The online version of this article (doi:10.1186/1476-4598-11-71) contains supplementary material, which is available to authorized users.
The authors declare that they have no competing interests.
Study concept and design: SGC, AHL, AK, LFH. Acquisition of data: SGC, AJ, BF, LB, LFH. Analysis and interpretation of data: SGC, AJ, AK, LFH. Drafting of the manuscript: SGC, LFH. Critical revision of the manuscript for important intellectual content: SGC, AK, AHL, LB, AJ, LFH. Statistical analysis: SGC, AJ. Obtained funding: SGC, LFH. Administrative, technical, or material support: SGC, BF, LB, LFH. Study supervision: LFH, AHL. All authors read and approved the final manuscript.
Gastric cancer is the second most common cause of cancer-related death in the world. Inflammatory signals originating from gastric cancer cells are important for recruiting inflammatory cells and regulation of metastasis of gastric cancer. Several microRNAs (miRNA) have been shown to be involved in development and progression of gastric cancer. miRNA-146a (miR-146a) is a modulator of inflammatory signals, but little is known about its importance in gastric cancer. We therefore wanted to identify targets of miR-146a in gastric cancer and examine its biological roles.
The expression of miR-146a was evaluated by quantitative PCR (qPCR) and found up-regulated in the gastrin knockout mice, a mouse model of gastric cancer, and in 73% of investigated human gastric adenocarcinomas. Expression of miR-146a by gastric cancer cells was confirmed by in situ hybridization. Global analysis of changes in mRNA levels after miR-146a transfection identified two transcripts, caspase recruitment domain-containing protein 10 (CARD10) and COP9 signalosome complex subunit 8 (COPS8), as new miR-146a targets. qPCR, Western blotting and luciferase assays confirmed these transcripts as direct miR-146a targets. CARD10 and COPS8 were shown to be part of the G protein-coupled receptor (GPCR) pathway of nuclear factor-kappaB (NF-kappaB) activation. Lysophosphatidic acid (LPA) induces NF-kappaB activation via this pathway and over-expression of miR-146a inhibited LPA-induced NF-kappaB activation, reduced LPA-induced expression of tumor-promoting cytokines and growth factors and inhibited monocyte attraction.
miR-146a expression is up-regulated in a majority of gastric cancers where it targets CARD10 and COPS8, inhibiting GPCR-mediated activation of NF-kappaB, thus reducing expression of NF-kappaB-regulated tumor-promoting cytokines and growth factors. By targeting components of several NF-kappaB-activating pathways, miR-146a is a key component in the regulation of NF-kappaB activity.
Additional file 1: Figure S1. Expression of miR-146a in the gastrin KO mice and in human gastric cancer. In situ hybridization detection of miR-146a expression. (A and B) miR-146a expression was absent in WT fundic mouse tissue, while miR-146a-positive cells (blue nuclei) were detected in the metaplastic fundic tissue from gastrin KO mice. (C and D) miR-146a expression was absent in normal human gastric tissue, while human gastric adenocarcinoma cells stained for miR-146a (blue nuclei). Original magnification x10, Scale bar = 100 μm. (PDF 1343 kb). Figure S2. Survival of gastric cancer patients with low or high tumor miR-146a expression. Kaplan-Meier overall survival curves according to miR-146a level. Although patients whose tumors have high expression of miR-146a seemed to have better survival than those with low expression, it was not significant, p = 0,31 (Mantel-Cox Test). Low expression was defined as those with a relative miR-146a expression below the median expression and high expression had relative expression above median. Figure S3. Relative expression of miR-146a in cell lines. The expression of miR-146a in cells lines determined by qPCR. Gastric cancer cell lines are indicated by red bars. Relative miR-146a expression was determined using TaqMan® MicroRNA Assay hsa-miR-146a according to the manufacturer’s protocol (Applied Biosystems). miR-146a levels were normalized to hsa-miR-191 (Applied Biosystems), which served as an endogenous control. Figure S4. Normal growth of SNU638 cells transfected with miR-146a in cell lines. 