Unknown,Transcriptomics,Genomics,Proteomics

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Transient transfection of miR-449 family in the hepatocellular carcinoma cell line HLE

ABSTRACT: In this study, we comparatively analyzed the members of the miR-449 family (miR-449a, miR-449b, and miR-449c) with regards to their target genes and functional effects in hepatocellular carcinoma (HCC). Microarray analysis after transient transfection of miR-449a, miR-449b, and/or miR-449c in the HCC cell line HLE identified putative target genes of miR-449a, miR-449b, and miR-449c. For transient overexpression of miRNAs, the HCC cell line HLE was transfected with 50 nM Allstars Negative Control or miScript miRNA mimics. MiRNA mimics for miR-449a, miR-449b, and miR-449c were either single transfected or cotransfected in equimolar amounts (16.7 nM each). Global mRNA expression profiling was performed utilizing pooled RNA of three biological replicates per microarray and two microarrays per condition.

ORGANISM(S): Homo sapiens

SUBMITTER: Maria Sandbothe

PROVIDER: E-GEOD-74710 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The transition of the endothelium to a pro-inflammatory state is key to progression of chronic inflammatory diseases including rheumatoid arthritis, chronic bowel disease and atherosclerosis. In atherosclerosis it is hypothesized that low density lipoproteins (LDL) that become trapped in the intima of the blood vessels are oxidized to minimally modified LDL (mmLDL) and that these serve as an important contributing factors to endothelial dysfunction. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine (OX-PAPC), a model of the active phospholipid components of mmLDL affects the expression of hundreds of genes involved in inflammatory and other biological processes in human aortic endothelial cells (HAECs). We hypothesized that microRNAs (miRNAs) partially regulate this response. Using next generation sequencing, we identified miR-21-3p and miR-27a-5p to be induced 4-fold and 3-fold, respectively in response to OX-PAPC treatment compared to control treatment in HAECs. To identify the targets, we performed whole genome transcript profiling following transient over-expression of these two miRNAs followed by. In total, 1254 genes were down-regulated with 925 of them overlapping between the two miRNAs. Functional enrichment analysis using Gene Ontology predicted that the two miRNAs were involved in the regulation of NF-κB signaling. We characterized the Toll/interleukin-1 receptor (TIR) domain-containing adaptor protein TICAM2 as a direct target of miR-21-3p and miR-27a-5p. Furthermore, we showed that over-expression of miR-21-3p and miR-27a-5p lead to decreased p65 translocation to the nucleus and decreased the expression of known NF-κB downstream target genes confirming both miRNAs’ role in negatively regulating NF-κB signaling in endothelial cells. mRNA expression profiling of human aortic endothelial cells from two separate donors that were transfected with 1 nM microRNA mimics and negative control. The miRIDIAN mimics used were miR-21-3p (Catalog Number:C-301023-01-0005), miR-27a-5p (Catalog No: C-301028-01-0005), negative control (Catalog No: CN-001000-01-05)

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Transient transfection of miR-449 family in the hepatocellular carcinoma cell line HLE

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