Lei Han

MicroRNA-21 inhibitor sensitizes human glioblastoma cells U251 (PTEN-mutant) and LN229 (PTEN-wild type) to taxol

BackgroundSubstantial data indicate that the oncogene microRNA 21 (miR-21) is significantly elevated in glioblastoma multiforme (GBM) and regulates multiple genes associated with cancer cell proliferation, apoptosis, and invasiveness. Thus, miR-21 can theoretically become a target to enhance the chemotherapeutic effect in cancer therapy. So far, the effect of downregulating miR-21 to enhance the chemotherapeutic effect to taxol has not been studied in human GBM.MethodsHuman glioblastoma U251 (PTEN-mutant) and LN229 (PTEN wild-type) cells were treated with taxol and the miR-21 inhibitor (in a poly (amidoamine) (PAMAM) dendrimer), alone or in combination. The 50% inhibitory concentration and cell viability were determined by the MTT assay. The mechanism between the miR-21 inhibitor and the anticancer drug taxol was analyzed using the Zheng-Jun Jin method. Annexin V/PI staining was performed, and apoptosis and the cell cycle were evaluated by flow cytometry analysis. Expression of miR-21 was investigated by RT-PCR, and western blotting was performed to evaluate malignancy related protein alteration.ResultsIC(50) values were dramatically decreased in cells treated with miR-21 inhibitor combine with taxol, to a greater extent than those treated with taxol alone. Furthermore, the miR-21 inhibitor significantly enhanced apoptosis in both U251 cells and LN229 cells, and cell invasiveness was obviously weakened. Interestingly, the above data suggested that in both the PTEN mutant and the wild-type GBM cells, miR-21 blockage increased the chemosensitivity to taxol. It is worth noting that the miR-21 inhibitor additively interacted with taxol on U251cells and synergistically on LN229 cells. Thus, the miR-21 inhibitor might interrupt the activity of EGFR pathways, independently of PTEN status. Meanwhile, the expression of STAT3 and p-STAT3 decreased to relatively low levels after miR-21 inhibitor and taxol treatment. The data strongly suggested that a regulatory loop between miR-21 and STAT3 might provide an insight into the mechanism of modulating EGFR/STAT3 signaling.ConclusionsTaken together, the miR-21 inhibitor could enhance the chemo-sensitivity of human glioblastoma cells to taxol. A combination of miR-21 inhibitor and taxol could be an effective therapeutic strategy for controlling the growth of GBM by inhibiting STAT3 expression and phosphorylation.

Co-delivery of as-miR-21 and 5-FU by Poly(amidoamine) Dendrimer Attenuates Human Glioma Cell Growth in Vitro

MicroRNAs have been demonstrated to be deregulated in different types of cancer. miR-21 is a key player in the majority of cancers. Down-regulation of miR-21 in glioblastoma cells leads to repression of cell growth, increased cellular apoptosis and cell-cycle arrest, which can theoretically enhance the chemotherapeutic effect in cancer therapy. In this study, the poly(amidoamine) (PAMAM) dendrimer was employed as a carrier to co-deliver antisense-miR-21 oligonucleotide (as-miR-21) and 5-fluorouracil (5-FU) to achieve delivery of as-miR-21 to human glioblastoma cells and enhance the cytotoxicity of 5-FU antisense therapy. The inhibitory effect toward brain tumors was evaluated by MTT assay, and measurements of cell apoptosis and invasion using the human brain glioma cell line U251. PAMAM could be simultaneously loaded with 5-FU and as-miR-21, forming a complex smaller than 100 nm in diameter. Both the chemotherapeutant and as-miR-21 could be efficiently introduced into tumor cells. The co-delivery of as-miR-21 significantly improved the cytotoxicity of 5-FU and dramatically increased the apoptosis of U251 cells, while the migration ability of the tumor cells was decreased. These results suggest that our co-delivery system may have important clinical applications in the treatment of miR-21-overexpressing glioblastoma.

HOTAIR, a cell cycle-associated long noncoding RNA and a strong predictor of survival, is preferentially expressed in classical and mesenchymal glioma.

BACKGROUND Long noncoding RNA Hox transcript antisense intergenic RNA (HOTAIR) has been characterized as a negative prognostic factor in breast and colon cancer patients. The clinical significance and function of HOTAIR in glioma remains unclear. METHODS We analyzed the clinical significance of HOTAIR in 3 different glioma cohorts with gene expression data, including correlation with tumor grade, prognosis, and molecular subtype. The function of HOTAIR in glioma was explored by performing gene set enrichment analysis and in vitro and in vivo experiments. RESULTS HOTAIR expression was closely associated with glioma grade and poor prognosis. Multivariate Cox regression analysis revealed that HOTAIR was an independent prognostic factor in glioblastoma multiforme patients. HOTAIR expression correlated with glioma molecular subtype, including those of The Cancer Genome Atlas. HOTAIR was preferentially expressed in the classical and mesenchymal subtypes compared with the neural and proneural subtypes. A gene set enrichment analysis designed to show gene set differences between patients with high and low HOTAIR expression indicated that HOTAIR expression was associated with gene sets involved in cell cycle progression. HOTAIR reduction induced colony formation suppression, cell cycle G0/G1 arrest, and orthotopic tumor growth inhibition. CONCLUSION Our data establish that HOTAIR is an important long noncoding RNA that primarily serves as a prognostic factor for glioma patient survival, as well as a biomarker for identifying glioma molecular subtypes, a critical regulator of cell cycle progression.

