MUC1 promotes glioblastoma progression and TMZ resistance by stabilizing EGFRvIII.

Epidermal growth factor receptor variant III (EGFRvIII) is a mutant isoform of EGFR with a deletion of exons 2-7 making it insensitive to EGF stimulation and downstream signal constitutive activation. However, the mechanism underlying the stability of EGFRvIII remains unclear. Based on CRISPR-Cas9 library screening, we found that mucin1 (MUC1) is essential for EGFRvIII glioma cell survival and temozolomide (TMZ) resistance. We revealed that MUC1-C was upregulated in EGFRvIII-positive cells, where it enhanced the stability of EGFRvIII. Knockdown of MUC1-C increased the colocalization of EGFRvIII and lysosomes. Upregulation of MUC1 occurred in an NF-κB dependent manner, and inhibition of the NF-κB pathway could interrupt the EGFRvIII-MUC1 feedback loop by inhibiting MUC1-C. In a previous report, we identified AC1Q3QWB (AQB), a small molecule that could inhibit the phosphorylation of NF-κB. By screening the structural analogs of AQB, we obtained EPIC-1027, which could inhibit the NF-κB pathway more effectively. EPIC-1027 disrupted the EGFRvIII-MUC1-C positive feedback loop in vitro and in vivo, inhibited glioma progression, and promoted sensitization to TMZ. In conclusion, we revealed the pivotal role of MUC1-C in stabilizing EGFRvIII in glioblastoma (GBM) and identified a small molecule, EPIC-1027, with great potential in GBM treatment.

[Retracted] Role of the AKT pathway in microRNA expression of human U251 glioblastoma cells.

Following the publication of the above article, a concerned reader drew to the Editor's attention that, regarding the western blots featured in Fig. 3B on p. 670, the bands featured in the U251 and U251­MC lanes for the miR­21 and U6 experiments appeared to be duplicates of each other. Moreover, certain of these data were strikingly similar to data that appeared in another article published at around the same time featuring some of the same authors (again, with apparent duplications of bands within the same gel slices, as they were presented). After having conducted an internal investigation of this matter, the Editor of International Journal of Oncology has judged that the apparently anomalous grouping of the data could not have been attributed to pure coincidence. Therefore, the Editor has decided that this article should be retracted from the publication on the grounds of an overall lack of confidence in the data. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive a reply. The Editor sincerely apologizes to the readership for any incovenience caused, and we thank the reader for bringing this matter to our attention. [International Journal of Oncology 36: 665­672, 2010; DOI: 10.3892/ijo_00000542].

MiR-221 and miR-222 target PUMA to induce cell survival in glioblastoma.

BACKGROUND: MiR-221 and miR-222 (miR-221/222) are frequently up-regulated in various types of human malignancy including glioblastoma. Recent studies have reported that miR-221/222 regulate cell growth and cell cycle progression by targeting p27 and p57. However the underlying mechanism involved in cell survival modulation of miR-221/222 remains elusive. RESULTS: Here we showed that miR-221/222 inhibited cell apoptosis by targeting pro-apoptotic gene PUMA in human glioma cells. Enforced expression of miR-22/222 induced cell survival whereas knockdown of miR-221/222 rendered cells to apoptosis. Further, miR-221/222 reduced PUMA protein levels by targeting PUMA-3'UTR. Introducing PUMA cDNA without 3'UTR abrogated miR-221/222-induced cell survival. Notably, knockdown of miR-221/222 induces PUMA expression and cell apoptosis and considerably decreases tumor growth in xenograft model. Finally, there was an inverse relationship between PUMA and miR-221/222 expression in glioma tissues. CONCLUSION: To our knowledge, these data indicate for the first time that miR-221/222 directly regulate apoptosis by targeting PUMA in glioblastoma and that miR-221/222 could be potential therapeutic targets for glioblastoma intervention.

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

BACKGROUND: Substantial 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. METHODS: Human 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. RESULTS: IC(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. CONCLUSIONS: Taken 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.

Overexpression of septin 7 suppresses glioma cell growth.

