Project description:The objective of the study was to examine the gene expression changes in glioma cell line U87 and U251 with LAMP2A knockdown. There were 15 samples in total- U87-1, U87-2, U87-3, U87-1812-1, U87-1812-2, U87-1812-3, U251-1, U251-2, U251-3, U251-1812-1, U251-1812-2, U251-1812-3, U251-1813-1, U251-1813-2, and U251-1813-3. U87-1 to U87-3 and U251--1 to U251-3 were used as the control groups (CON). U87-1812-1 to U871812-3, U251-1812-1 to U251-1812-3, and U251-1813-1 to U251-1813-3 were used as the experimental groups (shLAMP2A-1 and shLAMP2A-2). The total RNA of each sample was extracted from the stable transfected glioma cells by using TRIzol reagent. Then the RNA samples were processed for high throughput transcriptome sequencing on Illumina HiSeq 3000 platform. There were two types of libraries: the circRNA, mRNA, and lncRNA were all constructed by removing rRNA (one library, three types of RNA were analyzed together), and the insert fragment was about 300bp; the small RNA was constructed and analyzed separately, mainly microRNA of about 22bp. Results: among 60612 cleaned mRNAs, 781 were differentially expressed in U87-1812 group compared with U87 group, 146 were differentially expressed in U251-1812 group compared with U251 group, 43 were differentially expressed in U251-1813 group compared with U251 group (padj ≤ 0.05 and expression change ≥2 fold). The differential expressed genes distributed in all chromosomes. Functional annotation with GO and KEGG enrichment revealed the top functional groups including inflammation, DNA replication, cell adhesion, TNF, IL17, and axon guidance signaling pathways.
Project description:Overexpression of miR-31 inhibits the migration and invasion ability of glioma cell. We sought to obtain the genes regulated by mir-31 in glioma cell line. The gene expression of U251-mir-31 (U251 over expressing mir-31) and U251-control. U251-Control and U251-mir-31 cells were cultured in DMEM cell culture media for RNA extraction and hybridization on Affymetrix.GeneChip.HG-U133_Plus_2.
Project description:BRD4 is a member of the bromodomain and extra-terminal (BET) family, which has become a promising drug target for numerous cancers. It has been reported that BRD4 is deregulated in gliomas; however, the precise molecular pathways regulated by BRD4 are still elusive. In this study, we knocked down BRD4 expression in glioma cell line U251 by lentivirus-mediated short hairpin RNA (shRNA) approach. BRD4 downregulation attenuated cell proliferation and promoted cell apoptosis. Genome-wide analysis of BRD4-regulated transcripts in U251 cells was performed using microarray to reveal the possible molecular mechanism
Project description:We identified a rare coding variant (p.K420Q) in the complement component 7 (C7) gene affecting the risk of Alzheimer's disease. To investigate the cellular effects of the mutant, we performed RNA-seq in cell line overexpression wilt-type and mutant C7. U251 glioma cells with stable expression of mutant APP (K670N/M671L) (U251-APP cells), which produce Aβ42 under Dox inducing, were used as the model cell. Total RNA of U251-APP cells overexpressing wild type and mutant C7 proteins were subjected to transcriptome sequencing using Illumina Hiseq 4000 platform.
Project description:Glioma stem cells (GSCs) have been identified in glioma tissues and suggested to play important roles in the tumorigenesis of glioblastoma multiform (GBM). We established a novel cellular bioinformative pipeline that consisted of principal component analysis (PCA) with factor loading, intracellular pathway analysis, and immunopathway analysis and attempted to clarify the differences in gene expression profiles comprehensively among GSCs, a glioma cell line (U251), and a human GBM tissue (hGBM). To this end, we extracted total RNAs from the GSCs, U251, and the hGBM, performed microarray, and applied the data to the bioinformatics analyses described above. As the results, PCA clearly distinguished the three groups. Moreover, the second principal component (PC2) distinguished the GSCs from the hGBM and U251; it reflected the characteristics of stemness. The factor loading for PC2 suggested MYCN, DPP4, and, MIF as contributing factors to the stemness of GSCs. We clarify the similarities and differences among samples such as the GSCs, U251, and hGBM.
