Expert opinion on translational research for advanced glioblastoma treatment

Malignant gliomas are known to be one of the most difficult diseases to diagnose and treat because of the infiltrative growth pattern,rapid progression,and poor prognosis.Many antitumor drugs are not ideal for the treatment of gliomas due to the blood-brain barrier.Temozolomide(TMZ)is a DNA alkylating agent that can cross the blood-brain barrier.As the only first-line chemotherapeutic drug for malignant gliomas at present,TMZ is widely utilized to provide a survival benefit;however,some patients are inherently insensitive to TMZ.In addition,patients could develop acquired resistance during TMZ treatment,which limits antitumor efficacy.To clarify the mechanism underlying TMZ resistance,numerous studies have provided multilevel solutions,such as improving the effective concentration of TMZ in tumors and developing novel small molecule drugs.This review discusses the in-depth mechanisms underlying TMZ drug resistance,thus aiming to provide possibilities for the establishment of personalized therapeutic strategies against malignant gliomas and the accelerated development and transformation of new targeted drugs.

MicroRNA and Brain Tumors

MicroRNAs （miRNAs） were first described in 1993 by Lee and colleagues, and the term microRNA was only introduced in 2001 in a set of three articles in Science. One of the biggest surprises in the past few years has been the emergence of miRNAs as a major new class of gene expression regulators. Recent studies suggest that miRNA alterations are involved in the initiation and progression of human cancer. The brain tumor, glioblastoma multiforme, is the most malignant and deadly form of gliomas. The prognosis is poor and the median survival with combined radiotherapy and chemotherapy is only 14.6 months. With the discovery of miRNA, the miRNA profiles may become useful biomarkers for brain tumor diagnostics, and miRNA therapy could be a powerful tool for brain tumor prevention and therapeutics. This review outlines the background of miRNA and its expression and therapeutic potential for brain tumors.

siRNA epidermal growth factor receptor silencing in U251 glioma cells

BACKGROUND： Dicer, a large multidomain ribonuclease, is responsible for processing double-stranded RNAs （dsRNAs） to 20-bp-long small interfering RNAs （siRNAs）, which act as effectors during RNA interference （RNAi）. OBJECTIVE： To observe the efficacy of siRNA cocktails generated by recombinant human Dicer on the down-regulation of epidermal growth factor receptor （EGFR） expression in human glioma cells. DESIGN, TIME AND SETTING： The following in vitro experiment was performed at the Department of Neurosurgery, Tianjin Medical University General Hospital and Laboratory of Neuro-Oncology, Tianjin Neurological Institute. MATERIALS： Mini-RNA isolation kit, human placenta complimentary DNA （cDNA） was produced by Tiangen Biotech （Beijing, China）, human glioblastoma U251-MG cells were produced by the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. METHODS： A PCR product from the human EGFR, which corresponded to the tyrosine kinase domain of the 3＇-end fragment, was used as the T7-promotor for in vitro transcription. siRNA cocktails were generated by in vitro dicing of double stranded RNA. A total of 500, 250 and 125 μg siRNA cocktails were transiently transfected into U251 glioma cells through the use of the GeneSilencer. MAIN OUTCOME MEASURE： Expression of EGFR was detected by real-time PCR. RESULTS： The total PCR product of the human EGFR, corresponding to the tyrosine kinase domain, is approximately 680 bp in length. The PCR transcriptants included GCC leader sequences and a T7 promoter sequence, with a fragment of EGFR cDNA at the center. The T7 promoter was prepared for in vitro transcription of dsRNA. After dicing for 24 hours, the 21-nt siRNA cocktails were verified by 4% agarose gel. The difference between threshold cycle of a sample assay and threshold cycle of the corresponding endogenous reference （ΔCt） among parental U251 cells and cells transfected with different doses of siRNA cocktails were determined to be 3.06, 7.35, and 10.31 cycles, respectively. These results indicated effective silencing of EGFR expression by siRNA cocktails through transient transfection. CONCLUSION： The siRNA cocktails significantly suppressed exogenous expression of EGFR in U251 glioblastoma cells in a dose-dependent manner, thereby providing a more promising RNAi methodology for glioma therapy.

