Hypermethylation of the Promoter of miR-338-5p Mediates Aberrant Expression of ETS-1 and Is Correlated With Disease Severity Of Astrocytoma Patients

The pro-oncogene ETS-1 (E26 transformation-specific sequence 1) is a key regulator of the proliferation and invasion of cancer cells. The present work examined the correlation of the aberrant expression of ETS-1 with histological or clinical classification of astrocytoma: grade I (pilocytic astrocytoma), grade II (diffuse astrocytoma), grade III (anaplastic astrocytoma), and grade IV (glioblastoma multiforme). MicroRNA, miR-338-5p, was predicted by an online tool (miRDB) to potentially target the 3’ untranslated region of ETS-1; this was confirmed by multi-assays, including western blot experiments or the point mutation of the targeting sites of miR-338-5p in ETS-1’s 3’untralation region (3’UTR). The expression of miR-338-5p was negatively associated with that of ETS-1 in astrocytoma, and deficiency of miR-338-5p would mediate aberrant expression of ETS-1 in astrocytoma. Mechanistically, hypermethylation of miR-338-5p by DNA methyltransferase 1 (DNMT1) resulted in repression of miR-338-5p expression and the aberrant expression of ETS-1. Knockdown or deactivation of DNMT1 decreased the methylation rate of the miR-338-5p promoter, increased the expression of miR-338-5p, and repressed the expression of ETS-1 in astrocytoma cell lines U251 and U87. These results indicate that hypermethylation of the miR-338-5p promoter by DNMT1 mediates the aberrant expression of ETS-1 related to disease severity of patients with astrocytoma.

Berberine Photo-Activation Potentiates Cytotoxicity in Human Astrocytoma Cells through Apoptosis Induction

Photodynamic therapy (PDT) has recently attracted interest as an innovative and adjuvant treatment for different cancers including malignant gliomas. Among these, Glioblastoma (GBM) is the most prevalent neoplasm in the central nervous system. Despite conventional therapeutic approaches that include surgical removal, radiation, and chemotherapy, GBM is characterized by an extremely poor prognosis and a high rate of recurrence. PDT is a physical process that induces tumor cell death through the genesis and accumulation of reactive oxygen species (ROS) produced by light energy interaction with a photosensitizing agent. In this contribution, we explored the potentiality of the plant alkaloid berberine (BBR) as a photosensitizing and cytotoxic agent coupled with a PDT scheme using a blue light source in human established astrocytoma cell lines. Our data mainly indicated for the combined BBR-PDT scheme a potent activation of the apoptosis pathway, through a massive ROS production, a great extent of mitochondria depolarization, and the sub-sequent activation of caspases. Altogether, these results demonstrated that BBR is an efficient photosensitizer agent and that its association with PDT may be a potential anticancer strategy for high malignant gliomas.

Synthesis and Functionalisation of Superparamagnetic Nano-Rods towards the Treatment of Glioblastoma Brain Tumours

The complete removal of glioblastoma brain tumours is impossible to achieve by surgery alone due to the complex finger-like tentacle structure of the tumour cells and their migration away from the bulk of the tumour at the time of surgery; furthermore, despite aggressive chemotherapy and radiotherapy treatments following surgery, tumour cells continue to grow, leading to the death of patients within 15 months after diagnosis. The naturally occurring carnosine dipeptide has previously demonstrated activity against in vitro cultured glioblastoma cells; however, at natural physiological concentrations, its activity is too low to have a significant effect. Towards realising the full oncological potential of carnosine, the dipeptide was embedded within an externally triggered carrier, comprising a novel nano rod-shaped superparamagnetic iron oxide nanoparticle (ca. 86 × 19 × 11 nm) capped with a branched polyethyleneimine, which released the therapeutic agent in the presence of an external magnetic field. The new nano-carrier was characterized using electron microscopy, dynamic light scattering, elemental analysis, and magnetic resonance imaging techniques. In addition to cytotoxicity studies, the carnosine carrier’s effectiveness as a treatment for glioblastoma was screened in vitro using the U87 human glioblastoma astrocytoma cell line. The labile carnosine (100 mM) suppresses both the U87 cells’ proliferation and mobility over 48 h, resulting in significant reduction in migration and potential metastasis. Carnosine was found to be fully released from the carrier using only mild hyperthermia conditions (40 °C), facilitating an achievable clinical application of the slow, sustained-release treatment of glioblastoma brain tumours that demonstrates potential to inhibit post-surgery metastasis with the added benefit of non-invasive monitoring via MRI.

