Project description:Little is known about genes that promote melanoma cell growth and proliferation. siRNAs may be used to address the role of individual genesin these processes. RNAi library screens were used in the past to gain a comprehensive overview of all genes involved in cell growth, proliferation, migration and other cellular processes. A large-scale loss-of-function screen for eight different melanoma cell lines was performed using a pooled lentiviral shRNA library (GeneNet Human 50K lentiviral shRNA Library,cat#SI206B-1, System Biosciences) to identify genes relevant for melanoma cell growth and proliferation. shRNAs that lead to cell death or reduced growth of transduced melanoma cells are negatively selected and thereby underrepresented in the final cellular shRNA pool and vice versa. The shRNAs of the shRNA library (3-5 per gene) have complementary sequences to probes on the custom Affymetrix microarray HG-U133Plus2 and were analysed using this array.
Project description:Little is known about genes that promote melanoma cell growth and proliferation. siRNAs may be used to address the role of individual genesin these processes. RNAi library screens were used in the past to gain a comprehensive overview of all genes involved in cell growth, proliferation, migration and other cellular processes. A large-scale loss-of-function screen for eight different melanoma cell lines was performed using a pooled lentiviral shRNA library (GeneNet Human 50K lentiviral shRNA Library,cat#SI206B-1, System Biosciences) to identify genes relevant for melanoma cell growth and proliferation. shRNAs that lead to cell death or reduced growth of transduced melanoma cells are negatively selected and thereby underrepresented in the final cellular shRNA pool and vice versa. The shRNAs of the shRNA library (3-5 per gene) have complementary sequences to probes on the custom Affymetrix microarray HG-U133Plus2 and were analysed using this array. Well-known melanoma cell lines SK-Mel-103, A375, SK-Mel-147, SK-Mel-19, SK-Mel-28, SK-Mel-29, SK-Mel-5, WM3523cln6 were transduced with the lentiviral shRNA library and grown for 10 days under puromycin selection (day 10), control cells of respective cell lines were transduced and frozen immediately after transduction and genomic integration of shRNAs (day 0). Totel DNA was extracted and genomically integrated shRNAs were hybridized to Affymetrix microarrays (HG-U133Plus2.0 array).
Project description:Cancer is a disease of both genetic and epigenetic changes. While increasing evidence demonstrates that oncogenic progression entails chromatin-mediated changes such as DNA methylation, the role of histone variants in cancer initiation and progression currently remains unexplored. Here, we report that the histone variant macroH2A (mH2A) suppresses tumour progression of malignant melanoma. We hypothesized that loss of mH2A could contribute to melanoma progression by relieving repression of cell cycle- and metastasis-related genes. To gain insight into the transcriptional state of mH2A1 and mH2A2-deficient cells, we examined their gene expression profiles using Affymetrix microarrays. Murine B16-F1 cells with lentiviral shRNAs against mH2A1 and mH2A2 were generated along with control shRNA (against GFP) and used for the microarray analysis. Two independent biological replicates were used.
Project description:We have found that cyclophilin B (CypB) expression is important for malignant glioblastoma multiforme (GBM) cell proliferation. To identify molecular mechanisms that could explain CypB-dependent survival in human GBM cells, a microarray analysis was performed using RNA prepared from U251MG GBM cells transduced with lentiviral CypB shRNA. These data revealed that about 130 genes were more than 2-fold affected by CypB depletion. Significant alterations in the expression of genes related to cell death, cell proliferation and cell migration were found in the shCypB cells. U251 glioblastoma cells transduced with lentivirus expressing non-target control shRNA (shCON) were compared to cells transduced with lentivirus expressing shRNA sequence against cyclophilin B (shCypB). Cells transduced with the lentiviral shRNA (shCON VS shCypB) for 5 days before total RNA extraction. Three biological replicates of cells treated with each shRNA (shCON or shCypB) were profiled.
Project description:We FACS purified GFP+ cells 2 days post-transduction of LSK (Lin-Negative c-Kit+ Sca1+) cells with miR-99 KD and Scr lentiviral vectors and performed RNA-sequencing; this allowed us to identify potential miR-99 target genes for inclusion in the shRNA library
Project description:We have found that cyclophilin B (CypB) expression is important for malignant glioblastoma multiforme (GBM) cell proliferation. To identify molecular mechanisms that could explain CypB-dependent survival in human GBM cells, a microarray analysis was performed using RNA prepared from U251MG GBM cells transduced with lentiviral CypB shRNA. These data revealed that about 130 genes were more than 2-fold affected by CypB depletion. Significant alterations in the expression of genes related to cell death, cell proliferation and cell migration were found in the shCypB cells.
Project description:TRIM28 is a bromodomain protein implicated in the regulation of tumor growth. TRIM28 regulates gene expression by recruiting epigenetic writers including HDACs, SETDB1 and HP1. To determine the role of TRIM28 in melanoma cells we knocked down TRIM28 using lentiviral shRNA constructs followed by gene expression analysis using HTA 2.0 GeneChip.
Project description:PAR-1 is known to be involved in the transition from non-metastatic to metastatic melanoma. We sought to determine the downstream target genes regulated by PAR-1 to determine how PAR-1 is contributing to the metastatic melanoma phenotype. The A375SM metastatic melanoma cell line was subjected to stable transduction of lentiviral-delivered small hairpin RNA (shRNA) targeting PAR-1. As a control, a non-targeting shRNA sequence (no homology to any human gene) was also transduced into A375SM cells.