Our previous study demonstrated that SEPT7 was downregulated at mRNA level in human gliomas. This study is to further examine the expression of SEPT7 in glioma samples and characterizes its role on cell cycle progression and growth of glioma cells. mRNA and protein expression of SEPT7 were detected by RT-PCR, immunohistochemical staining, and western blot analysis in human glioma specimens and normal brain tissues. A pcDNA3-SEPT7 expression plasmid was constructed and transfected into human glioblastoma cell line U251, and cell proliferation and apoptosis were examined. The growth of established U251 and TJ905 subcutaneous xenograft gliomas was measured in nude mice treated with pcDNA3-SEPT7 and U251 xenograft tumors treated with SEPT7 siRNA. SEPT7 expression is negatively correlated with the increase of glioma grade. Overexpression of SEPT7 is able to inhibit cell proliferation and arrest cell cycle progression in the G0/G1 phase both in vitro and in vivo. Knocking down further the already low endogenous expression of SEPT7 in U251 xenograft tumors with siRNA leads to faster tumor growth compared with control tumors. This study demonstrates that SEPT7 is involved in gliomagenesis and suppresses glioma cell growth.

[Inhibitory effect of knocking down microRNA-221 and microRNA-222 on glioma cell growth in vitro and in vivo].

OBJECTIVE: To study the inhibitory effect of knocking down microRNA(miR)-221 and miR-222 on human glioma cell growth and its possible mechanism. METHODS: miRNA-221/222 antisense oligonucleotides (antisense miR221/222) were transfected into human glioma U251 cells by lipofectamine. Northern blot analysis was conducted to detect the mRNA expression of miR-221/222 in the control and transfected cell groups. The proliferation activity of cells was determined by MTT assay. Cell invasion ability was examined by transwell assay, and cell cycle kinetics and apoptosis were detected with flow cytometry. The expression of relevant proteins was analyzed by Western blotting. The therapeutic efficacy of antisense miR221/222 on the growth of xenograft tumors in nude mice were also observed. RESULTS: In the antisense miR-221/222-transfected cells, the expression of miR-221/222 was significantly reduced; the cell invasion ability was suppressed, cell cycle was blocked at G(0)/G(1) phase, and apoptotic cells were increased. The growth of xenograft tumors treated with antisense miR-221/222 was also inhibited. In antisense miR-221/222 treated tumor cells, the expression of bcl-2 was down-regulated while connexin43, p27, PUMA, caspase-3, PTEN, TIMP3 and Bax up-regulated, and p53 expression not changed. CONCLUSION: There is a significant inhibitory effect of antisense miR-221/222 on the growth of human glioma U251 cells. miR-221/222 may be considered as a candidate target for gene therapy of human gliomas.

[The effect of silencing Dicer by small interference RNA on the biological characteristics of human glioma cells].

OBJECTIVE: To study the effect of silencing Dicer by small interference RNA (siRNA) to suppress the global microRNA (miRNAs) expression on the biological characteristics of TJ905 glioblastoma cells. METHODS: The silencing effect of RNA interference on Dicer expression was evaluated by reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis and immunofluorescence staining. The cell proliferation rate and cell cycle kinetics were detected by MTT assay and flow cytometry respectively, and the cell invasive ability was evaluated by transwell assay. RESULTS: The siRNA targeting Dicer suppressed the expression of Dicer in TJ905 cells. Meanwhile, the proliferation activity and invasive ability were significantly enhanced in cells transfected with Dicer siRNA compared to those cells transfected with scrambled siRNA and the control cells. CONCLUSION: Suppression of Dicer expression renders the glioma cells harboring more aggressive phenotype. This preliminary finding suggests that global lower expression of miRNAs may play an oncogenic role.

[Study on the anti-invasion effect of SEPT7 gene for U251MG glioma cell in vitro].

OBJECTIVE: To study the anti-invasion effect of SEPT7 gene on U251MG glioma cells and its possible molecular mechanism. METHODS: Recombinant adenovirus vector carrying SEPT7 gene (rAd5-SEPT7) was transduced to human glioma cell line U251MG, and empty adenovirus vector was used as control. Tumor invasion was examined by Transwell method and 3 D-Matrigel assay, and tumor cell migration by wound-healing method and 2 D-Matrigel assay. Three major molecular events associated with cell motility and migration, including changes of expression in MMP2, MMP9, MT1-MMP, TIMP1 and TIMP2, the alteration of integrin alpha(v)beta(3) expression, and the structural change of cytoskeleton protein, tubulin-alpha, in U251 cells transduced with rAd5-SEPT7 were studied by Western blotting, immunofluorescence and laser scanning confocal microscope, respectively. RESULTS: The invasive and migratory capabilities of cells transduced with rAd5-SEPT7 were inhibited. The expression of extracellular matrix metalloproteinases MMP-2, MMP-9, MT1-MMP and integrin alpha(v)beta(3) was significantly decreased, while the expression of matrix metalloproteinase inhibitor TIMP1, TIMP2 was upregulated. Intracellular cytoskeleton protein-tubulin-alpha in U251 cells exhibited prominent morphological changes which including the appearance of distortion and aggregation resulting from redistribution of tubulin-alpha, and this feature of alteration was similar to the tubulin-alpha structure in normal non-tumor cells. CONCLUSION: SEPT7 gene can inhibit the invasion and migration ability of U251 glioma cells. Its molecular mechanism may include that SEPT7 gene reverses the imbalanced state of MMPs/TIMPs, downregulates the expression of integrin alpha(v)beta(3) and alters the structure of tubulin-alpha of U251MG glioma cells. It is suggested that SEPT7 gene could be a good candidate for gene therapy of gliomas.