AKT1 and AKT2 Promote Malignant Transformation in Human Brain Glioma LN229 Cells

OBJECTIVE To confirm the role played by AKT1 and AKT2 in the β- catenin/Tcf-4 signaling pathway in promoting malignant transfor- mation of glioma cells. METHODS LN229 cells were divided into five groups： a control group, acetone （ACE）group, acetylsalicylic acid （ASA; aspirin） group, ASA＋AKT1 plasmid group and ASA＋AKT2 plasmid group. Western blot and PCR were used to detect the expression of AKT1 and AKT2 after dealing with ASA and transferring AKTI/2 genes into LN229 cells. Cell proliferation was determined by flow cytometry, cell invasion was evaluated by transwell assay and cell apoptosis was detected with annexin V staining. The molecules regulating proliferation and invasion were examined by western blot analysis. RESULTS Aspirin down-regulates AKT1 and AKT2 expression by modulating β-cateninfrcf-4 activity. AKT1 and AKT2 can enhance cell proliferation and invasion by up-regulating the expression of cyclin-D and matrix metalloprotein-9 （MMP-9） in LN229 glioma cells. CONCLUSION AKT1 and AKT2 play an important role in the β- catenin/Tcf-4 signaling pathway promoting malignant transformation; AKT1 is more effective than AKT2. AKT1 and AKT2 may be potential targets for brain glioma therapy and an effective way to prevent metastasis of gliomas.

Glioma-conditioned Medium Blocks Endothelial Cells' Apoptosis Induced by Hypoxia and Promotes Its Angiogenesis via Up-regulation of u-PA/u-PAR

Objective： To investigate the role of urokinase-type plasminogen activator/urokinase-type plasminogen receptor（u-PA/u-PAR） system in glioma angiogenesis under hypoxic conditions, we studied the effect of glioma-conditioned medium on the hypoxia induced changes in human endothelial-like ECV304 cells proliferation, apoptosis, cord formation in vitro and u-PA/u-PAR expression. Methods： MTT assay was used to examine the changes in cell proliferation. Cell apoptosis was analyzed by Hoechst 33258 staining. Matrigel cord-like formation assay was used to evaluate the angiogenesis ability of ECV304 cells in vitro. Expressions of u-PA/u-PAR mRNA were detected by quantitative real-time RT-PCR. Results： Hypoxia inhibited ECV304 cells proliferation and induced cell apoptosis. Hypoxic conditioned medium（H-CM） while not normoxic conditioned medium（N-CM） of U251 glioma ceils partially blocked the effect of hypoxia on ECV304 cells proliferation and apoptosis. H-CM of U251 glioma ceils also promoted the cord formation of ECV304 cells seeded on matrigel. When u-PA or u-PAR monoclonal antibodies were added into ECV304 cells culturing medium, cord formation ability was partially inhibited. H-CM of U251 glioma cells induced uPA and uPAR expression in ECV304 cells. Conclusion： These suggest that u-PA/u-PAR system is involved in glioma angiogenesis trigged by hypoxic microenviroment.

Role of Dicer on tumorigenesis in glioma cells

Micro RNAs（miRNAs）are non-coding,single-stranded RNAs that regulate target gene expression by repressing translation or promoting RNA cleavage.Recent studies show that miRNA expression is globally decreased in some human tumors.Dicer is an essential component of the miRNA processing machinery.To determine whether global reduction of miRNA effects tumorigenesis,small interfering RNA were designed to target Dicer to restrain whole miRNA expression in the glioblastoma cell line-TJ905.With effective knock-down of Dicer,tumor cells were invasive and proliferative,and globally impaired miRNA processing enhanced proliferation and invasiveness of glioma cells in vitro.Suppression of Dicer expression resulted in a more aggressive glioma phenotype,which suggests that global reduction of miRNA expression could have an oncogenic role in glioblastoma cells.

Up-regulation of SEPT7 Gene Inhibits Migration and Invasion of Human Glioma Cells in vitro and in vivo

