MicroRNA-564 is downregulated in glioblastoma and inhibited proliferation and invasion of glioblastoma cells by targeting TGF-?1.
ABSTRACT: Increasing evidence has indicated that aberrant expression of miRNAs has been shown to be strongly implicated in the initiation and progression of glioblastoma. Here, we identified a novel tumor suppressive miRNA, miR-564, and investigated its role and therapeutic effect for glioblastoma. We showed that miR-564 was down-regulated in human glioblastoma tissues and cell lines. Introduction of miR-564 dramatically inhibited cell growth and invasion in glioblastoma cells. Subsequent experiments revealed that Transforming growth factor-?1 (TGF-?1) was a direct and functional target of miR-564 in glioblastoma cells. Furthermore, overexpression of miR-564 decreased p-SMAD and SMAD4 expression, which are the downstream signaling molecules of TGF-?. Meanwhile, ectopic of miR-564 reduced the messenger RNA (mRNA) and protein expression of epidermal growth factor receptor (EGFR) and MMP9. Furthermore, the upregulation of miR-564 suppressed TGF-?-mediated U87 proliferation and migration. The expression of EGFR and MMP9 was upregulated in glioblastoma tissues compared to their normal tissues. The EGFR and MMP9 expression levels were inverse correlated with the expression of miR-564. miR-564 suppressed the growth of U87-engrafted tumors. These findings reveal that miR-564/TGF-?1 signaling that may be required for glioblastoma development and may consequently serve as a new therapeutic target for the treatment of glioblastoma.
Project description:Glioblastoma multiformae (GBM) is the most aggressive type of malignant brain tumor with complex molecular profile. Overexpression of Na+/H+ Exchanger isoform 9 (NHE9) promotes tumor progression and correlates positively with insensitivity to radiochemotherapy and poor prognosis. However, molecular mechanisms responsible for increase in NHE9 levels beyond a critical threshold have not been identified.Bioinformatics analysis, luciferase reporter assays, real-time PCR and western blotting were conducted to examine the expression profiles and identify microRNAs (miRNA) that target NHE9. Cell proliferation and migration assays were conducted in U87 glioblastoma cells to determine the consequence of miRNA mediated targeting of NHE9. Endosomal pH measurements, immunofluorescence microscopy and surface biotinylation experiments were conducted to characterize the mechanistic basis of regulation.We show that microRNA 135a (miR-135a) targets NHE9 to downregulate its expression in U87 cells. MiR-135a levels are significantly lower in glioblastoma cells compared to normal brain tissue. Downregulation of NHE9 expression by miR-135a affects proliferative and migratory capacity of U87 cells. Selectively increasing NHE9 expression in these cells restored their ability to proliferate and migrate. We demonstrate that miR-135a takes a two-pronged approach affecting epidermal growth factor receptors (EGFRs) to suppress tumor cell growth and migration. EGFR activity is a potent stimulator of oncogenic signaling. While miR-135a targets EGFR transcripts to decrease the total number of receptors made, by targeting NHE9 it routes the few EGFRs made away from the plasma membrane to dampen oncogenic signaling. NHE9 is localized to sorting endosomes in glioblastoma cells where it alkalinizes the endosome lumen by leaking protons. Downregulation of NHE9 expression by miR-135a acidifies sorting endosomes limiting EGFR trafficking to the glioblastoma cell membrane.We propose downregulation of miR-135a as a potential mechanism underlying the high NHE9 expression observed in subset of glioblastomas. Future studies should explore miR-135a as a potential therapeutic for glioblastomas with NHE9 overexpression.
