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.

Up-regulated microRNA-299 corrected with poor prognosis of glioblastoma multiforme patients by targeting ELL2.

BACKGROUND: An increasing understanding of the genes and molecular pathways of glioblastoma multiforme (GBM) can provide us a useful insight for the development of more effective targeted therapeutic. METHODS: To investigate the expression and clinical significance of miR-299 and its target genes in GBM, the expression levels of miR-299 and its target gene in human normal brain tissues and GBM were analyzed in silico using genes microarray and hierarchical clustering analysis followed by validation with quantitative RT-PCR. RESULTS: Our results show that miR-299 is up-regulated in GBM patients. Moreover, patients with low miR-299 expression had longer overall survival (OS) compared with those with high miR-299 expression. RNA polymerase II elongation factor, ELL2, was identified as a miR-299 direct target. High expression of ELL2 together with miR-299 down-regulation correlated with a shorter median OS. CONCLUSIONS: Our results provide the first evidence that ELL2 is a direct target of miR-299 and increased ELL2 expression and down-regulation of miR-299 are associated with GBM progression and poor prognosis in patients, suggesting that ELL2 and miR-299 might have potential prognostic value and be used as tumor biomarkers for the diagnosis of patients with GBM.

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.

Edaravone protects HT22 neurons from H2O2-induced apoptosis by inhibiting the MAPK signaling pathway.

AIMS: Oxidative stress is frequently implicated in the pathology of neurodegenerative diseases. This study aimed to investigate the effects and their underlying mechanism(s) of edaravone upon hydrogen peroxide (H2O2)-induced oxidative stress and apoptosis in HT22 cells, a murine hippocampal neuronal model. METHODS: HT22 cells were treated with H2O2 in the presence of various concentrations of edaravone or in its absence. A CCK-8 assay, Hoechst 33342 staining, and flow cytometry were used to detect cytotoxicity and apoptosis. In addition, the levels of reactive oxygen species (ROS) and the expression of Bcl-2, Bax, p-ERK 1/2, p-JNK, and p-P38 proteins in HT22 cells were examined. RESULTS: Exogenous H2O2 decreased cell viability in a concentration-dependent manner and was associated with increased apoptosis and ROS production. Moreover, H2O2 significantly activated and upregulated the expression of p-ERK 1/2, p-JNK, and p-P38, while edaravon protected HT22 cells against H2O2-induced injury by inhibiting the production of ROS and activating the MAPK signaling pathway. CONCLUSIONS: Our results provide the first evidence that edaravone can protect H2O2-induced cell injury in HT22 neurons via its antioxidant action. These findings suggest that edaravone may be useful in the treatment of neurodegenerative disorders in which oxidative stress has been principally implicated.

[A related analysis for alpha, beta fibrinogen gene haplotypes and nucleotide polymorphisms associated with the ischemic stroke in Hainan Han population].

OBJECTIVE: To analyze the association of that the polymorphisms and haplotypes of Taq I site in beta fibrinogen gene and the single nucleotide sites -455 G/A, -249 C/T, -148 C/T, +1689T/G, Bsm A I G/C, 448 G/A, Bcl I G/A, Hinf I A/C in beta-fibrinogen gene are linked up with the ischemic stroke(IS). METHODS: Turbidmetric assay was used to measure the plasma fibrinogen level of one hundred and sixty cases with ischemic stroke and one hundred and thirty healthy individuals from Hainanese Han population. The polymorphisms and genotypes were characterized by PCR-RFLP. Hardy-Weinberg equilibrium and statistical differences of allelic, genotype and haplotype frequencies were obtained by Chi-square test. Pairwise linkage disequilibrium was calculated and haplotypes of nine or four polymorphisms were estimated by the EH + program. RESULTS: There were highly significant differences in genotype frequencies and allelic frequencies of the polymorphisms -455 G/A, -148 C/T, 448 G/A, which happened between the IS group and control subjects (P< 0.01). However, the significant differences of the allelic frequencies in the other six polymorphisms were not found between the IS group and the control (P> 0.05). The odds ratio(OR) with the rare alleles of A -455, T -148 and A 448 is 2.46, 2.30 and 2.08 (95% confidence interval 1.153%-3.924%, 1.429%-3.694% and 1.298%-3.329%) respectively. No definite haplotype block was found by linkage disequilibrium analysis in the control group and the IS group. Association of haplotypes constructed from the nine polymorphisms with IS was not found. Among the haplotypes constructed from four polymorphisms including -455 G/A, -148 C/T, 448 G/A alleles, haplotype differences were found between the control group and the IS group. Haplotypes with G -455, C -148, G448 alleles appeared more frequently in control group(P< or = 0.01), whereas haplotypes with A -455, T -148, A 448 occurred more frequently in the IS group(P< 0.01). CONCLUSION: The results of multi-allele and haplotype analysis indicated that the polymorphisms -455 G/A, -148 C/T, 448 G/A in beta fibrinogen gene were the possible risk factors associated with the occurrence of ischemic stroke in Hainan Han population.