miR-146a suppresses 5-lipoxygenase activating protein (FLAP) expression and Leukotriene B4 production in lung cancer cells

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.

Keywords: arachidonic acid; gene expression; lipoxygenase pathway; microRNA; promoter methylation.

Figure 1. 5-Lipoxygenase Activating Protein (FLAP) expression may have prognostic value in lung adenocarcinoma

The Non-Small Cell Lung Cancer (NSCLC) KM Plotter Tool (http://www.kmplot.com) was used to generate survival curves based on a patient's overall survival in months and their FLAP expression level (low or high) relative to the median value. (A) No significant correlation between FLAP expression and overall survival in 1,926 NSCLC patients (P = 0.15). (B) No significant correlation between FLAP expression and overall survival in 524 lung squamous cell carcinoma patients (P = 0.34). (C) Highly significant correlation between FLAP expression and overall survival in 720 lung adenocarcinoma patients (P = 3.1 × 10^-7).

Figure 2. FLAP expression levels in lung cell lines

(A) Western blot analysis of Beas2B (normal lung), A549 (NSCLC), H1299 (NSCLC), and H1975 (NSCLC) cell lysates. Western blots were repeated at least three times. (B) Quantification of relative FLAP protein levels was performed using the gel analysis tool on ImageJ software and normalized to α-tubulin protein levels. Analysis indicated significant overexpression of FLAP protein in A549 (lane 2) and H1299 cells (lane 3), but not H1975 cells (lane 4) with respect to Beas2B cells (lane 1). (^*) P < 0.04, n=3. (C) ΔΔCT qRT-PCR analysis indicated significantly increased expression of FLAP mRNA in Beas2B cells compared to A549 and H1299 cells, but not H1975 cells. FLAP expression was normalized to GAPDH mRNA. (^*) P < 0.01, n=3.

Figure 3. miR-146a is predicted to target the FLAP 3′ UTR

(A) Schematic illustration of the FLAP 3′ UTR (not drawn to scale). The stop sign and red triangle represent the stop codon and polyadenylation site, respectively. Green lines indicate putative miRNA binding sites as predicted by the microRNA.org and TargetScan algorithms. Purple lines indicate miRNA binding sites validated in the literature. (B) Predicted alignment of miR-146a binding to the FLAP 3′ UTR. The 7-mer seed sequence is highlighted in red and bold. Red lines indicate perfect complementarity and light blue lines indicate low-affinity U-G pairing. Numbers represent the base position within the 3′ UTR. (C) Diagram displaying the sequence conservation of the miR-146a seed sequence (highlighted in red) in the FLAP 3′ UTR across various vertebrate species. Uppercase italic letters indicate conservation over at least 12 vertebrate species; lowercase italic letters indicate conservation over at least 9 vertebrate species. Sequences were obtained from TargetScan.

Figure 4. Synthetic miR-146a represses endogenous FLAP mRNA and protein in A549 cells

A549 cells were transiently transfected with miRNA mimics. Cells were lysed and RNA and protein were isolated 48 hours post-transfection. (A) ΔΔCT qRT-PCR analysis indicated decreased FLAP mRNA expression in A549 cells transfected with 50 nM miR-146a. FLAP expression was normalized to GAPDH mRNA. FLAP mRNA levels were significantly different in cells transfected with miR-146a from levels in mock-treated cells and cells transfected with 50 nM non-targeting miRNA. (^*) P < 0.01, n=3. (B) Western blot analysis of cell lysates indicated decreased FLAP protein expression in A549 cells transfected with 50 nM miR-146a (lane 1) compared to mock-treated A549 cells (lane 2) and A549 cells transfected with 50 nM non-targeting miRNA (lane 3). A representative blot is shown of three independent experiments. (C) Quantification of relative FLAP protein levels was performed using the gel analysis tool on ImageJ software and normalized to α-tubulin protein levels. (^*) P < 0.01, n=3. (D) Western blot analysis of cell lysates indicated dose-dependent decreased FLAP protein expression in A549 cells transfected with 50 and 100 nM miR-146a (lanes 2 and 3) compared to mock-treated A549 cells (lane 1). A representative blot is shown of three independent experiments. (E) Quantification of relative FLAP protein levels was performed using the gel analysis tool on ImageJ software and normalized to GAPDH protein levels. (^*) P < 0.035, (^**) P < 0.025, n=3.

Figure 5. Doxycycline-induced expression of miR-146a in H1299 cells represses endogenous FLAP protein

(A) Schematic illustration of methodology used for stable cell line development (see methods section for more details). Where indicated, H1299 Tet/TRE-empty and H1299 Tet/TRE-miR-146a cells were cultured in 1 μg/mL doxycycline. (B) Left: ΔΔCT qRT-PCR analysis indicated successful induction of mature miR-146a expression in H1299 Tet/TRE-miR-146a cells. miR-146a expression was normalized to U6 snRNA expression. Right: Focused graph showing miR-146a expression in control cell lines. (^*) P < 0.03, n=3. (C) Western blot analysis of cell lysates indicated decreased FLAP protein expression only in H1299 Tet/TRE-miR-146a cells cultured with doxycycline. A representative blot is shown of three independent experiments. (D) Quantification of relative FLAP protein levels was performed using the gel analysis tool on ImageJ software and normalized to α-tubulin protein levels. (^*) P < 0.037, n=3.

