Introduction: Glioblastoma (GBM) is the most common and lethal of the central nervous system (CNS) malignancies. The initiation, progression, and infiltration ability of GBMs are attributed in part to the dysregulation of microRNAs (miRNAs). Thus, targeting dysregulated miRNAs with RNA oligonucleotides (RNA interference, RNAi) has been proposed for GBM treatment. Despite promising results in the laboratory, RNA oligonucleotides have clinical limitations that include poor RNA stability and off-target effects. RNAi therapies against GBM confront an additional obstacle, as they need to cross the blood-brain barrier (BBB). Methods: Here, we developed gold-liposome nanoparticles conjugated with the brain targeting peptides apolipoprotein E (ApoE) and rabies virus glycoprotein (RVG). First, we functionalized gold nanoparticles with oligonucleotide miRNA inhibitors (OMIs), creating spherical nucleic acids (SNAs). Next, we encapsulated SNAs into ApoE, or RVG-conjugated liposomes, to obtain SNA-Liposome-ApoE and SNA-Liposome-RVG, respectively. We characterized each nanoparticle in terms of their size, charge, encapsulation efficiency, and delivery efficiency into U87 GBM cells in vitro. Then, they were administered intravenously (iv) in GBM syngeneic mice to evaluate their delivery efficiency to brain tumor tissue. Results: SNA-Liposomes of about 30-50 nm in diameter internalized U87 GBM cells and inhibited the expression of miRNA-92b, an aberrantly overexpressed miRNA in GBM cell lines and GBM tumors. Conjugating SNA-Liposomes with ApoE or RVG peptides increased their systemic delivery to the brain tumors of GBM syngeneic mice. SNA-Liposome-ApoE demonstrated to accumulate at higher extension in brain tumor tissues, when compared with non-treated controls, SNA-Liposomes, or SNA-Liposome-RVG. Discussion: SNA-Liposome-ApoE has the potential to advance the translation of miRNAbased therapies for GBM as well as other CNS disorders.
Glioblastoma multiforme (GBM) is the most common and deadliest type of primary brain tumor with a prognosis of 14 months after diagnosis. Current treatment for GBM patients includes “total” tumor resection, temozolomide-based chemotherapy, radiotherapy or a combination of these options. Although, several targeted therapies, gene therapy, and immunotherapy are currently in the clinic and/or in clinical trials, the overall survival of GBM patients has hardly improved over the last two decades. Therefore, novel multitarget modalities are urgently needed. Recently, RNA interference (RNAi) has emerged as a novel strategy for the treatment of most cancers, including GBM. RNAi-based therapies consist of using small RNA oligonucleotides to regulate protein expression at the post-transcriptional level. Despite the therapeutic potential of RNAi molecules, systemic limitations including short circulatory stability and low release into the tumor tissue have halted their progress to the clinic. The effective delivery of RNAi molecules through the blood-brain barrier (BBB) represents an additional challenge. This review focuses on connecting the translational process of RNAi-based therapies from in vitro evidence to pre-clinical studies. We delineate the effect of RNAi in GBM cell lines, describe their effectiveness in glioma mouse models, and compare the proposed drug carriers for the effective transport of RNAi molecules through the BBB to reach the tumor in the brain. Furthermore, we summarize the most important obstacles to overcome before RNAi-based therapy becomes a reality for GBM treatment.
Glioblastoma (GBM) is the most common and aggressive of all brain tumors, with a median survival of only 14 months after initial diagnosis. Novel therapeutic approaches are an unmet need for GBM treatment. MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene expression at the post-transcriptional level. Several dysregulated miRNAs have been identified in all cancer types including GBM. In this study, we aimed to uncover the role of miR-143 in GBM cell lines, patient samples, and mouse models. Quantitative real-time RT-PCR of RNA extracted from formalin-fixed paraffin-embedded (FFPE) samples showed that the relative expression of miR-143 was higher in GBM patients compared to control individuals. Transient transfection of GBM cells with a miR-143 oligonucleotide inhibitor (miR-143-inh) resulted in reduced cell proliferation, increased apoptosis, and cell cycle arrest. SLC30A8, a glucose metabolism-related protein, was identified as a direct target of miR-143 in GBM cells. Moreover, multiple injections of GBM tumor-bearing mice with a miR-143-inh-liposomal formulation significantly reduced tumor growth compared to control mice. The reduced in vitro cell growth and in vivo tumor growth following miRNA-143 inhibition suggests that miR-143 is a potential therapeutic target for GBM therapy.
