Genome-wide miRNA response to anacardic acid in breast cancer cells.

MicroRNAs are biomarkers and potential therapeutic targets for breast cancer. Anacardic acid (AnAc) is a dietary phenolic lipid that inhibits both MCF-7 estrogen receptor α (ERα) positive and MDA-MB-231 triple negative breast cancer (TNBC) cell proliferation with IC50s of 13.5 and 35 μM, respectively. To identify potential mediators of AnAc action in breast cancer, we profiled the genome-wide microRNA transcriptome (microRNAome) in these two cell lines altered by the AnAc 24:1n5 congener. Whole genome expression profiling (RNA-seq) and subsequent network analysis in MetaCore Gene Ontology (GO) algorithm was used to characterize the biological pathways altered by AnAc. In MCF-7 cells, 69 AnAc-responsive miRNAs were identified, e.g., increased let-7a and reduced miR-584. Fewer, i.e., 37 AnAc-responsive miRNAs were identified in MDA-MB-231 cells, e.g., decreased miR-23b and increased miR-1257. Only two miRNAs were increased by AnAc in both cell lines: miR-612 and miR-20b; however, opposite miRNA arm preference was noted: miR-20b-3p and miR-20b-5p were upregulated in MCF-7 and MDA-MB-231, respectively. miR-20b-5p target EFNB2 transcript levels were reduced by AnAc in MDA-MB-231 cells. AnAc reduced miR-378g that targets VIM (vimentin) and VIM mRNA transcript expression was increased in AnAc-treated MCF-7 cells, suggesting a reciprocal relationship. The top three enriched GO terms for AnAc-treated MCF-7 cells were B cell receptor signaling pathway and ribosomal large subunit biogenesis and S-adenosylmethionine metabolic process for AnAc-treated MDA-MB-231 cells. The pathways modulated by these AnAc-regulated miRNAs suggest that key nodal molecules, e.g., Cyclin D1, MYC, c-FOS, PPARγ, and SIN3, are targets of AnAc activity

miRNAs downregulated by AnAc in MDA-MB-231 cells.

<p>The genomic location of each miRNA was identified in miRAD <a href="http://bmi.ana.med.uni-muenchen.de/miriad/" target="_blank">http://bmi.ana.med.uni-muenchen.de/miriad/</a> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184471#pone.0184471.ref034" target="_blank">34</a>]. Verified targets are those experimentally validated targets of the indicated miRNA as demonstrated by 3’-UTR luciferase reporter assay. Since many publications do not include whether the 5p or 3p arm of the miRNA was studied, if the sequence of the miRNA was provided, it was searched in miRBase.org to identify which arm was used in the target gene 3’-UTR luciferase reporter assay.</p

miRNAs upregulated by AnAc in both MCF-7 and MDA-MB-231 cells.

<p>The genomic location of each miRNA was identified in miRAD <a href="http://bmi.ana.med.uni-muenchen.de/miriad/" target="_blank">http://bmi.ana.med.uni-muenchen.de/miriad/</a> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184471#pone.0184471.ref034" target="_blank">34</a>]. Verified targets are those experimentally validated targets of the indicated miRNA as demonstrated by 3’-UTR luciferase reporter assay. Since many publications do not include whether the 5p or 3p arm of the miRNA was studied, if the sequence of the miRNA was provided, it was searched in miRBase.org to identify which arm was used in the target gene 3’-UTR luciferase reporter assay.</p

miRNAs upregulated by AnAc MCF-7 cells.

<p>The genomic location of each miRNA was identified in miRAD <a href="http://bmi.ana.med.uni-muenchen.de/miriad/" target="_blank">http://bmi.ana.med.uni-muenchen.de/miriad/</a> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184471#pone.0184471.ref034" target="_blank">34</a>]. Verified targets are those experimentally validated targets of the indicated miRNA as demonstrated by 3’-UTR luciferase reporter assay in the cited reference. Since many publications do not include whether the 5p or 3p arm of the miRNA was studied, if the sequence of the miRNA was provided, it was searched in miRBase.org to identify which arm was used in the target gene 3’-UTR luciferase reporter assay.</p

Enrichment analysis of miRNA-seq data.

<p>Differentially expressed genes were identified in pairwise comparisons: MCF-7 AnAc vs. MDA-MB-231 AnAc using the tuxedo suite of programs including cufflink-cuffdiff2. The Venn diagrams show the number of common and differentially expressed genes significantly downregulated (A) and upregulated (B). Pathway analysis was performed using GeneGo Pathways Software (MetaCoreTM). The pathways identified for each comparison are listed in the order provided by MetaCoreTM analysis.</p

Heat map of miRNAs significantly altered in AnAc-treated MCF-7 and MDA-MB-231 cells.

<p>miRNAs significantly affected by AnAc were analyzed using Partek Genomic Suite^™ to generate the heat map.</p

Overexpression of miR-612 inhibits cell viability and inhibition of miR-612 inhibits AnAc’s anti-proliferative activity.

<p>MCF-7 and MDA-MB-231 cells were transfected with miR-Control (negative control), miR-612 mimic, anti-miR-Control (negative control), or anti-miR-612 for 24 h prior to 48 h treatment with EtOH (vehicle control) or 13.5 μM (MCF-7) or 35 μM (MDA-MB-231) AnAc. miR-612 expression was measured by qPCR relative to RNU48 in the transfected, untreated cells 72 h after transfection to match the time of the MTT assay (B). Values are the average of triplicate determinations ± SEM in one transfection and are relative to the appropriate transfection control as indicated. Cell viability was evaluated by MTT assay (B). Values for the MTT assay are relative to negative controls and are the avg ± SEM of 2 separate experiments. AnAc is proposed to affect cell viability through miR-612 (C).</p

miRNAs upregulated by AnAc in MDA-MB-231 cells.

<p>The genomic location of each miRNA was identified in miRAD <a href="http://bmi.ana.med.uni-muenchen.de/miriad/" target="_blank">http://bmi.ana.med.uni-muenchen.de/miriad/</a> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184471#pone.0184471.ref034" target="_blank">34</a>]. Verified targets are those experimentally validated targets of the indicated miRNA as demonstrated by 3’-UTR luciferase reporter assay. Since many publications do not include whether the 5p or 3p arm of the miRNA was studied, if the sequence of the miRNA was provided, it was searched in miRBase.org to identify which arm was used in the target gene 3’-UTR luciferase reporter assay.</p

miRNAs downregulated by AnAc in MCF-7 cells.

<p>The genomic location of each miRNA was identified in miRAD <a href="http://bmi.ana.med.uni-muenchen.de/miriad/" target="_blank">http://bmi.ana.med.uni-muenchen.de/miriad/</a> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184471#pone.0184471.ref034" target="_blank">34</a>]. Verified targets are those experimentally validated targets of the indicated miRNA as demonstrated by 3’-UTR luciferase reporter assay. Since many publications do not include whether the 5p or 3p arm of the miRNA was studied, if the sequence of the miRNA was provided, it was searched in miRBase.org to identify which arm was used in the target gene 3’-UTR luciferase reporter assay.</p