Lentiviral shRNA library screening of tumor growth associated genes in CAPAN2 cells
ABSTRACT: Our aim is to identify genes that play important role to cell proliferation in PDAC. Lentivirus shRNA library was transduced to CAPAN2 cells. The cells were orthotopically injected into nude the pancreas of the mice. Tumors were allowed to grow and subsequently total RNA was extracted from the tumor tissues. The differential level of shRNA between tumor cells and uninjected cells were compared.
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: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.
Project description:We hypothesized that tissue genome-wide gene expression analysis, coupled with gene network analyses of differentially expressed genes, would provide novel insights into the pathogenesis of pulmonary sarcoidosis. Keywords: Disease state analysis Genome-wide gene expression profiles were compared in tissues derived from subjects with active pulmonary sarcoidosis (n=6) and those with normal lung anatomy (n=6). Differentially expressed genes were analyzed by gene network analysis
Project description:EGFR degradation is delayed in Cbl, Cbl-b double-deficient MCF10A but EGF stimulation does not enhance their growth. We performed a transcriptome analysis to gain insights into biological consequences of Cbl, Cbl-b loss in mammary epithelial cells. We compared the transcriptome of MCF10A cells expressing shRNA against Cbl and Cbl-b with control shRNA-expressing cells using Affymetrix U133 Plus 2.0. Each sample was prepared in duplicates. Data were analyzed by GSEA.
Project description:Merm1/Wbscr22 is one of genes in chromosomal region deleted in Williams-Beuren syndrome, a multisystem developmental disorder. Wbscr22 contains a nuclear localization signal and an S-adenosyl-L-methionine-dependent methyltransferase fold, but its real function is completely unknown. In this study, to examine the function, we compared the gene expression profiles between control and Merm1/Wbscr22 knock-downed tumor cells. Four established cell lines were selected for RNA extraction and hybridization on Affymetrix microarrays: (1) control LM8 cells, designated as LM8/control, (2) Merm1/Wbscr22 knock-downed LM8 cells, designated as LM8/shRNA, (3) control A375M cells, designated as A375M/control, and (4) Merm1/Wbscr22 knock-downed A375M cells, designated as A375M/shRNA.
Project description:So far, we have found PMA induced USP2b isoform in myeloid leukemia cell lines such as HL60, THP-1, and U937. To explore molecular function of USP2 in the cells, we assess expression profiles of HL60-derivatives continuously expressing shRNA for USP2 and control shRNA. HL60 derivatives continuously expressing shRNA for USP2 and control shRNA were treated with PMA (30nM) for 0, 1, 2, and 3 days. Total RNA was extracted from the cells and subjected to microarray experiments using Human Genome U133 GeneChips.
Project description:We aimed to analyze the effects of Wnt-1 overexpression on the mRNA expression profile of human melanoma in a mouse xenograft model and correlated the results with then presence or absence of lymphangiogenesis and metastasis. Affymetrix gene expression analysis revealed activation of canonical and non-canonical targets genes in response to Wnt-1 as compared with controls. In regard to lymphangiogenic factors, the amount of VEGF-C was the single best marker to correlate with the amount of lymph-angiogenesis. mRNA expression array of human melanoma orthotopically grown in SCID mice. Comparison includes mRNA expression profile of two melanoma cell-lines (A375 and M24met) stably overexpressing control vector or Wnt-1 treated with or without CsA. Comparison #1 comprised Wnt-1 versus control in A375 and M24met melanoma, respectively. Comparison #2 comprised Wnt-1 + Cyclosporine A (CsA) versus Wnt-1 without CsA.
Project description:Proline rich 15 (PRR15) is a small nuclear protein required for normal conceptus development in the sheep. We used microarrays to assess the changes in the trophoblast transcriptome when PRR15 expression was diminished. The human first-trimester trophoblast cell line, ACH-3P, was infected with control lentivirus expressing no shRNA or lentivirus expressing shRNA to target PRR15 mRNA for degradation, with three biological replicates per treatment.
Project description:Downregulations of TCAM1P-004 and RP11-598D14.1 were frequently observed in HCC tumors as compared to adjacent non-tumor tissues. To further study the molecular functions of TCAM1P-004 and RP11-598D14.1, we attempted to identify the gene targets regulated by either lncRNAs. Knockdown of TCAM1P-004 or RP11-598D14.1 were achieved by transduction of lentivirus carrying respective shRNAs in non-tumor hepatocyte MIHA cells. Diffferentially expressed genes after knockdown of the lncRNAs were compared to cells tranduced with lentivirus carrying scramble shRNAs.
Project description:Pancreatic ductal adenocarcinoma (PDAC) has a characteristically dense stroma comprised predominantly of cancer associated fibroblasts (CAFs). CAFs promote tumor growth, metastasis and treatment resistance. We aimed to investigate the molecular changes and functional consequences associated with chemotherapy treatment of PDAC CAFs. Chemoresistant immortalized CAFs (R-CAFs) were generated by continuous incubation in 100nM gemcitabine. Gene expression differences between treatment naïve CAFs (N-CAFs) and R-CAFs were compared by array analysis. Immortalized human pancreatic CAFs were grown for 30 days in either control media or media containing 100nM gemcitabine. RNA was then isolated and hybidized on U133 Plus 2.0 Affymetrix arrays.