Platelets play a vital role in hemostasis and inflammation. The membrane receptor TREM-like transcript-1 (TLT-1) is involved in platelet aggregation, bleeding, and inflammation, and it is localized in the α-granules of platelets. Upon platelet activation, TLT-1 is released from α-granules both in its transmembrane form and as a soluble fragment (sTLT-1). Higher levels of sTLT-1 have been detected in the plasma of patients with acute inflammation or sepsis, suggesting an important role for TLT-1 during inflammation. However, the roles of TLT-1 in hemostasis and inflammation are not well understood. We are developing the mouse model of TLT-1 to mechanistically test clinical associations of TLT-1 in health and disease. To facilitate our studies, monoclonal murine TLT-1 (mTLT-1) antibodies were produced by the immunization of a rabbit using the negatively charged region of the mTLT-1 extracellular domain PPVPGPREGEEAEDEK. In the present study, we demonstrate that two selected clones, 4.6 and 4.8, are suitable for the detection of mTLT-1 by western blot, immunoprecipitation, immunofluorescent staining, flow cytometry and inhibit platelet aggregation in aggregometry assays. In addition, we found that the topical administration of clone 4.8 delayed the wound healing process in an experimental burn model. These results suggest that TLT-1 plays an important role in wound healing and because both clones specifically detect mTLT-1, they are suitable to further develop TLT-1 based models of inflammation and hemostasis in vivo.
Glioblastoma is the most aggressive type of primary brain tumor with an overall survival of 14.6 months with standard care treatment (surgery, radiotherapy, and Temozolomide chemotherapy). About 90% of patients develop recurrent tumors due to acquired resistance to temozolomide. Therefore, new multimodal treatments that target more than one molecular pathway are needed. MicroRNAs (miRNAs) are small (18–22 nucleotides) non‐coding RNAs that regulate more than 60% of protein coding genes post‐transcriptionally. Their dysregulation is implicated in many diseases including diabetes, heart disease, and cancer. MicroRNA‐92b (miR92b) is highly upregulated in glioblastoma tumors and its expression has an inverse correlation with patient overall survival. This study aims to determine the biological effect of targeting miR92b in glioblastoma. We hypothesized that its inhibition would lead to cell death and decreased proliferation by allowing the expression of messenger RNA (mRNA) targets needed for normal cell function. To determine miR92b targeting effect in vitro, we transfected glioblastoma cell lines with an anti‐miR92b and evaluated cellular apoptosis, clonogenicity, and proliferation. Results showed that by inhibiting miR92b there was an increase in apoptotic cells and reduction of cell proliferation. We next evaluated the miR92b targeting effect in vivo, administering intraperitoneal injections of anti‐miR92b encapsulated liposomes to glioblastoma‐xenograft mice. Inhibiting miR92b in mice xenografts led to a significant decrease in tumor volume and tumor weight. Furthermore, we performed bioinformatic analysis to identify mRNA potential targets of miR92b. We both inhibited and overexpressed miR92b in glioblastoma cells (U87 and CRL1620 respectively) and determined the expression of more than 90 potential mRNA targets by quantitative PCR. This analysis lead to the identification of five potential miR92b mRNA targets‐ASB5, TEF, KIAA1024, ZNF776, FBXW7 and BAZ2‐ which are currently under further validation using Western blot analysis. Our results show that miR92b is a good therapeutic target, since its inhibition leads to increased cell death and decreased cellular proliferation both in vitro and in vivo. MiR92b is a potential target for glioblastoma patients and its targeting could lead to the regulation of tumor suppressive protein coding genes.Support or Funding InformationNIGMS‐RISE Grant Number R25‐GM061838, RCMI grant U54 MD007600 (National Institute of Health and Health Disparities), and UPR Comprehensive Center Seed Funds.