2,5 106 SNU638 cells were seeded in 10 cm petri dishes and transfected the following day with 50 nM miR-146a, miCURY miR-146a inhibitor or Ctrl using Lipofectamine 2000 (Invitrogen). The following day the cell were transferred to 24-well plates where the cells were fixed at indicated time points in 4% paraformaldehyde, stained in a 0.1% crystal violet solution, and resuspended in 10% acetic acid. Sample absorbance was measured at 620 nm, and normalized to that of siGlo control transfected cells. Mean ± S.D. n = 4 /day. Figure S5. Expression of the mammalian signalosome 8 subunits (GPS1 and COPS2-8) in SNU638 cells transfected with miR-146a. Alteration in the expression of one subunit has been reported to affect the expression of the others. Using qPCR the expression of the subunits (GPS1 and COPS2-8) was therefore examined in SNU638 cells transfected with miR-146a. Only COPS8 mRNA is a direct target of miR-146a, indicated by open bars. The absence of miR-146a target sites is indicated by closed bars. The expression of COPS8 and COPS2 was reduced following miR-146a transfection. Mean ± S.D. * = p < 0.05, n = 4. COPS8 mRNA is directly targeted by miR-146a, while we assume that the expression of COPS2 is indirectly affected by miR-146a since the mRNA does not contain a miR-146a target site. Figure S6. Up-regulation of miR-146a in response to cancer-related cytokines. SNU638 cells were grown in media with 1% FCS overnight and subsequently stimulated with either LPA (10 μM), IL-1β (10 ng/ml) or vehicle for 6 h. The expression of miR-146a was measured by qPCR and normalized to the expression of U44. miR-146a expression is shown relative to the average expression in the control. Data are the mean ± S.D. (n = 4). Figure S7. Monocyte migration Boyden chamber assay. Monocyte migration against conditioned medium from control- or miR-146a-transfected SNU638 cells that were left untreated or stimulated with 25 μM LPA was followed over 6 hours. Monocyte migration against medium from LPA-stimulated SNU638 cells was increased compared to migration against medium from untreated cells, while monocyte migration against medium from miR-146a-transfected, LPA-stimulated cells was decreased compared to medium from control-transfected, LPA-stimulated cells. Data are shown as mean ± S.D, n = 3, * = P < 0.05. Monocytes were isolated as described. Monocytes were seeded in the upper chambers of Boyden chamber assay (Becton Dickinson, Heidelberg, Germany), 2.5x104 cells/well were seeded in RPMI1640 medium containing 1%. The lower chambers contained conditioned medium from SNU638 cells that had been transfected with Ctrl, miR-146a, or miCURY-miR-146a inhibitor and the following day left untreated or treated with 25 μM LPA (Avanti Polar Lipids) for 6 hours (n = 3). After 120-minute incubation in 5% CO2 at 37°C, non-migrating cells were scraped from the upper surface of the filter using a cotton plug. Cells on the lower surface were fixed with methanol and stained with methylene blue. The number of cells on the lower surface of the filter was determined microscopically by counting 3 high-power (x400) fields of constant area per well. To normalize all experiments, values were expressed as the fraction of number of cells that migrated through in the unconditioned control. Mean ± S.D. Table S1 mRNA qRT-PCR primers (TAG Copenhagen, Copenhagen, Denmark). Table S2 3’UTR luciferase primers. Restriction sites are indicated with lower case lettters (TAG Copenhagen). Table S3 Mutagenesis primers. Table 4 Transcripts with predicted 3’UTR miR-146a target sites that were significantly down-regulated upon miR-146a transfection. (PDF 1 MB)12943_2012_1047_MOESM1_ESM.pdf
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- microRNA-146a inhibits G protein-coupled receptor-mediated activation of NF-κB by targeting CARD10 and COPS8 in gastric cancer
Stephanie Geisler Crone
Anders H Lund
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