Project description:Glioblastoma multiforme (GBM) is the most common and highly malignant primary brain tumor, which is virtually incurable due to its therapeutic resistance to radiation and chemotherapy. To develop novel therapeutic approaches for treatment of GBM, we examined the role of miR-378 on tumor growth, angiogenesis, and radiation response in ectopic and orthotopic U87 glioblastoma models. Cell and tumor growth rates, in vitro and in vivo radiation sensitivities, and tumor vascular density were evaluated in U87-GFP and U87-miR-378 tumor lines. Ectopic tumor response to radiation was evaluated under normal blood flow and clamp hypoxic conditions. Results show that in vitro, miR-378 expression moderately increased cell growth rate and plating efficiency, but did not alter radiation sensitivity. U87-miR-378 tumors exhibited a higher transplantation take rate than U87-GFP tumors. In vivo, under oxygenated condition, subcutaneous U87-miR-378 tumors receiving 25 Gy showed a tendency for longer tumor growth delay (TGD) than control U87-GFP tumors. In contrast, under hypoxic condition, U87-miR-378 xenografts exhibited substantially shorter TGD than U87-GFP tumors, indicating that under normal blood flow conditions, U87-miR-378 tumors were substantially more oxygenated than U87-GFP tumors. Intracranial multi-photon laser-scanning microscopy demonstrated increased vascular density of U87-miR-378 versus control U87-GFP tumors. Finally, miR-378 increased TGD following 12 Gy irradiation in U87 intracranial xenografts, and significantly prolonged survival of U87-miR-378 tumor-bearing mice (P?=?0.04). In conclusion, higher miR-378 expression in U87-miR-378 cells promotes tumor growth, angiogenesis, radiation-induced TGD, and prolongs survival of orthotopic tumor-bearing hosts. Regulation of VEGFR2 by miR-378 significantly increased vascular density and oxygenation in U87 xenografts.
Project description:Paeoniflorin (PF) is a polyphenolic compound derived from Radix Paeoniae Alba thathas anti-cancer activities in a variety of human malignancies including glioblastoma. However, the underlying mechanisms have not been fully elucidated. Epithelial to mesenchymal transition (EMT), characterized as losing cell polarity, plays an essential role in tumor invasion and metastasis. TGF?, a key member of transforming growth factors, has been demonstrated to contribute to glioblastoma aggressiveness through inducing EMT. Therefore, the present studies aim to investigate whether PF suppresses the expression of TGF? and inhibits EMT that plays an important role in anti-glioblastoma. We found that PF dose-dependently downregulates the expression of TGF?, enhances apoptosis, reduces cell proliferation, migration and invasion in three human glioblastoma cell lines (U87, U251, T98G). These effects are enhanced in TGF? siRNA treated cells and abolished in cells transfected with TGF? lentiviruses. In addition, other EMT markers such as snail, vimentin and N-cadherin were suppressed by PF in these cell lines and in BALB/c nude mice injected with U87 cells. The expression of MMP2/9, EMT markers, are also dose-dependently reduced in PF treated cells and in U87 xenograft mouse model. Moreover, the tumor sizes are reduced by PF treatment while there is no change in body weight. These results indicate that PF is a potential novel drug target for the treatment of glioblastoma by suppression of TGF? signaling pathway and inhibition of EMT.
Project description:MicroRNAs (miRNAs) act an important role in the progression of tumor. In this study, we showed that the serum expression of miR-365 was downregulated in the glioblastoma compared with in the healthy controls. We also demonstrated that miR-365 expression was downregulated in glioblastoma tissues compared with the adjacent normal tissues. Overexpression of miR-365 suppressed the glioblastoma cell proliferation and migration. Moreover, ectopic expression of miR-365 promoted the expression of Ecadherin while inhibited the expression of N-cadherin and Vimentin in U87 cell. Furthermore, we identified PAX6 as a direct target gene of miR-365 in U87 cell. Overexpression of miR-365 suppressed glioblastoma cell proliferation and migration and epithelial-to-mesenchymal transition through inhibiting PAX6 expression. These results suggested that miR-365 played a tumor suppressor in glioblastoma.