[Differential expression of Notch1 and Notch2 in astrocytoma and medulloblastoma].

OBJECTIVE: To detect the differential expression of Notch1 and Notch2 in human astrocytoma and medulloblastoma; and to study the role of Notch1 and Notch2 in the development of both tumors. METHODS: Immunohistochemical staining (SP method) and Western blot analysis were used to detect Notch1 and Notch2 expression in tissue arrays and freshly resected samples of normal brain tissue, astrocytoma and medulloblastoma. RESULTS: Notch1 and Notch2 were negative in normal human brain tissue. Notch1 was highly expressed (total positive rate 80.0%, 48/60) while Notch2 was not detected in grade IV astrocytomas and sporadically observed in lower grade astrocytomas (total positive rate 10.0%, 6/60). The percentage of positive tumor cells and expression level of Notch1 increased with higher histologic grade (r = 0.859, P < 0.05). On the other hand, overexpression of Notch2 was detected in medulloblastoma (9/10) in contrast with lower expression of Notch1 (2/10). CONCLUSIONS: Notch1 and Notch2 show differential expression in astrocytoma and medulloblastoma. This may be related to their different functional activities during the process of brain development.

[Influence of SEPT7 on biological characters of glioma cell line TJ905].

OBJECTIVE: To investigate the influence of SEPT7 on biological characters of gliomas cells TJ905. METHODS: Recombinant SEPT7 constructs was transfected to human glioblastoma cell line TJ905 in which SEPT7 expression is absent. The positive clones were identified by RT-PCR and Western blot analysis. The cell proliferation was determined by MTT assay and flow cytometry, cell apoptosis was detected with Annexin V staining and cell invasion was evaluated by motility in three-dimensional culture. Moreover, the molecules regulating the cell cycle progression were examined by immunofluorescence staining and Western blot analysis. RESULTS: When SEPT7 was successfully transfected to TJ905 cells, the cell proliferation activity of TJ905 cell was inhibited, the cell cycle was arrested in G0/G1 phase and S phase fraction (SPF) was lowered, the positive regulatory molecules for cell cycle progression including cyclin D1, CDk4, cyclin E and CDk2 were downregulated while the negative modulators including p16 and p21 were upregulated, apoptotic cells were increased and cell invasive ability was attenuated. CONCLUSIONS: Transfection of SEPT7 construct into the glioma cells TJ905 is able to inhibit the proliferation activity and invasive ability of TJ905 cell and to induce cell apoptosis. These results revealed that SEPT7 exerted the suppressive effect on the glioma cell growth and invasion, and induced apoptosis, and suggested that SEPT7 as a gene of glioma suppressor.

[Study on the expression of epidermal growth factor receptor and p53 in astrocytic gliomas: evidence for a distinct genetic pathway].

OBJECTIVE: To study further the most important and frequent genetic alterations of p53 and epidermal growth factor receptor (EGFR) in astrocytic gliomas. METHODS: (1) EGFR expression was examined in samples collected from 37 astrocytic gliomas and 6 normal brain tissue using reverse transcriptase polymerase chain reaction and immunohistochemical staining. (2) p53 gene mutation and accumulation were detected simultaneously in the same specimens using PCR-SSCP, DNA sequencing and immunohistochemical staining. RESULTS: The frequency of p53 mutation in diffuse astrocytomas, anaplastic astrocytomas, primary glioblastomas and secondary glioblastomas was 1/10, 4/19 (21.1%), 4/6 and 2/2, respectively and the frequency of EGFR overexpression was 5/10, 10/19 (52.6%), 5/6 and 2/2, respectively. Both p53 accumulation and EGFR overexpression increased accompanied by a successive increase of degree of the glioma malignancy. CONCLUSIONS: EGFR overexpression is not infrequently seen, however, p53 mutation is rarely seen in the low grade gliomas. Both p53 gene mutation and EGFR overexpression are often associated with primary and secondary glioblastoma. Consequently, EGFR overexpression and p53 gene mutation are not mutually exclusive in astrocytic gliomagenesis but synergistically to promote the glioma progression.

