Scope: The molecular basis underlying the anti-inflammatory and anticarcinogenic properties of cranberries is incompletely understood. The effects of a cranberry proanthocyanidin-rich extract (PAC) and two of its gut microbial metabolites, 3,4-dihydroxyphenylacetic acid (DHPAA) and 3-(4-hydroxyphenyl)-propionic acid (HPPA), on intestinal epithelial cells microRNA (miRNA) expression and their downstream pathways at homeostasis and in inflammatory conditions, are investigated. Methods and Results: The expression of 799 miRNAs is quantitatively assessed in differentiated Caco-2BBe1 cells pre-treated with PAC, DHPAA, or HPPA and stimulated with interleukin (IL)-1𝜷 or not. PAC, DHPAA, and HPPA generate subsets of shared and distinct miRNA responses. At homeostasis, miRNAs affected by the metabolites, but not PAC, targeted genes enriched in kinase, Wnt, and growth factor signaling, cell growth and proliferation, apoptosis, and specific cancer pathways. In an inflammatory environment, PAC and DHPAA, but not HPPA, reverses the expression of 16 and two IL-1𝜷-induced miRNAs, respectively, regulating inflammatory and cancer pathways. Conclusion: miRNA modulation is a novel mechanism for PAC bioactivity in the gut. The gut microbiota may be necessary to unlock these effects at homeostasis and partially in inflammation.

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Objectives Polyphenols are emerging as novel prebiotic compounds. Cranberries are a rich source of polyphenols, such as proanthocyanidin (PAC), which has known benefits including anti-cancer properties. In the colon, PACs are catabolized by the gut microbiota into 3,4-dihydroxyphenylacetic acid (DHPAA) and 3-(4-hydroxyphenyl) propionic acid (HPPA), which may mediate prebiotic effects. Mechanisms are unknown but may involve host microRNA (miRNA). The objective of this study was to investigate the effects of nutritionally relevant doses of cranberry PAC, DHPAA, and HPPA on the human intestinal miRNome. Methods Differentiated Caco-2BBe1 colonic epithelial cells, a morphologically homogenous subclone of Caco-2 cells, were treated with cranberry extract containing 94% PAC (Ocean Spray Cranberries, 50 μg/ml), DHPAA, HPPA (5 μg/ml) or control vehicle (Dulbecco's Modified Eagle Medium) for 24 hours. Experiments were repeated 3 times. Cell viability was assessed by fluorescence microscopy. Cell RNA was extracted and used for miRNA profiling via NanoString Technology. Data were processed and normalized in nSolverTM 4.0, statistics and hierarchical clustering were done with R and ClustVis. Gene targets were predicted with miRNet and pathway enrichment analysis was done in PathDIP. Results The treatments had no effect on cell viability. Of the 829 miRNAs assessed, 248 were expressed. Five miRNAs were differentially expressed among groups (ANOVA, P < 0.01; FDR < 5%). Unsupervised hierarchical clustering with these miRNAs revealed perfect separation based on treatment. These miRNAs were found to target 686 genes, enriched in 116 KEGG pathways including “miRNAs in cancer” and “pathways in cancer”. Treatment-specific responses included increased miR-2116-5p, miR-6721-5p, and miR-1290 for PAC, DHPAA, and HPPA, respectively. Pathways in glucagon signaling and central carbon metabolism in cancer were enriched in response to DHPPA only. Conclusions Cranberry PAC and polyphenol metabolites at concentrations representing dietary intakes elicit different miRNA signatures in colonic cells, providing a novel mechanism to explain their effects on intestinal health. Cranberry-mediated miRNA modulation may represent a potential strategy for preventing chronic disease. Funding Sources NSERC, Ocean Spray Cranberries, Inc., NSERC Graduate Scholarship.

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