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The purpose of this study is to assess the biological role of the microRNA-143 (miR-143) in Glioblastoma multiforme (GBM). GBM is the most common and lethal of all brain tumors. In the United States, the incidence of GBM is about 17% of all primary brain tumors and about 60-75% of all Astrocytomas (American Brain Tumor Association, 2014). The standard therapy is surgical tumor removal followed by chemotherapy and radiotherapy. However, many patients recur after treatment and the median survival rate for GBM has remained 15 months for the past 20 years. Thus, novel therapies for GBM treatment are urgently necessary. MicroRNAs (miRNAs) are a class of small non-coding RNAs (18-22 nucleotides in length) that regulate gene expression at the post-transcriptional level. MiRNAs bind to the 3’-untranslate region (UTR) of messenger RNAs (mRNAs) and regulate protein synthesis. Several deregulated miRNAs have been identified in all cancer types including GBM. In this study we aim to thoroughly uncover the role of miR-143 by using GBM cell lines, mouse models and patient samples. Total RNA was isolated from FFPE samples from brain tumor patients. TaqMan-based Real-time PCR showed that the relative expression of miR-143 was higher in GBM patients compared to control individuals, and with paired surrounding non-cancerous tissue. Furthermore, GBM cells transiently transfected with a miR-143 oligonucleotide inhibitor showed reduced cell proliferation (68.5%) (clonogenicity assay), increased apoptosis and cell cycle arrest of GBM cells in the S phase (Flow cytometry and Western blots). In vivo studies using primary GBM cells injected in the flank of nude mice showed that repeated doses of miR-143-inhibitor liposomal formulation increased the tumor size compared with control mice. These contradictory results could be due to effects of the microenvironment where the tumor is growing. Further studies will be made using intracranial injections in an orthotopic xenograft mouse model to confirm this hypothesis. Western blot analysis and luciferase reporter assays are also underway to identify novel miR-143 target genes in GBM cells. This research project is being supported by: PRCTRC: NCRR (U54 RR 026139-01A1), NIMHD (8U54 MD 007587-03), and RCMI: MBRS-RISE, NCRR (2G12-RR003051) and NIMHD (8G12-MD007600) from the NIH. Citation Format: Eunice Lozada-Delgado, Fatma Valiyeva, Maria Marcos, Pablo Vivas. Targeting microRNA-143 in glioblastoma in vivo increases tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3437. doi:10.1158/1538-7445.AM2017-3437
The purpose of this study is to assess the biological role of microRNA-143 (miR-143) in Glioblastoma multiforme (GBM). In the United States, the incidence of GBM is about 17% of all primary brain tumors and about 60-75% of all Astrocytomas. Many patients recur even after therapy with a median survival rate of 15 months. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression post-transcriptionally. Deregulated miRNAs have been identified in cancer including GBM. In this study, we aim to thoroughly uncover the role of miR-143 by using GBM cell lines, mouse models, and patient samples. Previous qPCR data from FFPE samples of Puerto Rican GBM patients showed a higher relative expression of miR-143 in GBM patients compared to control individuals and paired surrounding non-cancerous tissue suggesting an oncogenic role. Moreover, in vitro data of transient transfections of a miR-143 oligonucleotide inhibitor in GBM cells showed reduced cell proliferation (clonogenic assay), increased apoptosis and cell cycle arrest in the G0/G1-S phase (flow cytometry and western blot analysis). Furthermore, we want to identify novel target mRNAs of miR-143 in GBM cells that may explain these results. Using in silico analysis tools and a qPCR analysis we identified 5 candidate genes as potential miR-143 targets. Western blot analysis of the proteins encoded by these genes using miR-143 overexpressing cells showed that the Integral membrane protein 2B (ITM2B) and Zinc transporter 8 (SLC30A8) protein levels were reduced and when miR-143 was transiently inhibited their protein levels where increased. These results suggest that miR-143 targets these mRNAs. Ongoing luciferase reporter assays will determine direct binding of miR-143 to the 3'UTR of these mRNAs in GBM cells. Citation Format: Eunice Lozada-Delgado, Fatma Valiyeva, Maria Marcos, Pablo Vivas. Identification of novel target mRNAs of microRNA-143 in glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 527.
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