[Preliminary study on the mechanism of connexin 43 gene transfection in the control of glioma cell proliferation].

OBJECTIVE: To study the mechanism involved in the control of glioma cell proliferation with transfection of connexin (Cx) 43 gene. METHODS: C6 rat glioma and TJ905 human glioblastoma cell lines without Cx43 gene expression were transfected with Cx43cDNA mediated by lipofectamine. Northern blot, in situ hybridization and immunohistochemical technology were used to detect the expression of Cx43mRNA and its protein with MTT assay and silver colloid stain for the detection of cell proliferation, TUNEL method for determination of cell apoptosis, scrape loading dye transfer (SLDT) for GJIC, Western blot and immunohistochemical technology for bFGF, PDGF, EGFR, IGF-I and IGFBP3 expression. RESULTS: Cx 43 gene transfected glioma cells showed decreased proliferation, restored GJIC and decreased bFGF, PDGF, IGFBP3, except EGFR expression and cell apoptosis which showed no change. CONCLUSION: The mechanism of Cx 43 gene inhibiting gliomas cell proliferation is the restoration of GJIC and decreased autocrine growth factors.

[Inhibitory effects of siRNA targeting epidermal growth factor receptor on proliferation and invasion of human glioblastoma cells].

OBJECTIVE: To study the inhibitory effects of siRNA targeting epidermal growth factor receptor (EGFR) on the proliferation and invasion of human glioblastoma cells. METHOD: Two siRNA expression constructs using psiRNA-NeoG2 vector, that targeted sequences of human EGFR receptor L domain (516 - 536) and catalytic domain (2400 - 2420) respectively, were constructed. Human malignant glioma cells of the line TJ905 were cultured in vitro and transfected with pcDNA3-hEGFR, anti-sense RNA, blank vector psiRNA-NeoG2 (as negative control), psiRNA-NeoG2-516, and psiRNA-NeoG2-2400 respectively mediated by LipofectAMINE. Immunofluorescence assay and Western blotting were used to detect the EGFR expression. Cell apoptosis was detected by apoptotic index (AI) using TUNEL method. Cell cycle was analyzed by flow cytometry, and cell proliferative activities were measured by MTT. The expression and enzymatic activities of matrix metalloproteinase 9 (MMP-9) were measured by Western blotting and gelatin zymography, and cell invasive capabilities were evaluated by Transwell-ECM method. RESULT: Immunofluorescence assay and Western blotting showed that the expression of EGFR was down-regulated by 90% and 92% respectively in the siRNA constructs transfected groups, while down-regulated by 82% in the antisense EGFR RNA transfected cells in comparison with the TJ905 cells and the cells transfected with blank vector. TUNEL assay showed that almost no apoptotic cell was found in the parental cells or the cells transfected with blank vector, however, apoptosis was increased in antisense EGFR transfected cells (AI = 7.2) and siRNA constructs transfected cells (AI = 13.7 and 14.7; chi(2) = 31.549, P < 0.001). Flow cytometric analysis showed that the S phase fraction (SPF) was lowered in both siRNA constructs transfected cells than that in the parental cells, the cells transfected with blank vector, and the antisense EGFR transfected cells. MTT assay indicated that compared to the parental cells and the cells transfected with blank vector, the survival rates of transfected cells dramatically dropped down from the first day after implantation (P < 0.05), the siRNA transfected cells demonstrated much lower survival rate than the antisense EGFR transfected cells. Meanwhile, the expression and enzymatic activities of MMP-9 decreased significantly after the transfection of antisense EGFR into the TJ905 cells compared to the TJ905 cells and the cells transfected with blank vector, and were much lower in the siRNA groups than that in the antisense groups (P < 0.05). Cell invasive capability assay demonstrated the similar inhibitory results in the Transwell ECM-Matrigel study. CONCLUSION: Compared with antisense approach, siRNA expression constructs targeting EGFR specifically suppresses the EGFR expression, induces gene silencing, and inhibits cell growth and invasion. The plasmid-based siRNA approach should be a new strategy in glioma gene therapy targeting EGFR.

[Preliminary study on the gene expression profiles of ependymomas with cDNA array].