LncRNA profile of glioblastoma reveals the potential role of lncRNAs in contributing to glioblastoma pathogenesis.

Long non-coding RNAs (lncRNAs) have recently emerged as a major class of regulatory molecules involved in a broad range of biological processes and complex diseases. Our aim was to identify important lncRNAs that might play an important role in contributing to glioblastoma (GBM) pathogenesis by conducting lncRNA and mRNA profile comparison between GBM and normal brain tissue. The differentially expressed lncRNA and mRNA profiles of the tissue between GBM patient and age-matched donor without GBM diseases were analyzed using microarrays. We propose a novel model for the identification of lncRNA-mRNA targeting relationships that combine the potential targets of the differentially expressed lncRNAs with the differentially expressed mRNA abundance data. Bioinformatic analysis of the predicted target genes (gene ontology, pathway and network analysis) was performed for further research. The lncRNA microarray reveals differentially expressed lncRNAs between GBM and normal brain tissues. In the GBM group, 654 lncRNAs were upregulated and 654 were downregulated (fold change ≥4.0 or ≤0.25, P<0.01). We found 104 matched lncRNA-mRNA pairs for 91 differentially expressed lncRNAs and 84 differentially expressed genes. Target gene-related pathway analysis showed significant changes in PPAR pathways in the GBM group compared with the normal brain group (P<0.05). By further conducting lncRNA gene network analysis, we found that ASLNC22381 and ASLNC2081 were likely to play roles in the regulation of glioma signaling pathways. In conclusion, our results indicated that the lncRNA expression profile in GBM tissue was significantly altered. These results may provide important insights into the mechanisms responsible for GBM progression and pathogenesis. This study also suggests that ASLNC22381 and ASLNC20819 may play important roles via their target IGF-1 in the recurrence and malignant progression of GBM.

AC1MMYR2, an Inhibitor of Dicer-Mediated Biogenesis of Oncomir miR-21, Reverses Epithelial–Mesenchymal Transition and Suppresses Tumor Growth and Progression

The extensive involvement of miRNAs in cancer pathobiology has opened avenues for drug development based on oncomir inhibition. Dicer is the core enzyme in miRNA processing that cleaves the terminal loop of precursor microRNAs (pre-miRNAs) to generate mature miRNA duplexes. Using the three-dimensional structure of the Dicer binding site on the pre-miR-21 oncomir, we conducted an in silico high-throughput screen for small molecules that block miR-21 maturation. By this method, we identified a specific small-molecule inhibitor of miR-21, termed AC1MMYR2, which blocked the ability of Dicer to process pre-miR-21 to mature miR-21. AC1MMYR2 upregulated expression of PTEN, PDCD4, and RECK and reversed epithelial-mesenchymal transition via the induction of E-cadherin expression and the downregulation of mesenchymal markers, thereby suppressing proliferation, survival, and invasion in glioblastoma, breast cancer, and gastric cancer cells. As a single agent in vivo, AC1MMYR2 repressed tumor growth, invasiveness, and metastasis, increasing overall host survival with no observable tissue cytotoxicity in orthotopic models. Our results offer a novel, high-throughput method to screen for small-molecule inhibitors of miRNA maturation, presenting AC1MMYR2 as a broadly useful candidate antitumor drug.

High level of miR-221/222 confers increased cell invasion and poor prognosis in glioma

BackgroundMiR-221 and miR-222 (miR-221/222), upregulated in gliomas, can regulate glioma cell cycle progression and apoptosis, respectively. However, the association of miR-221/222 with glioma cell invasion and survival remains unknown.MethodsInvasion capability of miR-221/222 was detected by mutiple analyses, including diffusion tensor imaging (DTI), transwell, wound healing and nude mouse tumor xenograft model assay. Further, the target of miR-221/222 was determined by luciferase reporter, western blot and gene rescue assay. The association of miR-221/222 with outcome was examined in fifty glioma patients.ResultsMiR-221/222 expression was significantly increased in high-grade gliomas compared with low-grade gliomas, and positively correlated with the degree of glioma infiltration. Over-expression of miR-221/222 increased cell invasion, whereas knockdown of miR-221/222 decreased cell invasion via modulating the levels of the target, TIMP3. Introduction of a TIMP3 cDNA lacking 3’ UTR abrogated miR-221/222-induced cell invasion. In addition, knockdown of miR-221/222 increased TIMP3 expression and considerably inhibited tumor growth in a xenograft model. Finally, the increased level of miR-221/222 expression in high-grade gliomas confers poorer overall survival.ConclusionsThe present data indicate that miR-221 and miR-222 directly regulate cell invasion by targeting TIMP3 and act as prognostic factors for glioma patients.

