Identification of the E2F1-RAD51AP1 axis as a key factor in MGMT-methylated GBM TMZ resistance

Objective:Epidermal growth factor receptor variant III(EGFRvIII)is a constitutively-activated mutation of EGFR that contributes to the malignant progression of glioblastoma multiforme(GBM).Temozolomide(TMZ)is a standard chemotherapeutic for GBM,but TMZ treatment benefits are compromised by chemoresistance.This study aimed to elucidate the crucial mechanisms leading to EGFRvIII and TMZ resistance.Methods:CRISPR-Cas13a single-cell RNA-seq was performed to thoroughly mine EGFRvIII function in GBM.Western blot,realtime PCR,flow cytometry,and immunofluorescence were used to determine the chemoresistance role of E2F1 and RAD51-associated protein 1(RAD51AP1).Results:Bioinformatic analysis identified E2F1 as the key transcription factor in EGFRvIII-positive living cells.Bulk RNA-seq analysis revealed that E2F1 is a crucial transcription factor under TMZ treatment.Western blot suggested enhanced expression of E2F1 in EGFRvIII-positive and TMZ-treated glioma cells.Knockdown of E2F1 increased sensitivity to TMZ.Venn diagram profiling showed that RAD51AP1 is positively correlated with E2F1,mediates TMZ resistance,and has a potential E2F1 binding site on the promoter.Knockdown of RAD51AP1 enhanced the sensitivity of TMZ;however,overexpression of RAD51AP1 was not sufficient to cause chemotherapy resistance in glioma cells.Furthermore,RAD51AP1 did not impact TMZ sensitivity in GBM cells with high O6-methylguanine-DNA methyltransferase(MGMT)expression.The level of RAD51AP1 expression correlated with the survival rate in MGMT-methylated,but not MGMT-unmethylated TMZ-treated GBM patients.Conclusions:Our results suggest that E2F1 is a key transcription factor in EGFRvIII-positive glioma cells and quickly responds to TMZ treatment.RAD51AP1 was shown to be upregulated by E2F1 for DNA double strand break repair.Targeting RAD51AP1 could facilitate achieving an ideal therapeutic effect in MGMT-methylated GBM 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.

Fabrication of submicron barium sulfate aggregates in the presence of ethylenediaminetetraacetic acid anions

Barium sulfate aggregates with an average size of 0.5μm were synthesized at pH 7, directed by ethylenediaminetetraacetic acid （EDTA） anions. The particle morphology, chemical composition, and size distribution of the BaSO4 aggregates were characterized. The as-synthesized BaSO4 particles were spherical and comprised many interconnected nanoballs, of which the surface properties were affected by the EDTA anions. The adsorption of EDTA anions reversed the charge and weakened the surface polarity of BaSO4, instigating the formation of aggregates by a self-assembly and transformation process. The resulting BaSO4 particles at pH 9-10 were ellipsoidal and featured smooth surfaces. Based on the zeta potential of BaSO4, variations in the morphology induced by changes in pH were closely related to the adsorption of mono- and multi-valent anions onto the electrical double layer of BaSO4.