Lentiviral shRNA library screen to identify growth-related genes in malignant melanoma
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE46675 | GEO | 2014/01/24
SECONDARY ACCESSION(S): PRJNA201490
REPOSITORIES: GEO
Similar Datasets
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).
2014-01-24 | E-GEOD-46675 | biostudies-arrayexpress
Project description:Lentiviral shRNA library screen to identify growth-related genes in malignant melanoma
| PRJNA201490 | ENA
Project description:To find new genes that might be targeted for reduction of Copper (Cu) toxicity in Wilson disease we employed genome-wide shRNA screening in ATP7B-KO HepG2 cells, a bona fide WD cell model. Cells were screened using the Dharmacon™ Decode™ Pooled Lentiviral shRNA Screening Library targeting 18205 human genes (Dharmacon #RHS6083). The Lentiviral RNAi screening library is made of 10 pools of 9570 GIPZ short hairpin RNAs (shRNAs) packaged into high-titer lentiviral particles. Genomic DNA was isolated from the cells and read counts for each shRNAs were obtained by deep sequencing to determine the abundance of each shRNA in the Cu-treated cells compared to controls. This analysis allowed the identification of genes whose downregulation promoted survival of ATP7B-KO cells upon Cu overload. To exclude that some shRNAs would have been responsible for ATP7B-KO survival by suppressing generic proapoptotic genes, we run the same screening with the proapoptotic drug vinblastine instead of CuCl2. Genes, whose suppression allowed cell to resist Cu but not vinblastine were considered as Cu-specific hits and through this approach we identified PrP as a suitable target for reduction of Cu toxicity in WD. Indeed, we found that Cu accumulation in ATP7B-KO cells stimulates expression of PrP, which brings to toxic Cu overload while PrP suppression significantly reduces Cu accumulation and toxicity in ATP7B-deficient cells. In sum our data highlight an important regulatory role of PrP in copper metabolism and as a therapeutic tool for Wilson disease.
2024-12-09 | GSE277865 | GEO
Project description:To find new genes that might be targeted for reduction of Copper (Cu) toxicity in Wilson disease we employed genome-wide shRNA screening in ATP7B-KO HepG2 cells, a bona fide WD cell model. Cells were screened using the Dharmacon™ Decode™ Pooled Lentiviral shRNA Screening Library targeting 18205 human genes (Dharmacon #RHS6083). The Lentiviral RNAi screening library is made of 10 pools of 9570 GIPZ short hairpin RNAs (shRNAs) packaged into high-titer lentiviral particles. Genomic DNA was isolated from the cells and read counts for each shRNAs were obtained by deep sequencing to determine the abundance of each shRNA in the Cu-treated cells compared to controls. This analysis allowed the identification of genes whose downregulation promoted survival of ATP7B-KO cells upon Cu overload. To exclude that some shRNAs would have been responsible for ATP7B-KO survival by suppressing generic proapoptotic genes, we run the same screening with the proapoptotic drug vinblastine instead of CuCl2. Genes, whose suppression allowed cell to resist Cu but not vinblastine were considered as Cu-specific hits and through this approach we identified PrP as a suitable target for reduction of Cu toxicity in WD. Indeed, we found that Cu accumulation in ATP7B-KO cells stimulates expression of PrP, which brings to toxic Cu overload while PrP suppression significantly reduces Cu accumulation and toxicity in ATP7B-deficient cells. In sum our data highlight an important regulatory role of PrP in copper metabolism and as a therapeutic tool for Wilson disease.
2024-12-09 | GSE260454 | GEO
Project description:Short hairpin RNA library-based functional screening identified ribosomal protein L31 that modulates prostate cancer cell growth Vehicle vs. bicalutamide treated LNCaP cells infected with lentiviral shRNA library. Biological replicates: 2 vehicle treatments, 2 bicalutamide treatments.
2015-05-15 | E-GEOD-60382 | biostudies-arrayexpress
Project description:Recent work has shown that cytotoxic T cells play a central role in immune-mediated control of cancers1-3, and monoclonal antibodies that target inhibitory receptors on T cells can induce significant clinical benefit in patients despite advanced disease4-6. However, many of the regulatory pathways that result in loss of T cell function within immunosuppressive tumors remain unknown. Here we show that such regulatory mechanisms can be systematically discovered in vivo in the tumor microenvironment. We devised a pool shRNA screening approach aimed at identifying genes that block the function of tumor-infiltrating CD8 T cells. We postulated that shRNAs targeting key inhibitors would enable robust T cell infiltration and proliferation in tumors, despite multiple inhibitory signals. Candidate shRNAs were discovered by transfer of shRNA-transduced T cells into tumor-bearing mice, followed by deep sequencing to quantify the representation of all hairpins in tumors and lymphoid organs. A subset of shRNAs induced T cell accumulation in tumors but not the spleen, demonstrating feasibility of discovering shRNAs with differential action across tissues. One of the targets was Ppp2r2d, a regulatory subunit of the family of PP2A phosphatases7. Control shRNA-transduced T cells underwent apoptosis upon recognition of melanoma cells, while Ppp2r2d shRNA-transduced T cells accumulated in tumors due to enhanced proliferation and reduced apoptosis. Ppp2r2d shRNAexpressing T cells also significantly delayed tumor growth. This in vivo approach has wide applications to dissect complex immune functions in relevant tissue microenvironments.