The aim of this study was to explore the potential role of SEPT7 in glioma cell invasion. From in vitro experiments, we observed that the migratory and invasive abilities were inhibited in human glioblastoma U251MG and TJ899 cells after transfection with SEPT7 recombinant adenovirus constructs (Ad-SEPT7) as evaluated by Transwell assay, 3D Matrigel growth, 2D Matrigel growth and scratch assays. We further investigated the molecular events associated with the alteration of cell migration and invasion by immunohistochemistry and immuno uores-cence staining, Western blot and laser scanning confocal micros-copy analyses, and found the decreased expression of MMP2, MMP9, MT1-MMP and integrin αvβ3, increased expression of TIMP1 and TIMP2, and redistribution of intracellular cytoskel-eton tubulin-α. From in vivo study, it was demonstrated that the tumor growth rate in nude mice bearing xenograft subcutaneous U251 gliomas treated with Ad-SEPT7 was signi cantly slowed during the observation period of 4 weeks and the tumor volumes were much smaller than those in control and empty vector treated group. The expression of MMP2, MMP9, MT1-MMP and integrin αvβ3 was markedly inhibited while the expression of SEPT7, TIMP1, TIMP2 was upregulated in tumors treated with Ad-SEPT7. Taken together, these results suggest that SEPT7 plays an important role in glioma cell invasion and growth and it may be a candidate target for gene therapy of invasive gliomas.

Blockage of EGFR/AKT and mevalonate pathways synergize the antitumor effect of temozolomide by reprogramming energy metabolism in glioblastoma

Background Metabolism reprogramming plays a vital role in glioblastoma(GBM)progression and recurrence by producing enough energy for highly proliferating tumor cells.In addition,metabolic reprogramming is crucial for tumor growth and immune-escape mechanisms.Epidermal growth factor receptor(EGFR)amplification and EGFR-vIII mutation are often detected in GBM cells,contributing to the malignant behavior.This study aimed to investigate the functional role of the EGFR pathway on fatty acid metabolism remodeling and energy generation.Methods Clinical GBM specimens were selected for single-cell RNA sequencing and untargeted metabolomics analysis.A metabolism-associated RTK-fatty acid-gene signature was constructed and verified.MK-2206 and MK-803 were utilized to block the RTK pathway and mevalonate pathway induced abnormal metabolism.Energy metabolism in GBM with activated EGFR pathway was monitored.The antitumor effect of Osimertinib and Atorvastatin assisted by temozolomide(TMZ)was analyzed by an intracranial tumor model in vivo.Results GBM with high EGFR expression had characteristics of lipid remodeling and maintaining high cholesterol levels,supported by the single-cell RNA sequencing and metabolomics of clinical GBM samples.Inhibition of the EGFR/AKT and mevalonate pathways could remodel energy metabolism by repressing the tricarboxylic acid cycle and modulating ATP production.Mechanistically,the EGFR/AKT pathway upregulated the expressions of acyl-CoA synthetase short-chain family member 3(ACSS3),acyl-CoA synthetase long-chain family member 3(ACSL3),and long-chain fatty acid elongation-related gene ELOVL fatty acid elongase 2(ELOVL2)in an NF-κB-dependent manner.Moreover,inhibition of the mevalonate pathway reduced the EGFR level on the cell membranes,thereby affecting the signal transduction of the EGFR/AKT pathway.Therefore,targeting the EGFR/AKT and mevalonate pathways enhanced the antitumor effect of TMZ in GBM cells and animal models.Conclusions Our findings not only uncovered the mechanism of metabolic reprogramming in EGFR-activated GBM but also provided a combinatorial therapeutic strategy for clinical GBM management.

A flexible metal-organic framework with double interpenetration for highly selective CO_2 capture at room temperature

MicroRNAs （miRNAs） are small non-coding RNAs that regulate gene expression at the post-transcriptional level, and their aberrant expression occurs during the development of malignant diseases. Recently, miRNAs have been proposed as potential prognostic and predictive biomarkers for early diagnosis. However, a major obstacle in rapid miRNA analysis from real samples is the lack of ultrasensitive and quantitative techniques. In this regard, the use of chemiluminescence （CL） system offers a highly sensitive strategy for detecting miRNAs. In this article, an ultrasensitive approach has been established for the quantification ofmiRNAs, using magnetic beads （MBs） and alkaline phosphatase （AP）-based CL system. This technique depends on sandwich hybridization among MBs-labeled capture probes, target miRNAs and biotin-labeled reporter probes, conjugation of streptavidin-alkaline phosphatase （SA-AP） to biotin-labeled reporter probes, and CL detection of AP-linked targets. Detection of miR-21 with this technique demonstrated a high selectivity and an ultralow limit of detection （LOD） of 60 fM with an extraordinarily wide range of six orders of magnitudes. The quantitation could be achieved by direct detecting target miRNA in serum samples within a total time of 1.5 h and did not require reverse transcription and polymerase chain reaction （PCR） amplification. Therefore, this developed method shows great potential for early cancer diagnosis based on miRNAs as biomarkers.