Expression of Remyelination-modulating Genes in Astrocytes is Regulated by MALAT1 and Lnc-DC Long Non-coding RNAs

Astrocyte-secreted factors play multifunctional roles in central nervous system (CNS) in health and disease. Here, we examined the regulatory machinery of long non-coding RNAs (lncRNAs) on gene expression of several factors of great importance in remyelination - CNTF, NT-3, FGF2 and PDGF-C - in human astrocytoma cell line via in silico and experimental studies. To know any expression correlations among these genes as well as their changes in inflammatory conditions, their expression was measured under H2O2 induction. Using available databases, a computational screening was performed to collect lncRNAs having the potentiality of regulating expression of the target genes. MALAT1 and Lnc-DC were selected as high potential expression regulators of four genes of the study among 40 lncRNAs that were evaluated bioinformatically. Downregulation of remyelination-modulating genes of interest under DNAzyme-induced suppression of MALAT1 or Lnc-DC verified the regulatory role of these lncRNAs. More detailed information on expression regulatory machinery of lncRNAs and remyelination-modulating genes in inflammatory conditions could pave the way for understanding the reasons of their inefficiency in demyelinating diseases such as Multiple Sclerosis (MS).

A Functional SNP rs895819 on Pre-miR-27a is Associated with Bipolar Disorder by Targeting ICAM-1.

Aberrant expression or genomic mutations of microRNA have been reported to be associated with a variety of human diseases. This study is aimed to analyze the association between genetic variations of miRNA and schizophrenia or bipolar disorder. We performed case-control studies for ten SNPs in a total sample of 1584 Chinese subjects. All these ten SNPs are on or near mature microRNAs which are expressed in the brain. To illustrate the function of miR-27a, we constructed a miR-27a knockout (KO) astrocytoma cell line U-251MG. By comparing the differentially expressed genes in wild-type and KO U-251MG, we determined several candidate target genes of miR-27a and then verified ICAM-1 as a target gene of miR-27a. We identified the association between the T/C polymorphism at rs895819 and bipolar disorder. Ectopic expression of miR-27a in NPCs showed that the T to C mutation at rs895819 reduced the suppressive effects of miR-27a on the expression of its target genes. Further studies revealed that the T/C polymorphism on miR-27a led to differential expression of mature and precursor miR-27a without affecting the expression of primary miR-27a. Our study highlighted the importance of miR-27a and its polymorphism at rs895819 in bipolar disorder.

Sulfatide-Rich Liposome Uptake by a Human-Derived Neuroblastoma Cell Line

Liposomes are bilayer membrane vesicles that can serve as vehicles for drug delivery. They are a good alternative to free drug administration that provides cell-targeted delivery into tumors, limiting the systemic toxicity of chemotherapeutic agents. Previous results from our group showed that an astrocytoma cell line exhibits selective uptake of sulfatide-rich (SCB) liposomes, mediated by the low-density lipoprotein receptor (LDL-R). The goal of this study was to assess the uptake of liposomes in a neuroblastoma cell line. For this purpose, we used two types of liposomes, one representing a regular cell membrane (DOPC) and another rich in myelin components (SCB). An astrocytoma cell line was used as a control. Characterization of liposome uptake and distribution was conducted by flow cytometry and fluorescence microscopy. Similar levels of LDL-R expression were found in both cell lines. The uptake of SCB liposomes was higher than that of DOPC liposomes. No alterations in cell viability were found. SCB liposomes were located near the cell membrane and did not colocalize within the acidic cellular compartments. Two endocytic pathway inhibitors did not affect the liposome uptake. Neuroblastoma cells exhibited a similar uptake of SCB liposomes as astrocytoma cells; however, the pathway involved appeared to be different than the hypothesized pathway of LDL-R clathrin-mediated endocytosis.

Extracellular Alpha-Synuclein Promotes a Neuroinhibitory Secretory Phenotype in Astrocytes

Multiple system atrophy (MSA) and dementia with Lewy bodies (DLB) are α-synucleinopathies that exhibit widespread astrogliosis as a component of the neuroinflammatory response. Munc18, a protein critical to vesicle exocytosis, was previously found to strongly mark morphologically activated astrocytes in brain tissue of MSA patients. Immunofluorescence of MSA, DLB and normal brain tissue sections was combined with cell culture and co-culture experiments to investigate the relationship between extracellular α-synuclein and the transition to a secretory astrocyte phenotype. Increased Munc18-positive vesicles were resolved in activated astrocytes in MSA and DLB tissue compared to controls, and they were also significantly upregulated in the human 1321N1 astrocytoma cell line upon treatment with α-synuclein, with parallel increases in GFAP expression and IL-6 secretion. In co-culture experiments, rat primary astrocytes pretreated with α-synuclein inhibited the growth of neurites of co-cultured primary rat neurons and upregulated chondroitin sulphate proteoglycan. Taken together, these results indicate that the secretory machinery is significantly upregulated in the astrocyte response to extracellular α-synuclein and may participate in the release of neuroinhibitory and proinflammatory factors in α-synucleinopathies.