OBJECTIVE: To investigate the differential gene expression of ependymomas. METHODS: Four fresh samples of ependymomas and 1 of normal brain tissue were collected during operation. The extracted total RNAs were converted as (32)P tagged cDNA probes, which were then hybridized with the Atlas Human Cancer Array, producing the array based hybridization maps following the protocol provided with the kit. A set of special software was applied to the analysis and RT-PCR was performed to test the result. RESULT: In comparison with the normal brain tissue, there were 31 upregulated gene and 1 downregulated gene in ependymomas, most of which were firstly found to be differentially expressed in this kind of tumor. CONCLUSION: The discrepancy of gene expression profiles between ependymomas and normal brain tissues is highly put through and effectively detected with cDNA array, which provides new information for the further research on the molecular mechanisms of this lesion.

Downregulation of Dicer enhances tumor cell proliferation and invasion.

miRNAs are non-coding, single-stranded RNAs that regulate target gene expression by repressing translation or promoting RNA cleavage. Dicer is an essential component of the miRNA processing machinery. To identify a role for miRNAs in tumorigenesis, we designed an adenovirus expressing small hairpin RNA (shRNA) to silence Dicer and globally suppress the maturation of miRNAs. We identified that the impairment of miRNA processing conferred an enhanced proliferative activity and invasive ability on each of three tumor cell lines in vitro. Inhibition of Dicer was associated with activation of p-Akt and enhanced expression of the cell cycle associating molecules, cyclin A and PCNA, as well as MMP-2 and MMP-9, proteins involved in tumor cell invasion. Adenoviral gene silencing of Dicer in subcutaneous MCF-7 xenografts significantly increased tumor growth in vivo compared to tumors infected with non-loading adenovirus. Increased tumor growth was associated with p-Akt activation and upregulation of cyclin A, PCNA MMP-2 and MMP-9. These findings demonstrate that global reduction of miRNA processing by silencing Dicer enhances tumor proliferation and invasion, and the p-Akt pathway may contribute to this phenotype via the downstream molecules, cyclin A, PCNA, MMP-2 and MMP-9.

Role of the AKT pathway in microRNA expression of human U251 glioblastoma cells.

Activation of the AKT (serine-threonine kinase) pathway is a common feature in glioblastoma cells. Downstream factors of the AKT pathway are involved in cell proliferation, apoptosis, cellular migration and angiogenesis. Micro-RNAs (miRNAs) are highly conserved small non-coding RNAs that block targeted mRNA expression at the post-transcriptional level. The aim of this study was to investigate the role of the AKT pathway in regulating miRNA. The changes of miRNA expression profile in human glioblastome U251 cells after AKT small interfering RNA transfection were examined by a microarray, and confirmed by Northern blotting. Down-regulation of AKT expression by siRNA decreased the activity of AKT pathway in U251 cells. Interruption of AKT pathway suppressed the expression of NF-kappaB and c-Myc, furthermore, the expression of a set of miRNAs was also changed after AKT siRNA transfection. There are putative binding sites of NF-kappaB and c-Myc in the promoters of several up-regulated miRNAs, indicating these transcription factors may also be involved in the regulation of miRNA expression, thus affecting the activity of AKT pathway in tumorigenesis. We provide new components of the regulatory function of AKT pathway to better understand the regulatory network mediated by downstream transcription factors. The understanding of the regulatory function of AKT pathway is crucial in tailored therapy of gliomas.

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.

Combination gene therapy with PTEN and EGFR siRNA suppresses U251 malignant glioma cell growth in vitro and in vivo.

The over-expression/amplification of the epidermal growth factor receptor (EGFR) gene and mutation/deletion of tumor suppressor PTEN gene are main genetic changes identified in glioblastomas. These two genetic changes play a critical role in the formation of many malignant tumors and have been shown to be the important therapeutic targets. In this study, we used an expression plasmid that expresses small hairpin RNA-targeting sequences of human EGFR and wild-type PTEN cDNA to examine the growth inhibitive effects in U251 glioma cells. It was found that down-regulation of EGFR expression and up-regulation of PTEN expression resulted in the suppression of cell proliferation, arrest of cell cycle, reduction in cell invasion and promotion of cell apoptosis in vitro. In addition, the growth of the subcutaneous U251 glioma in the nude mice treated with expression plasmid was significantly inhibited. Our results demonstrated that the expression plasmid could exert proliferation and invasion inhibition effects on U251 cells in vitro and in vivo. It suggested that combinatory gene therapy targeting EGFR and PTEN would be a new strategy in gene therapy of glioblastoma.

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.