EGFR/EGFRvIII remodels the cytoskeleton via epigenetic silencing of AJAP1 in glioma cells.

EGFR amplification and mutations are the most common oncogenic events in GBM. EGFR overexpression correlates with GBM invasion, but the underlying mechanisms are poorly understood. In a previous study, we showed that AJAP1 is involved in regulating F-actin to inhibit the invasive ability of GBM. In addition, in a GBM cell line, the AJAP1 promoter was highly bound by H3K27me3 and, through bioinformatics analysis, we found that AJAP1 expression was negatively correlated with EGFR. In this study, we found that the pathway downstream of EGFR had a higher activation level in GBM cell lines, which led to excessive tumor suppressor silencing. Therefore, we deduced that in glioma cells, the pathway downstream of EGFR remodels the cytoskeleton via AJAP1 epigenetic silencing to enhance invasion. Furthermore, MK2206 reversed AJAP1 downregulation by inhibiting the EGFR pathway. In vivo, MK2206 also inhibited the proliferation and local invasion of 87-EGFRvIII. These data suggest that activation of the EGFR signal transduction pathway genetically silences anti-oncogenes to enhance GBM malignancy. MK2206 might be a promising therapeutic for EGFR/EGFRvIII-positive GBMs.

HOTAIR upregulates an 18-gene cell cycle-related mRNA network in glioma.

HOTAIR is a tumor promoting long non-coding RNA (lncRNA) with roles in multiple cancers. However, the role of HOTAIR in glioma has not been well charaterized. Genes that positively correlated with HOTAIR were identified from the Chinese Glioma Genome Atlas and constructed into an interacting network. In total, 18 genes with P-values <0.01 were further extracted and constructed into a subnetwork. Real-time PCR, western blot and immunofluorescence analyses were employed to examine the expression of the genes after HOTAIR overexpression or knockdown. Intracranial glioblastoma multiform (GBM) models were used to test the potential of HOTAIR as a glioma therapy target. It was discovered that the 18 genes that most significantly correlated with HOTAIR expression formed a cell cycle-related mRNA network, which is positively regulated by HOTAIR. Furthermore, HOTAIR knockdown inhibited mouse intracranial GBM model formation. HOTAIR positively regulates a cell cycle-related mRNA network in glioma, and could be a potential therapeutic target for treating glioma.

The CRISPR/Cas9 system targeting EGFR exon 17 abrogates NF-κB activation via epigenetic modulation of UBXN1 in EGFRwt/vIII glioma cells.

Worldwide, glioblastoma (GBM) is the most lethal and frequent intracranial tumor. Despite decades of study, the overall survival of GBM patients remains unchanged. epidermal growth factor receptor (EGFR) amplification and gene mutation are thought to be negatively correlated with prognosis. In this study, we used proteomics to determine that UBXN1 is a negative downstream regulator of the EGFR mutation vIII (EGFRvIII). Via bioinformatics analysis, we found that UBXN1 is a factor that can improve glioma patients' overall survival time. We also determined that the down-regulation of UBXN1 is mediated by the upregulation of H3K27me3 in the presence of EGFRvIII. Because NF-κB can be negatively regulated by UBXN1, we believe that EGFRwt/vIII activates NF-κB by suppressing UBXN1 expression. Importantly, we used the latest genomic editing tool, CRISPR/Cas9, to knockout EGFRwt/vIII on exon 17 and further proved that UBXN1 is negatively regulated by EGFRwt/vIII. Furthermore, knockout of EGFR/EGFRvIII could benefit GBM in vitro and in vivo, indicating that CRISPR/Cas9 is a promising therapeutic strategy for both EGFR amplification and EGFR mutation-bearing patients.

Chemiluminescent Detection for Estimating Relative Copy Numbers of Porcine Endogenous Retrovirus Proviruses from Chinese Minipigs Based on Magnetic Nanoparticles.

Chinese Bama minipigs could be potential donors for the supply of xenografts because they are genetically stable, highly inbred, and inexpensive. However, porcine endogenous retrovirus (PERV) is commonly integrated in pig genomes and could cause a cross-species infection by xenotransplantation. For screening out the pigs with low copy numbers of PERV proviruses, we have developed a novel semiquantitative analysis approach based on magnetic nanoparticles (MNPs) and chemiluminescence (CL) for estimating relative copy numbers (RCNs) of PERV proviruses in Chinese Bama minipigs. The CL intensities of PERV proviruses and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were respectively determined with this method, and the RCNs of PERV proviruses were calculated by the equation: RCN of PERV provirus = CL intensity of PERV provirus/CL intensity of GAPDH. The results showed that PERVs were integrated in the genomes of Bama minipigs at different copy numbers, and the copy numbers of PERV-C subtype were greatly low. Two Bama minipigs with low copy numbers of PERV proviruses were detected out and could be considered as xenograft donor candidates. Although only semiquantitation can be achieved, this approach has potential for screening out safe and suitable pig donors for xenotransplantation.

Ultrasensitive Detection and Subtyping of Porcine Endogenous Retrovirus Provirus Based on Magnetic Nanoparticles and Chemiluminescence.

Porcine endogenous retrovirus (PERV) is commonly integrated in pig genomes, and could cause a cross-species infection by xenotransplantation. In this study, we developed a rapid and ultrasensitive approach for detection and subtyping of PERV provirus based on magnetic nanoparticles (MNPs) and chemiluminescence (CL). The carboxylated MNPs (CMNPs) were covalently coupled with aminated probes for capturing biotinylated target fragments of PERV, the product of polymerase chain reaction (PCR). Agarose gel electrophoresis analysis approved the reliability of biotinylated fragments. The MNPs composites were incubated with streptavidin-alkaline phosphatase (SA-ALP) and CL signal intensities were determined by subsequently adding 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy) phenyl-1,2-dioxetane (AMPPD). The optimal assay conditions of this approach were 1 mM for SA modification, 10 µM for probe modification, 55 (PERV), 54 (PERV-A), 50 (PERV-B), and 56 °C (PERV-C) for hybridization temperatures respectively, and 30 min for hybridization time. This approach was specific and highly sensitive, and the limit of detection (LOD) was 100 amol, which has the potential for screening out safe pig donors for xenotransplantation as well as to examine clinical samples from human patients treated with porcine xenotranplantation.

Extracellular miRNAs: origin, function and biomarkers in hepatic diseases.

MicroRNAs (miRNAs), a class of 19-24 nucleotides non-coding RNAs, regulate gene expression by inhibiting both translation and stability of specific mRNAs at the post-transcriptional level. The existence of miRNAs in a series of mammalian body fluids as extracellular nuclease-resistant entities, together with the aberrant expression of miRNAs during the occurrence and development of a wide range of diseases, triggers the possibility that miRNAs as promising noninvasive diagnostic biomarkers are applied to predict the pathological status of the body. However, the origin and biological function of extracellular miRNAs have not been systematically elucidated. In this review, we summarize the current literature on the biogenesis and post-transcriptional regulation of miRNAs, discuss available evidence regarding the possible origin and release of extracellular miRNAs, and collect novel views on their potentials as key mediators in cell-cell communication processes. Finally, we shed light on the accumulating knowledge about their utilities as diagnostic biomarkers in hepatic diseases.