Abstract
Arachidonic acid (AA) can be converted into prostaglandins (PGs) or leukotrienes (LTs) by the enzymatic actions of cyclooxygenases (COX-1 and COX-2) or 5-lipoxygenase (5-LO), respectively. PGs and LTs are lipid signaling molecules that have been implicated in various diseases, including multiple cancers. 5-LO and its activating protein (FLAP) work together in the first two conversion steps of LT production. Previous work has suggested a role for LTs in cancer development and progression. MicroRNAs (miRNAs) are small RNA molecules that negatively regulate gene expression post-transcriptionally, and have previously been shown to be involved in cancer. Here, we show that high FLAP expression is associated with lower overall survival in lung adenocarcinoma patients, and FLAP protein is overexpressed in lung cancer cells compared to normal lung cells. Our lab has previously shown that miR-146a regulates COX-2 in lung cancer cells, and this miRNA is also predicted to target FLAP. Transient and stable transfections of miR-146a repress endogenous FLAP expression in lung cancer cells, and reporter assays show this regulation occurs through a direct interaction between the FLAP 3′ untranslated region (UTR) and miR-146a. Restoration of miR-146a also results in decreased cancer cell Leukotriene B4 (LTB4) production. Additionally, methylation analysis indicates the miR-146a promoter is hypermethylated in lung cancer cell lines. Taken together, this study and previous work from our lab suggest miR-146a is an endogenous dual inhibitor of AA metabolism in lung cancer cells by regulating both PG and LT production through direct targeting of the COX-2 and FLAP 3’ UTRs.
Highlights
Arachidonic acid (AA) metabolism and subsequent eicosanoid production is an important biochemical pathway that is more complex than is apparent at first glance
Analysis of data from 1,926 non-small cell lung cancer (NSCLC) patients showed a trend of high Figure 1: 5-Lipoxygenase Activating Protein (FLAP) expression correlating with a lower overall probability of survival, but this was not statistically significant (P = 0.15, hazard ratios (HR) = 1.1) (Figure 1A). 1,244 of these patients had an NSCLC subtype associated with their data
Protein and RNA were isolated from the cells 48 hours post-transfection. quantitative reverse transcriptase PCR (qRT-PCR) was performed for FLAP mRNA expression and ΔΔCT data analysis revealed a significant decrease in FLAP mRNA expression in A549 cells transfected with miR-146a (Figure 4A)
Summary
Arachidonic acid (AA) metabolism and subsequent eicosanoid production is an important biochemical pathway that is more complex than is apparent at first glance. There are two main arms of the metabolic pathway: AA is converted by the actions of COX-1 and COX-2 into prostaglandins (PGs) and thromboxanes (TXs) in one arm and the actions of 5-LO and its associated protein, 5-Lipoxygenase Activating Protein (FLAP/ALOX5AP), create leukotrienes (LTs) in the other arm of the pathway [1,2,3]. To determine the effect of exogenous miR-146a on endogenous FLAP mRNA and protein, A549 cells were transfected with 50 nM synthetic mature miR-146a. Another subset of A549 cells was transfected with 50 nM of a commercially available miRNA mimic with a sequence predicted to not target the human transcriptome. It is clear that endogenous FLAP can be downregulated by miR-146a in A549 lung cells
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