2014-03-14 | GSE53388 | GEO
Project description:Sub-genomewide shRNA libraries were constructed using the current RNAi consortium constructs as well as using the DSIR (siRNA algoirthm) and a novel shRNA specific algorithm (shERWOOD). All libraries were placed into mir30 expression vectors. The shERWOOD libraries were also placed in a vector harboring an optimized mir cassette (ultramir). Each library was screened using the pancreatic cell line A385. A concensus set of essential genes identified as the set for which two shRNAs depleted in each of the libries. For these genes, a great percentage of shERWOOD seletected shRNA depleted. In addition the placement of shERWOOD selected constructs into ultramir scaffoled increased the rate of shRNA depletion for essential genes further. Purpose: shRNA screens were carried out using various library construction strategies to identify the strategy that provides the best shRNA screening results. Method: Libraries were constructed using the TRC shRNA set as well as shRNAs identified using the DSIR and shERWOOD algorithms. shRNA libraries were cloned into mir30 expression vectors. shERWOOD shRNAs were also cloned into an expression vector harboring an optimized microRNA scaffold termed ultramir. Each resultant library was screened using the pancreatic cell line A385. Each library was analyzed separately to identify a set of genes where at least two shRNAs depleted. These gene sets were intersected to develop a set of essential genes. Results: The shERWOOD shRNA libraries provided the highest number depleting shRNAs for each essential gene. Further these shRNAs depleted to a greater extent than did the shRNAs from the other libraries. When shERWOOD libraries were placed into the ultramir cassette a greater number of shRNAs per essential gene depleted.
2014-11-24 | E-GEOD-62185 | biostudies-arrayexpress
Project description:Recent work has shown that cytotoxic T cells play a central role in immune-mediated control of cancers1-3, and monoclonal antibodies that target inhibitory receptors on T cells can induce significant clinical benefit in patients despite advanced disease4-6. However, many of the regulatory pathways that result in loss of T cell function within immunosuppressive tumors remain unknown. Here we show that such regulatory mechanisms can be systematically discovered in vivo in the tumor microenvironment. We devised a pool shRNA screening approach aimed at identifying genes that block the function of tumor-infiltrating CD8 T cells. We postulated that shRNAs targeting key inhibitors would enable robust T cell infiltration and proliferation in tumors, despite multiple inhibitory signals. Candidate shRNAs were discovered by transfer of shRNA-transduced T cells into tumor-bearing mice, followed by deep sequencing to quantify the representation of all hairpins in tumors and lymphoid organs. A subset of shRNAs induced T cell accumulation in tumors but not the spleen, demonstrating feasibility of discovering shRNAs with differential action across tissues. One of the targets was Ppp2r2d, a regulatory subunit of the family of PP2A phosphatases7. Control shRNA-transduced T cells underwent apoptosis upon recognition of melanoma cells, while Ppp2r2d shRNA-transduced T cells accumulated in tumors due to enhanced proliferation and reduced apoptosis. Ppp2r2d shRNAexpressing T cells also significantly delayed tumor growth. This in vivo approach has wide applications to dissect complex immune functions in relevant tissue microenvironments. OT-I derived T cells were transduced with shRNA's and adoptively transferred to B16 tumor bearing mice. Following incubation the T cells were purified from either tumors or spleens. The shRNA's targeted either a control gene (LacZ) or one of 5 selected genes found to regulate the presence of T cells in tumors vs in spleen
2014-03-14 | E-GEOD-53388 | biostudies-arrayexpress
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.
2010-12-23 | E-GEOD-19181 | biostudies-arrayexpress
Project description:Purpose: Assess the transcriptional changes induced upon RAB7 knock-down in melanoma (SK-Mel-28 and UACC-62) and in colon cancer (HCT-116) cell lines. Methods: mRNA profiles of tumor cell lines (SK-Mel-28, UACC-62, HCT-116) stably expressing scrambled shRNA or RAB7 shRNA (harvested at day 3 after lentiviral infection) were generated by deep sequencing, using three biological replicates per condition. The sequence reads that passed quality filters were analyzed with TopHat and Cufflinks. Validation of induced / silenced genes was performed by western blot. Results show a differential impact of RAB7 expression in the transcriptomic profile of melanoma vs non-melanoma cell lines, and support a lineage-specific role of this small GTPase in melanoma. Examination of the mRNA profiles RAB7-depleted vs wild type cells, performed in parallel in 3 different tumor cell lines (Melanomas: SK-Mel-28 and UACC-62, Non-melanoma: HCT-116) harvested at day 3 after lentiviral infection.
2014-04-20 | E-GEOD-42735 | biostudies-arrayexpress