Guangxiu Wang

Downregulation of Wnt2 and β-catenin by siRNA suppresses malignant glioma cell growth

Increasing evidence suggests that aberrant activation of Wnt signaling is involved in tumor development and progression. Our earlier study on gene expression profile in human gliomas by microarray found that some members of Wnt family were overexpressed. To further investigate the involvement of Wnt signaling in gliomas, the expression of core components of Wnt signaling cascade in 45 astrocytic glioma specimens with different tumor grades was examined by reverse transcription-PCR and immunohistochemistry. Wnt2, Wnt5a, frizzled2 and β-catenin were overexpressed in gliomas. Knockdown of Wnt2 and its key mediator β-catenin in the canonical Wnt pathway by siRNA in human U251 glioma cells inhibited cell proliferation and invasive ability, and induced apoptotic cell death. Furthermore, treating the nude mice carrying established subcutaneous U251 gliomas with siRNA targeting Wnt2 and β-catenin intratumorally also delayed the tumor growth. In both in vitro and in vivo studies, downregulation of Wnt2 and β-catenin was associated with the decrease of PI3K/p-AKT expression, indicating the interplay between Wnt/β-catenin and PI3K/AKT signaling cascades. In conclusion, the canonical Wnt pathway is of critical importance in the gliomagenesis and intervention of this pathway may provide a new therapeutic approach for malignant gliomas.

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.

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.

Suppression of EGFR expression by antisense or small interference RNA inhibits U251 glioma cell growth in vitro and in vivo

Epidermal growth factor receptor (EGFR) had been reported as one of the major responsible genes for malignant progression and phenotype reversion of gliomas, and has been used as one of the most important therapeutic targets. In the present study, small interference RNA (siRNA) and antisense EGFR expression constructs, which target sequences of human EGFR catalytic domain (2400–2420) and the 3′-coding region, respectively, were used to examine the growth inhibition effects on U251 glioma cells. Cell growth was significantly inhibited and G2/M arrest was observed in antisense- and siRNA-treated groups. Matrigel matrix demonstrated spotted cell clustering pattern in antisense- and siRNA-transfected U251 cells, indicating poor cell growth activities. In addition, the tumor volumes in U251 subcutaneous mice model treated with antisense and siRNA were significantly smaller than those treated with control siRNA and phosphate-buffered saline. Also, glial fibrillary acidic protein expression was upregulated in antisense- and siRNA-treated groups than the control groups. Our results demonstrated that antisense- or siRNA-targeting intracellular region of EGFR can inhibit EGFR expression, exerted growth inhibition effect on U251 glioma cells in vitro and in vivo. Consequently, siRNA expression plasmid-mediated gene therapy would be a new strategy in treatment of gliomas.

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.

The oncogenic roles of Notch1 in astrocytic gliomas in vitro and in vivo

Notch receptors play an essential role in cellular processes during embryonic and postnatal development, including maintenance of stem cell self-renewal, proliferation, and determination of cell fate and apoptosis. Deregulation of Notch signaling has been implicated in some genetic diseases and tumorigenesis. The function of Notch signaling in a variety of tumors can be either oncogenic or tumor-suppressive, depending on the cellular context. In this study, Notch1 overexpression was observed in the majority of 45 astrocytic gliomas with different grades and in U251MG glioma cells. Transfection of siRNA targeting Notch1 into U251 cells in vitro downregulated Notch1 expression, associated with inhibition of cell growth, arrest of cell cycle, reduction of cell invasiveness, and induction of cell apoptosis. Meanwhile, tumor growth was delayed in established subcutaneous gliomas in nude mice treated with Notch1 siRNA in vivo. These results suggest that Notch1 plays an important oncogenic role in the development and progression of astrocytic gliomas. Furthermore, knockdown of Notch1 expression by siRNA simultaneously downregulated the expression of EGFR and the important components of its downstream pathways, including PI3K, p-AKT, K-Ras, cyclin D1 and MMP9, indicating the crosstalk and interaction of Notch and EGFR signaling pathways.