MicroRNA roles in beta-catenin pathway

Abstractβ-catenin, a key factor in the Wnt signaling pathway, has essential functions in the regulation of cell growth and differentiation. Aberrant β-catenin signaling has been linked to various disease pathologies, including an important role in tumorigenesis. Here, we review the regulation of the Wnt signaling pathway as it relates to β-catenin signaling in tumorigenesis, with particular focus on the role of microRNAs. Finally, we discuss the potential of β-catenin targeted therapeutics for cancer treatment.

Molecular classification of gliomas based on whole genome gene expression: a systematic report of 225 samples from the Chinese Glioma Cooperative Group

Defining glioma subtypes based on objective genetic and molecular signatures may allow for a more rational, patient-specific approach to molecularly targeted therapy. However, prior studies attempting to classify glioma subtypes have given conflicting results. We aim to complement and validate the existing molecular classification system on a large number of samples from an East Asian population. A total of 225 samples from Chinese patients was selected for whole genome gene expression profiling. Consensus clustering was applied. Three major groups of gliomas were identified (referred to as G1, G2, and G3). The G1 subgroup correlates with a good clinical outcome, young age, and extremely high frequency of IDH1 mutations. Relative to the G1 subgroup, the G3 subgroup is correlated with a poorer clinical outcome, older age, and a very low rate of mutations in the IDH1 gene. Correlations of the G2 subgroup with respect to clinical outcome, age, and IDH1 mutation fall between the G1 and G3 subgroups. In addition, the G2 subtype was associated with a higher percentage of loss of 1p/19q when compared with G1 and G3 subtypes. Furthermore, our classification scheme was validated on 2 independent datasets derived from the cancer genome atlas (TCGA) and Rembrandt. With use of the TCGA classification system, proneural, neural, and mesenchymal, but not classical subtype, associated gene signatures were clearly defined. In summary, our results reveal that 3 main subtypes stably exist in Chinese patients with glioma. Our classification scheme may reflect the clinical and genetic alterations more clearly. Classical subtype-associated gene signature was not found in our dataset.

MicroRNA miR-451 downregulates the PI3K/AKT pathway through CAB39 in human glioma

The microRNA miR-451 is downregulated in gliomas, this has been suggested by several different research groups and is consistent with our data. Our previous study also confirmed that miR-451 has a repressive role in glioma by inhibiting cell growth, proliferation and by inducing cell apoptosis. In the present study, we identified a target gene of miR-451 in human glioma and investigated the mechanism for the glioma suppressive effect of miR-451 functions. Expression of miR-451 in gliomas was identified by quantitative real-time PCR and fluorescence in situ hybridization. Human glioma cell lines (U251, U87, LN229 and A172) were transfected with miR-451 mimics to restore miR-451 expression. The tumor suppressive effects of miR-451 were further verified by subcutaneous assays in nude mice, in addition to our previous in vitro data. A candidate target gene was tested by Western blotting and luciferase reporter assays. Some PI3K/AKT pathway factors were tested by Western blotting. We found that miR-451 expression was downregulated in glioma samples and was inversely correlated with WHO grades of gliomas. In vivo assays confirmed that miR-451 had tumor suppressive traits. CAB39-3′UTR luciferase reporter assay confirmed CAB39 as a direct target gene of miR-451. Significant alterations in the expression of PI3K/AKT pathway factors were observed by Western blot assays. We conclude that miR-451 represses glioma in vitro and in vivo, likely through targeting CAB39 directly and inhibiting the PI3K/AKT pathway indirectly.

Downregulation of miR-21 Enhances Chemotherapeutic Effect of Taxol in Breast Carcinoma Cells

The successful of anti-cancer treatment are often limited by the development of drug resistance. Recent work has highlighted the involvement of non-coding RNAs, microRNAs(miRNAs) in cancer development, and their possible involvement in the evolution of drug resistance has been proposed. In this study, we combine taxol chemotherapy and miR-21 inhibitor treatment via polyamidoamine (PAMAM) dendrimers vector to evaluate the effects of combination therapy on suppression of breast cancer cells. The 50% inhibitory concentration (IC50) values for taxol were significantly decreased to a greater extent in the cells transfected with miR-21 inhibitor compared with cells treated with taxol alone. Taxol treatment also increased the percentage of apoptotic breast cancer cells in miR-21 inhibitor transfected cells compared with control cells. Furthermore, treatment of the miR-21 inhibitor-transfected cells with the anti-cancer drugs taxol resulted in significantly reduced cell viability and invasiveness compared with control cells. These results indicated that the miR-21 plays an important role in the resistance of breast carcinoma cells to chemotherapeutic drugs. Therefore, miR-21 inhibitor gene therapy combined with taxol chemotherapy might represent a promising novel therapeutic approach for the treatment of breast malignancies.