Furthermore, overexpression of miR-203 led to similar outcomes as bufalin treatment with respect to the cell viability, colony formation, cell apoptosis and the phenotypes of glioma cells. While anti-miR-203 attenuated the inhibitory effects of bufalin as promoting cell proliferation, colony formation and CSC phenotyes and inhibiting cell apoptosis. In addition, we identified SPARC as a novel target gene of miR-203. CONCLUSIONS: These findings suggest that miR-203 plays an important role in bufalin's ability to inhibit the growth of glioma cells and the development of stem cell-like phenotypes.

Upregulation of miR-181a suppresses the formation of glioblastoma stem cells by targeting the Notch2 oncogene and correlates with good prognosis in patients with glioblastoma multiforme.

Glioblastoma stem-like cells (GSCs) are responsible for the initiation and progression of glioblastoma multiforme (GBM), and microRNAs (miRNAs) play an important role in this disease. However, the mechanisms underlying the role of miRNAs in the stemness of GSCs have not been completely elucidated. We previously showed that miR-181a is downregulated in GBM and may predict prognosis in patients with this disease. Here, we demonstrate that the upregulation of miR-181a suppressed GSC formation and inhibited GBM tumorigenesis by targeting the Notch2 oncogene. We found that miR-181a was downregulated in GSCs derived from human glioblastoma U87MG and U373MG cells. The high expression of miR-181a inhibited the levels of stemness-related markers CD133 and BMI1, attenuated sphere proliferation, promoted cell apoptosis, and reduced the tumorigenicity of GSCs. MiR-181a decreased the expression of Notch2 by targeting the 3'-untranslated region of its mRNA. Notch2 overexpression inhibited the effects of miR-181a downregulation on GSCs, and was negatively correlated with miR-181a expression. Moreover, high Notch2 expression together with low miR-181a expression was correlated with a shorter median overall survival for GBM patients. Together, these data show that miR-181a may play an essential role in GSC formation and GBM progression by targeting Notch2, suggesting that Notch2 and miR-181a have potential prognostic value as tumor biomarkers in GBM patients.

The correlation of microRNA-181a and target genes with poor prognosis of glioblastoma patients.

To investigate the expression and clinical significance of miR-181a and its target genes in glioblastoma multiforme (GBM), the expression levels of miR-181a and three target genes in human normal brain tissues and GBM were analyzed in silico using gene microarray, gene ontology, KEGG pathway and hierarchical clustering analysis followed by validation with quantitative RT-PCR. Our results show that miR-181a is down-regulated in GBM patients. The three target genes, ANGPT2, ARHGAP18 and LAMC1, are negatively correlated with the expression of miR-181a. Moreover, high expression of ANGPT2 or LAMC1 together with large size of GBM is correlated with a shorter median overall survival. In conclusion, our results showed that miR-181a and it targets ANGPT2 and LAMC1 might be predictors of prognosis in GBM patients.