Project description:We aimed to identify SAT1 regulated genes in U87MG cell by knockdown of SAT1 with two different shRNAs, and then compared knockdown cells to control cells with shGFP
Project description:Performed gene expression profiling of U87MG cells with two different shRNAs to SAT1 using Affymetrix microarrays covering 67,528 gene transcripts. Accordingly, we infected U87MG cells with lentiviruses expressing shRNAs to either the green fluorescent protein (GFP) or SAT1, performed a drug selection to ensure stable expression of the shRNAs, and expanded the cells for RNA harvest within 7 days of infection. RNA was reverse transcribed into cDNA, labelled with biotin and hybridized onto the arrays for analyses. Hierarchical clustering of duplicate experiments correctly segregated samples according to sample identity (i.e. shGFP clustered together, shSAT1-1 clustered together, and shSAT1-2 clustered together; with the latter two separately branching from shGFP)
Project description:Most patients affected by Glioblastoma multiforme (GBM) experience a recurrence of the disease because of the spreading of tumor-initiating cells (TICs) beyond surgical boundary. Unveiling and targeting molecular mechanisms causing this process is a logic goal to impair GBM killing ability. In an orthotopic xenograph model, we have noticed that GBM TICs isolated from several patients may fall into two classes of invasive behavior: nodular or diffuse. In order to identify genes responsible for the diffusive type of invasion, we have compared by genome expression analysis, cultured GBM TICs belonging to the two classes. This analysis allowed us to identify a small group of regulated genes in the diffusive type of GBM TICs. The gene ontology process of cell adhesion and the localization of the gene product functions to the plasmamembrane resulted significantly associated to this gene set. Real time RT-PCR and immunofluorescence analyses performed for a selected subgroup of regulated genes/gene products confirmed the results obtained by the expression analysis. Some of the genes that we found upregulated in our screening were already proven to be involved in Glioma cell invasion supporting our study. However, we have also identified genes that were not previously implicated in this process. To assess whether these are required to sustain TICs GBM invasion, we silenced a subset of them and evaluated in Boyden chamber the invasive ability of the cells. Our study provides novel target genes to be evaluated for the inhibition of GBM diffusion within the SNC. As we observed that GBM TICs may fall into two classes of M-bM-^@M-^\in vivoM-bM-^@M-^] invasive behavior in mouse orthotopic transplantation: expansive or highly diffusive, resulting in the hostM-bM-^@M-^Ys white and gray matters substitution, we decided to identify genes associated with the latter phenotype by microarray analysis. Three replicates of each class were analyzed.
Project description:An acidic tumor microenvironment plays a critical role in tumor progression. However, understanding of metabolic reprogramming of tumors in response to acidic extracellular pH has remained elusive. Using comprehensive metabolomic analyses, we demonstrated that acidic extracellular pH (pH 6.8) leads to the accumulation of N1-acetylspermidine, a pro-tumor metabolite, through upregulation of the expression of spermidine/spermine acetyl transferase 1 (SAT1). Inhibition of SAT1 expression suppressed the accumulation of intra- and extracellular N1-acetylspermidine at acidic pH. Conversely, overexpression 3 of SAT1 increased intra- and extracellular N1-acetylspermidine levels, supporting the proposal that SAT1 is responsible for accumulation of N1-acetylspermidine. While inhibition of SAT1 expression only had a minor effect on cancer cell growth in vitro, SAT1 knockdown significantly decreased tumor growth in vivo, supporting a contribution of the SAT1-N1-acetylspermidine axis to pro-tumor immunity. Immune cell profiling revealed that inhibition of SAT1 expression decreased neutrophil recruitment to the tumor, resulting in impaired angiogenesis and tumor growth. We showed that anti-neutrophil neutralizing antibodies suppressed growth in control tumors to a similar extent to that seen in SAT1 knockdown tumors in vivo. Further, a SAT1 signature was found to be correlated with poor patient prognosis. Our findings demonstrate that extracellular acidity stimulates recruitment of pro-tumor neutrophils via the SAT1-N1-acetylspermidine axis, which may represent a novel target for anti-tumor immune therapy.
Project description:To identify TGF-β regulated lncRNAs in glioblastoma, we performed a genome-wide microarray screen in T98G glioma cells. T98G cells were treated with 10 ng/ml TGF-β (24h) and differentially expressed lncRNAs were identified using microarray in comparison with control cells.
Project description:Identifying RBBP4-regulated transcriptome in T98G glioblastoma (GBM) cells using RNA sequencing, and epigenome screening for genes regulated by RBBP4 in GBM cells