Project description:Background:MicroRNAs (miRNAs) play an important role in cancer initiation, progression, and metastasis by directly regulating their target genes. Materials and methods:In this study, we observed that the miR-1225-5p expression level in glioblastoma tissues was significantly lower as compared with that in normal brain tissues, and its low expression was significantly associated with histopathological grade and poor patient prognosis. Results:Through establishing a miR-1225-5p overexpression glioblastoma cell line, we found that ectopic overexpression of miR-1225-5p inhibited the proliferation, migration, and invasion of glioblastoma cells in vitro. Moreover, the growth of a glioblastoma xenograft tumor was attenuated by overexpression of miR-1225-5p. Further integrative studies suggested that the insulin receptor substrate 1 (IRS1) was a direct functional target of miR-1225-5p in glioblastoma, and the mRNA and protein levels of IRS1 in six human glioblastoma cell lines (A172, SW1783, U87, LN-229, SW1088, and T98G) were significantly higher as compared with normal human astrocytes. Conclusion:These results suggest that miR-1225-5p may be a novel candidate for glioblastoma therapy.
Project description:Chloride intracellular channel 1 (CLIC1) is highly expressed and secreted by human glioblastoma cells and cell lines such as U87, initiating cell migration and tumor growth. Here, we examined whether CLIC1 could be transferred to human primary microvascular endothelial cells (HMEC). We previously reported that the oncogenic microRNA, miR-5096, increased the release of extracellular vesicles (EVs) by which it increased its own transfer from U87 to surrounding cells. Thus, we also examined its effect on the CLIC1 transfer. In homotypic cultures, miR-5096 did not increase the expression of CLIC1 in U87 nor in HMEC. However, the endothelial CLIC1 level increased after exposure to EVs released by U87, and even more by miR-5096-loaded U87. The EVs-transferred CLIC1 was active in HMEC, promoting endothelial sprouting in matrigel. Cell exposure to EVs induced cytosolic Ca2+ spikes which were dependent on the transient receptor potential melastatin member 7 (TRPM7). TRPM7 silencing prevented Ca2+ spikes and the subsequent CLIC1 delivery into HMEC. Our data suggest that the vesicular transfer of CLIC1 between cells requires TRMP7 expression in recipient endothelial cells. How the vesicular transfer of CLIC1 is modulated in cancer therapy is a future challenge.
Project description:The EGF receptor (EGFR) is amplified and mutated in glioblastoma, in which its common mutation (?EGFR, also called EGFRvIII) has a variety of activities that promote growth and inhibit death, thereby conferring a strong tumor-enhancing effect. This range of activities suggested to us that ?EGFR might exert its influence through pleiotropic effectors, and we hypothesized that microRNAs might serve such a function. Here, we report that ?EGFR specifically suppresses one such microRNA, namely miR-9, through the Ras/PI3K/AKT axis that it is known to activate. Correspondingly, expression of miR-9 antagonizes the tumor growth advantage conferred by ?EGFR. Silencing of FOXP1, a miR-9 target, inhibits ?EGFR-dependent tumor growth and, conversely, de-repression of FOXP1, as a consequence of miR-9 inhibition, increases tumorigenicity. FOXP1 was sufficient to increase tumor growth in the absence of oncogenic ?EGFR signaling. The significance of these findings is underscored by our finding that high FOXP1 expression predicts poor survival in a cohort of 131 patients with glioblastoma. Collectively, these data suggest a novel regulatory mechanism by which ?EGFR suppression of miR-9 upregulates FOXP1 to increase tumorigenicity.