Figure 6. miR-146a directly targets the FLAP 3′ UTR

(A, top) Schematic illustration of the Renilla luciferase 3′ UTR constructs used: pLightSwitch_GAPDH 3′ UTR, pLightSwitch_FLAP-WT 3′ UTR, and pLightSwitch_FLAP-146a MUT 3′ UTR. (A, bottom) Predicted alignment of miR-146a binding to the FLAP WT 3′ UTR. The 7-mer seed sequence is highlighted in bold. Lines indicate perfect complementarity and potential low-affinity U-G pairing. The alignment is also shown for miR-146a binding to the FLAP 3′ UTR containing a 4-nt mutation (UUCU-CCGC) in the FLAP-146a MUT 3′ UTR construct. (B) Renilla luciferase activity was measured in HeLa cells transfected with 50 nM synthetic miRNAs (miR-146a or non-targeting miR) and the pLightSwitch_FLAP-WT 3′ UTR construct. FLAP WT 3′ UTR luciferase activity was normalized to GAPDH 3′ UTR luciferase activity in HeLa cells exposed to the same miRNA condition. Luciferase activity was further normalized to total protein concentration of each sample. Renilla luciferase activity was significantly decreased only in samples treated with miR-146a. (^*) P < 0.002, n=6. (C) FLAP-WT 3′ UTR Renilla luciferase activity was measured in HeLa cells transfected with 50 nM miR-146a alone, 50 nM anti-miR-146a alone, or 50 nM of both miR-146a and anti-miR-146a. Luciferase activity was normalized as described above in B. Renilla luciferase activity in cells treated with miR-146a and its antagomiR together was significantly greater than in cells treated with miR-146a alone. (^*) P < 0.001, n=5. (D) FLAP-WT 3′ UTR and FLAP-146a MUT 3′ UTR Renilla luciferase activities were measured in HeLa cells transfected with 50 nM synthetic miRNAs (miR-146a or non-targeting miR). Luciferase activity was normalized as described above in B. FLAP-146a MUT 3′ UTR Renilla luciferase activity was significantly higher than FLAP-WT 3′ UTR Renilla luciferase activity in cells treated with miR-146a due to the mutation in the miR-146a binding site. (^*) P < 0.01, n=3.

Figure 7. Increased miR-146a levels result in decreased cellular production of LTB₄

(A) A549 cells were transiently transfected with 50 nM miRNA mimics. Cell-free, serum-free supernatants were collected 48 hours post-transfection. An enzyme-linked immunosorbent assay (ELISA) was used to measure Leukotriene B4 (LTB₄) concentrations. A549 cells transfected with miR-146a produced significantly less LTB₄ compared to mock-treated cells and cells transfected with a non-targeting miRNA. (^*) P < 0.025, n=3. (B) Where indicated, H1299 Tet/TRE-empty and H1299 Tet/TRE-miR-146a cells were cultured in 1 μg/mL doxycycline. Cell-free, serum-free supernatants were collected and an ELISA was used to measure LTB₄ concentrations. H1299 Tet/TRE-miR-146a cells cultured in doxycycline produced significantly less LTB₄. (^*) P < 0.04, n=3.

Figure 8. miR-146a expression is regulated by CpG methylation in lung cell lines

(A) Schematic illustration depicting the miR-146a promoter region (not drawn to scale). Vertical lines indicate CpG sites. Primer annealing locations for methylation-specific PCR (MSP) are shown as white arrows. (B) ΔΔCT qRT-PCR analysis indicated increased expression of mature miR-146a in A549 cells following 5-aza-2’-deoxycytidine (5-aza-dC) treatment. miR-146a expression was normalized to U6 snRNA expression. (^*) P < 0.03, n=3. (C) Left: MSP products for miR-146a promoter region 1 in four lung cell lines run on a 1% agarose gel. Representative gel of three independent experiments is shown. Right: Image J software was used to measure band intensities. The intensity of the methylated band divided by total band intensity for each cell line is shown. (^*) P < 0.006, n=3. (D) Left: MSP products for miR-146a promoter region 2 in four lung cell lines run on a 1% agarose gel. Representative gel of three independent experiments is shown. Right: Image J software was used to measure band intensities. The intensity of the methylated band divided by total band intensity for each cell line is shown. (^*) P < 0.025, (^**) P < 0.001, n=3. U: unmethylated state; M: methylated state.

Figure 9. miR-146a negatively regulates both the prostaglandin and leukotriene arms of the arachidonic acid metabolic pathway

(A) In normal lung cells, a hypomethylated promoter allows miR-146a to be expressed. miR-146a directly targets COX-2 and FLAP, which controls PGE₂ and LTB₄ production. (B) In lung cancer cells, a hypermethylated promoter results in reduced miR-146a expression. This leads to increased COX-2 and FLAP protein expression, and therefore higher levels of PGE₂ and LTB₄. Adapted in part from: [14].