miR-221/222 is the regulator of Cx43 expression in human glioblastoma cells

The miR-221/222 cluster is significantly upregulated in malignant glioma cells and regulates the expression of multiple genes associated with glioma cell proliferation, invasion and apoptosis, which was shown in our previous studies. Cx43 has been identified as a tumor suppressor and major component for the establishment of gap junction intercellular communication (GJIC) in glial cells, which is frequently reduced or deleted in high-grade gliomas. According to bioinformatic analysis, connexin 43 (Cx43) may be one of the target genes of miR-221/222. The aim of the present study was to validate Cx43 as a target gene of miR-221/222 and to determine whether overexpression of miR-221/222 is one of the molecular mechanisms for the reduced expression of Cx43 in malignant gliomas. We transfected miR-221/222 antisense oligonucleotides (AS-miR-221/222) into U251 human glioblastoma cells using a lipofectamine method. Northern blot analysis was conducted to detect the expression of the miR-221/222 cluster. Luciferase reporter assays were exploited to confirm Cx43 as a target gene of miR-221/222. Cx43 expression was assessed by western blotting and immunofluorescence staining. Scrape loading and dye transfer (SLDT) assays were used for examination of GJIC. Proliferation and invasion of U251 cells were evaluated by MTT and transwell assays, respectively. Cell cycle kinetics and apoptosis were determined with flow cytometry. We found that expression of the miR-221/222 cluster was significantly reduced while Cx43 expression was upregulated in U251 cells transfected with AS-miR-221/222, and the GJIC deficiency in parental U251 cells was re-established. Moreover, the luciferase activity determined by the luciferase reporter assay was enhanced in AS-miR-221/222-treated cells, and cell proliferation and invasion were suppressed while apoptosis was induced. We conclude that miR-221/222 function as oncogenic microRNAs in human gliomas, at least in part, by targeting Cx43.

MiR-30a-5p Antisense Oligonucleotide Suppresses Glioma Cell Growth by Targeting SEPT7

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by targeting the mRNAs of hundreds of human genes. Variations in miRNA expression levels were shown to be associated with glioma. We have previously found miR-30a-5p overexpression in glioma cell lines and specimens. Bioinformatics analyses predict that several miRNAs, including miR-30a-5p, are involved in the post-transcriptional regulation of SEPT7. SEPT7 is a member of the septin family, which is a highly conserved subfamily of GTPases implicated in exocytosis, apoptosis, synaptogenesis, neurodegeneration and tumorigenesis. Our previous study has also demonstrated that SEPT7 expression is decreased in astrocytic gliomas with different grades and plays a tumor suppressor role. In the present study, we knocked down miR-30a-5p with antisense oligonucleotide (miR-30a-5p AS) in LN229 and SNB19 glioblastoma(GBM) cells, and found that cell growth and invasion were inhibited, while apoptosis was induced. miR-30a-5p AS treated cells showed upregulation of SEPT7 and downregulation of PCNA, cyclin D1, Bcl2, MMP2 and MMP9. In contrast, when miR-30a-5p mimics were transfected into LN229 and SNB19 GBM cells, cell growth and invasion were promoted and the expression of relevant proteins increased. Meanwhile, the effect of miR-30a-5p mimics on glioma cells can be reversed by transfection of SEPT7 construct. Additionaly, miR-30a-5p directly targeting SEPT7 was identified by the reporter gene assay. Our study demonstrates,for the first time, that miR-30a-5p is a bona fide negative regulator of SEPT7 and the oncogenic activity of miR-30a-5p in human gliomas is at least in part through the repression of SEPT7.

The anti-glioma effect of suicide gene therapy using BMSC expressing HSV/TK combined with overexpression of Cx43 in glioma cells

The disseminated neoplastic foci of malignant gliomas are essentially responsible for the limited efficacy of current available therapeutic modalities. Bone marrow-derived stem cells (BMSCs) have the ability to migrate into these tumors and even track infiltrating tumor cells, making them to be promising cellular vehicles for delivering therapeutic agents to glioma cells. The herpes simplex virus thymidine kinase (HSV–TK)/ganciclovir (GCV) suicide gene therapy with a potent bystander effect has been considered as one of the most promising therapeutic strategies for malignant gliomas. In this study, we evaluate the anti-glioma effect of suicide gene therapy using BMSCs expressing HSV–TK combined with overexpression of connexin 43 (Cx43), which can restore the gap junction of intercellular communication and may enhance the bystander effect of suicide gene therapy. To assess the potential of BMSCs to track glioma cells, a spheroid co-culture system in matrigel was used to show that some BMSCs migrated to C6 glioma cell microspheres. Transwell assay showed the tumor tropic property of BMSCs. In addition, BrdU-labeled BMSCs injected directly into the cerebral hemisphere opposite to the established C6 rat gliomas were capable of migrating into the xenograft gliomas. C6 cell growth was more intensively inhibited by HSV–TK/GCV treatment mediated by BMSCs, and could be further enhanced by combination with Cx43 transfection into glioma cells. The same result was observed in vivo by the growth of C6 gliomas and the survival analysis of rats bearing C6 glioma. In conclusion, Cx43 combined with HSV–TK/GCV gene therapy using BMSCs as vehicles was highly effective in a rat glioma model and therefore hold great potential as a novel approach for the gene therapy of human malignant gliomas.