Unknown,Transcriptomics,Genomics,Proteomics

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MiR-196a is able to restore the aggressive phenotype of Annexin A1 CRISPR/Cas9 knock-out pancreatic cancer cells

ABSTRACT: Annexin A1 (ANXA1) is a Ca2+-binding protein involved in pancreatic cancer (PC) progression. It is able to mediate cytoskeletal organization maintaining a malignant phenotype. ANXA1 Knock-Out (KO) MIA PaCa-2 cells partially lost their migratory and invasive capabilities and also the metastatization process is affected in vivo. Here, we investigated the microRNA (miRNA) profile in ANXA1 KO cells. The analysis of the modification in miRNA expression remarked the significant involvement of ANXA1 in PC progression. In this study, we focused on miR-196a which is a well known oncogenic factor in several tumour models and it appeared down-modulated in absence of ANXA1. Furthermore, both ANXA1 and miR-196a are able to trigger the mechanisms of the epithelial to mesenchymal transition (EMT). Our results show that the reintroduction of miR-196a through the mimic sequence restored the early aggressive phenotype of MIA PaCa-2. Then, ANXA1 seems to support the expression of miR-196a and its role. On the other hand, this miRNA is able to mediate some of protein functions in PC progression. This work elucidates the correlation between ANXA1 and specific miRNA sequences, particularly miR-196a, and provides new knowledge about the protein intracellular role.

INSTRUMENT(S): Illumina HiSeq 2500

ORGANISM(S): Homo sapiens

SUBMITTER: Domenico Memoli

PROVIDER: E-MTAB-6599 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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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MiR-196a is able to restore the aggressive phenotype of Annexin A1 CRISPR/Cas9 knock-out pancreatic cancer cells

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