Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of effected genes by compound BIO,3G4 and knockdown of MED23

ABSTRACT: SW480 cells were cultured in Leibovitz's L-15 medium at 37oC. To profile the effected genes by SHPRH, cells were seeded in 6 well plates to allow 80% confluence the day after. Cells were transfected with control GFP vector or GFP-SHPRH vector 6 well plates by using lipofectamine 3000, 2.5ug DNA was used per well with triplicate repeats for both vectors. Cells were harvested in 24 hours. All the cells were harvested and total RNA was extracted using TRIZol method followed by Agilent DNA microarray analysis. This is related to E-MTAB-3438 (http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-3438)

ORGANISM(S): Homo sapiens

SUBMITTER: Anne Margrete Oyan

PROVIDER: E-MTAB-3652 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Polymorphisms in pre-miRNAs may affect its expression, then having effect on its target mRNAs and be associated with cancer susceptibility. In the current study, we evaluated the association of a 3-base pair (3-bp) indel polymorphism (rs57408770) in pre-miR-3131 with hepatocellular carcinoma (HCC) susceptibility in a Chinese population. Logistic regression analysis showed that the insertion allele of rs57408770 was significantly associated with an increased risk for HCC occurrence in both case-control studies. To further investigated the molecular mechanism underlying the correlation between rs57408770 and the risk of HCC, overexpression of pre-miR-3131 in HCC cell line was performed. Human HCC cell lines Sk-Hep-1 was obtained from Shanghai Cell Bank of Chinese Academy of Sciences on 9 December 2013.The fragment of 366bp including the insert allele of rs57408770 in pre-miR-3131 was directly synthesized and cloned into BamHI and EcoRI sites of pEGFP-N1 yielding the wild-type vector (pEGFP-N1-miR3131-WT). The mutant-type vector (pEGFP-N1-miR3131-MT) including the delete allele of rs57408770 was generated using QuikChange Lightening Site-Directed Mutagenesis Kit. The resulting constructs were verified by direct sequencing. For overexpression experiments, cells were cultured in 6-well plates at a density of 200 000 cells per well the day before transfection. 2.5Î¼g plasmids of empty vector pEGFP-N1, pEGFP-N1-miR3131-WT or pEGFP-N1-miR3131-MT were transfected into the growing cells (about 80%-90% confluent) using Lipofectamine2000. After 24 hours of the transfection, the cells were collected for subsequent experiments.And human genome-wide gene expression profile assay was used to screen the targets of miR-3131. For overexpression experiments, cells were cultured in 6-well plates at a density of 200 000 cells per well the day before transfection. 2.5Î¼g plasmids of empty vector pEGFP-N1, pEGFP-N1-miR3131-WT or pEGFP-N1-miR3131-MT were transfected into the growing cells (about 80%-90% confluent) using Lipofectamine2000 (Invitrogen). After 24 hours of the transfection, the cells were collected for subsequent experiments. There are 3 samples in this experiment.

Project description:GNAS, a gene encoding G-protein stimulating alpha subunit, is frequently mutated in intraductal papillary mucinous neoplasms (IPMNs), which is an indolent and slow-growing pancreatic neoplasm that secretes abundant mucin. GNAS mutation is not observed in conventional ductal adenocarcinomas of the pancreas. To determine the functional significance of GNAS mutation in pancreatic ductal cells, we examined in vitro phenotypes and gene expression profiles of cells of pancreatic ductal lineage, HPDE, PK-8, PCI-35, and MIA PaCa-2, with exogenous expression of either wild-type or mutated (R201H) GNAS. We found that exogenous GNAS upregulated intracellular cyclic-adenine monophosphate, particularly in the mutated GNAS transfectants. Exogenous GNAS induced no obvious cell-growth promotion, but induced suppression in some cells. The exogenous GNAS upregulated MUC2 and MUC5AC in HPDE and PK-8, and the latter was most sensitive to exogenous GNAS, exhibiting drastic alteration of the global gene expression that is consistent with that of IPMN. Hence, PK-8 expressing exogenous mutated GNAS may be an ideal in vitro model of IPMN. On the other hand, exogenous GNAS downregulated expression of mucin genes and produced modest alteration of gene expression profiles in PCI-35 and MIA PaCa-2, indicating lower sensitivity to exogenous GNAS. Furthermore, we showed diverse and cell-type specific mucin expression pathways with complicated interactions between signaling pathways of the G-protein coupled receptor (GPCR), the mitogen-activated protein kinase (MAPK), and the phosphatidylinositol 3 kinase (PI3K), in which the GPCR pathway appeared to be dominant in some and the MAPK pathway in others. In conclusion, mutated GNAS found in IPMNs may extensively alter gene expression profiles, including expression of mucin genes, with the interaction with MAPK and PI3K pathways in pancreatic ductal-lineage cells, which may determine the characteristic phenotype of the neoplasm. Cells of pancreatic cancer cell lines, PK-8, PCI-35,and MIA PaCa-2, were seeded at 4 M-CM-^W 10^5 cells/well in 6-well plates and incubated for 24 hours at 37M-BM-0C in 5% CO2 with humid atmosphere. Then the cells were transfected with either pcDNA 3.1/V5-His vector or pcDNA3.1-GNAS(R201H)-V5-His vector using Lipofectamine 2000 reagent (Life Technologies) according to the manufacturerM-bM-^@M-^Ys recommendations. The cells were incubated for 24 hours and collected by dissociation using trypsin. Total RNAs were isolated using the RNeasy Mini kit (Qiagen, Hilden, Germany). Serial analysis of gene expression (SAGE) library was constructed using a SOLiD SAGE Kit (Life Technologies) according to the manufactureM-bM-^@M-^Ys instruction. The constructed libraries were analysed by means of the massively parallel sequencing method using SOLiD 4 System (Life Technologies). The SAGE analysis was performed for samples obtained from single transfection experiment.

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Transcription profiling of effected genes by compound BIO,3G4 and knockdown of MED23

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