Project description:Glioblastoma is one of the common types of primary brain tumors with a median survival of 12-15 months. The receptor tyrosine kinase (RTK) pathway is known to be deregulated in 88% of the patients with glioblastoma. 45% of GBM patients show amplifications and activating mutations in EGFR gene leading to the upregulation of the pathway. In the present study, we demonstrate that a brain specific miRNA, miR-219-5p, repressed EGFR by directly binding to its 3'-UTR. The expression of miR-219-5p was downregulated in glioblastoma and the overexpression of miR-219-5p in glioma cell lines inhibited the proliferation, anchorage independent growth and migration. In addition, miR-219-5p inhibited MAPK and PI3K pathways in glioma cell lines in concordance with its ability to target EGFR. The inhibitory effect of miR-219-5p on MAPK and PI3K pathways and glioma cell migration could be rescued by the overexpression of wild type EGFR and vIII mutant of EGFR (both lacking 3'-UTR and thus being insensitive to miR-219-5p) suggesting that the inhibitory effects of miR-219-5p were indeed because of its ability to target EGFR. We also found significant negative correlation between miR-219-5p levels and total as well as phosphorylated forms of EGFR in glioblastoma patient samples. This indicated that the downregulation of miR-219-5p in glioblastoma patients contribute to the increased activity of the RTK pathway by the upregulation of EGFR. Thus, we have identified and characterized miR-219-5p as the RTK regulating novel tumor suppressor miRNA in glioblastoma.
Project description:Glioblastoma (GBM) is a highly heterogeneous type of tumor characterized by genomic and signaling abnormalities affecting pathways involved in control of cell fate, including tumor-suppressor- and growth factor-regulated pathways. An aberrant miRNA expression has been observed in GBM, being associated with impaired cellular functions resulting in malignant transformation, proliferation and invasion. Here, we demonstrate for the first time that platelet-derived growth factor-B (PDGF-B), a potent angiogenic growth factor involved in GBM development and progression, promotes downregulation of pro-oncogenic (miR-21) and anti-oncogenic (miR-128) miRNAs, as well as upregulation/downregulation of several miRNAs involved in GBM pathology. Retrovirally mediated overexpression of PDGF-B in U87 human GBM cells or their prolonged exposure, as well as that of F98 rat glioma cells to this ligand, resulted in decreased miR-21 and miR-128 levels, which was associated with increased cell proliferation. Furthermore, siRNA-mediated PDGF-B silencing led to increased levels of miR-21 and miR-128, while miRNA modulation through overexpression of miR-21 did not alter the levels of PDGF-B. Finally, we demonstrate that modulation of tumor suppressors PTEN and p53 in U87 cells does not affect the decrease in miR-21 levels associated with PDGF-B overexpression. Overall, our findings suggest that, besides its role in inducing GBM tumorigenesis, PDGF-B may enhance tumor proliferation by modulating the expression of oncomiRs and tumor suppressor miRNAs in U87 human GBM cells.
Project description:Inflammatory cytokines and transforming growth factor-? (TGF-?) are mutually inhibitory. However, hyperactivation of nuclear factor-?B (NF-?B) and TGF-? signaling both emerge in glioblastoma. Here, we report microRNA-148a (miR-148a) overexpression in glioblastoma and that miR-148a directly suppressed Quaking (QKI), a negative regulator of TGF-? signaling.We determined NF-?B and TGF-?/Smad signaling activity using pNF-?B-luc, pSMAD-luc, and control plasmids. The association between an RNA-induced silencing complex and QKI, mitogen-inducible gene 6 (MIG6), S-phase kinase-associated protein 1 (SKP1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was tested with microribonucleoprotein immunoprecipitation and real-time PCR. Xenograft tumors were established in the brains of nude mice.QKI suppression induced an aggressive phenotype of glioblastoma cells both in vitro and in vivo. Interestingly, we found that NF-?B induced miR-148a expression, leading to enhanced-strength and prolonged-duration TGF-?/Smad signaling. Notably, these findings were consistent with the significant correlation between miR-148a levels with NF-?B hyperactivation and activated TGF-?/Smad signaling in a cohort of human glioblastoma specimens.These findings uncover a plausible mechanism for NF-?B-sustained TGF-?/Smad activation via miR-148a in glioblastoma, and may suggest a new target for clinical intervention in human cancer.