Project description:Assess gene expression patterns upon HOXA9 ectopic expression in U87MG GBM cell line and hTERT/E6/E7 immortalized human astrocytes, and HOXA9 silencing in U251 and GBML18 GBM cell lines. U87MG and hTERT/E6/E7 were retrovirally-infected with an MSCV control vector (MSCV-Control) or with a construct containing the coding region of HOXA9 (MSCV-HOXA9), resulting in U87MG-Control, U87MG-HOXA9, hTERT/E6/E7-Control and hTERT/E6/E7-HOXA9 cell lines. GBML18 and U251 cells were transfected with HOXA9 gene-specific shRNA sequences (shHOXA9) or a non-efective shRNA (shControl) in pGFP-V-RS plasmid, resulting in U251-shControl, U251-shHOXA9, GBML18-shControl and GBML18-shHOXA9 cell lines. Four experimental replicates for HOXA9 overexpression cell lines, and three for HOXA9 silencing cell lines were performed.
Project description:Glioma is a malignant primary tumour that occurs in the central nervous system. TEA domain transcription factor (TEAD) family proteins are the Hippo pathway's ultimate effector molecules. The function of TEAD3 in gliomas is still unclear. Therefore, RNA sequencing was performed on TEAD3 knockdown U251 cells and normal U251 cells. The samples in this study included normal U251 control group and TEAD3 knockdown group. Each group contained three samples. Total RNA was extracted using Trizol reagent following the manufacturer's procedure. The total RNA quantity and purity were analysis of Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit, high-quality RNA samples with RIN number > 7.0 were used to construct sequencing library. Gene Set Enrichment Analysis (GSEA) based on the sequencing results showed that knockdown of TEAD3 in the U251 cell line caused changes in several important pathways, including CTLA4 inhibitory pathway, defective pyroptosis, signaling by Hippo, regulation of TP53 activity, E2F mediated regulation of DNA replication, and FCGR3A mediated phagocytosis.
Project description:Artesunate possesses the potential of intervening with glioma, however, its pharmacological mechanisms remain unclarified. In this research, we aim to explore the regulatory mechanism of artesunate acting on glioma through constructing the interaction network of therapeutic effects-related genes of artesunate based on transcriptome sequencing data of glioma cells, and then verified by apoptosis staining and molecular docking at cellular level. In vitro cell activity and proliferation assay of two human glioma cell lines indicated that artesunate exerted more obvious inhibitory effects on the cell activity and proliferation of U87 cells than that of U251 cells. It could significantly promote apoptosis in U87 cells (P<0.01), while not in U251 cells (P>0.05), detected by using Hoechst and TUNEL cell apoptosis staining. Further, the differential expression gene sets between artesunate-sensitive and non-sensitive cell line, as well the therapeutic effects-related genes of artesunate were obtained, respectively, by transcriptome sequencing and differential data analysis using the lysis buffer of U87 and U251 cells before and after artesunate treatment. Then, key putative targets that related to therapeutic effects were screened by constructing the interaction network of differential genes of three above comparison groups, and calculating their topological characteristics. Pathway enrichment analysis showed that those key putative targets were significantly enriched in several signaling pathways that were closely associated with the main pathological changes of glioma, among which ATF4-DDIT3-PARP1 signaling axis, that was related with cell apoptosis, was the most enriched in. Molecular docking indicated that artesunate had fine binding affinities with ATF4 and DDIT3. Above all, this study preliminarily revealed that artesunate may induce the apoptosis and inhibited cell proliferation of glioma cells through regulating the abnormal alterations of corresponding proteins in ATF4-DDIT3-PARP1 signaling axis, which provided the novel scientific basis for its potential application in treating glioma.
Project description:Whole gene methylation profiles of U87 and U251 glioma cells before and after artesunate treatment, Infinium MethylationEPIC BeadChip, samples including 3 U87 cell normal group, 3 U251 cell normal group, 3 artesunate treated U87 cell group, U251 cell group was treated with 3 artesunate.