ChIP\PCR analysis was performed using antibodies against H3K4me3 and p\NF\B (p\p65) and four pairs of genomic PCR primers specific for the E2F6 promoter (Figure S4e, Supporting Information). with paired primaryCrecurrent RNA sequencing data from 134 GBM patients who received TMZ treatment after surgery, it has been revealed that the E2F6 expression level is a predictive marker for TMZ response. Therefore, the inhibition of E2F6 is a promising strategy to conquer TMZ resistance in GBM. value less than 0.05 and fold change larger than 2 (Figure S2a, Supporting Information). Subsequently, we screened the sgRNAs that were depleted in response to TMZ treatment for 14 days (Figure S2b, Supporting Information). We speculated that cells with overexpression of these genes showed therapeutic\resistance to TMZ. Meanwhile, we conducted the RNA sequencing for U87\EGFRvIII and U87wt cells under TMZ or DMSO treatment. Principal component analysis (PCA) showed that the TMZ\induced differentially expressed genes were totally different from those in the DMSO\treated counterparts (Figure S2c, Supporting Information). By overlapping the differentially expressed sgRNAs, mRNAs, and EGFRvIII\regulated genes, E2F6 was the only hit induced by both the EGFRvIII mutation and TMZ treatment. 2.3. E2F6 Expression Is Correlated with Glioma Grade in Classical Subtype To gain insight into the expression profile of E2F6 in glioma samples, we employed The Cancer Genome Atlas (TCGA) RNA\seq data and microarray data of Rembrandt. Gliomas of World Health Organization (WHO) grade II, III, or IV were selected for analysis in our following study. As shown in Figure 3 a, the E2F6 expression level was significantly associated with tumor grade ( 0.0001). Hence, the E2F6 expression level was higher in classical subtype than proneural (PN) and mesenchymal (MES) subtypes (Figure ?(Figure3b,3b, 0.003). In addition, analyses of TCGA HG\U133A and Agilent 4502A microarrays and the RNA\seq data cohorts revealed that the E2F6 expression was significantly elevated in GBM than normal brain tissues (NBT) (Figure S2dCf, Supporting Information). Furthermore, the immumohistochemical (IHC) staining of 31 WHO II patients, 32 WHO III patients, and 54 WHO IV patients proved Tubercidin that elevated levels of E2F6 expression are associated with high grade of glioma (Figure ?(Figure3c).3c). Additionally, E2F6 was found to be a valuable gene for GBM diagnosis with a high sensitivity and specificity in these three datasets (Number S2gCi, Supporting Info). Thereafter, the correlated genes of E2F6 in the TCGA RNA\seq data were selected (Number ?(Figure3d).3d). KEGG analysis showed that these genes are associated with the cell cycle and DNA restoration (Number ?(Figure33e). Open in a separate window Number 3 E2F6 manifestation is definitely correlated with glioma grade in classical subtypes. a) Analysis of E2F6 Tubercidin manifestation in glioma TCGA RNA\seq and Rembrandt cohorts. Elevated levels of E2F6 manifestation levels are associated with high grade of glioma. b) Analysis of E2F6 in three Rabbit Polyclonal to Smad1 subtypes of glioblastoma, including classical (CL), mesenchymal (MES), and proneural (PN). E2F6 is definitely enriched in individuals with classical GBM. c) Representative IHC staining images of E2F6 in WHO II, III, and IV. Level pub, 200 m. d) Heatmap of E2F6\connected genes in the TCGA RNA\seq cohort. e) BiNGO pathway analysis of E2F6\related genes. 2.4. E2F6 Is definitely a TMZ\Resistant Gene We next investigated the effects of E2F6 on TMZ resistance. Four GBM cell lines (U87, N5, Tubercidin N9, and N33) were stably infected with lentiviruses expressing EGFRvIII or vector control and were consequently treated with TMZ or DMSO vehicle. Immunoblot analyses exposed that E2F6 level was significantly improved in EGFRvIII cells, as compared with.