MicroRNAs in Apple-Derived Nanoparticles Modulate Intestinal Expression of Organic Anion-Transporting Peptide 2B1/SLCO2B1 in Caco-2 Cells.

Plant-derived nanoparticles exert cytoprotective effects on intestinal cells by delivering their cargo to intestinal tissues. We previously reported that apple-derived nanoparticles (APNPs) downregulate the mRNA of the human intestinal transporter organic anion-transporting peptide 2B1 (OATP2B1)/SLCO2B1 and that the 3'-untranslated region (3'UTR) is required for the response to APNPs. Here, we investigated the involvement of microRNAs (miRNAs) in APNPs in suppressing OATP2B1 expression to demonstrate that APNP macromolecules directly interact with intestinal tissues. Using in silico analysis, seven apple miRNAs were predicted as candidate miRNAs that interact with the SLCO2B1-3'UTR. The APNP-mediated decrease in luciferase activity of pGL3/SLCO2B1-3'UTR was abrogated by inhibitors of mdm-miR-160a-e, -7121a-c, or -7121d-h. Each miRNA mimic reduced the endogenous expression of SLCO2B1 mRNA in Caco-2 cells. The luciferase activity of the truncated pGL3/SLCO2B1-3'UTR, which contains approximately 200 bp around each miRNA recognition element (MRE), was decreased by the miR-7121d-h mimic but decreased little by the other mimics. APNP also reduced the luciferase activity of truncated pGL3/SLCO2B1-3'UTR containing an MRE for miR-7121d-h. Thus, we demonstrated that mdm-miR-7121d-h contributes to the APNP-mediated downregulation of intestinal OATP2B1. Accordingly, plant macromolecules, such as miRNAs, may directly interact with intestinal tissues via nanoparticles. SIGNIFICANCE STATEMENT: This study demonstrates that mdm-miR7121d-h contained in apple-derived nanoparticles downregulated the mRNA expression of SLCO2B1 by interacting with SLCO2B1-3'-untranslated region directly and that SLCO2B1 mRNA might also be decreased by mdm-miR160a-e and -7121a-c indirectly. This finding that the specific apple-derived microRNAs influence human intestinal transporters provides a novel concept that macromolecules in foods directly interact with and affect the intestinal function of the host.

Apple-Derived Nanoparticles Modulate Expression of Organic-Anion-Transporting Polypeptide (OATP) 2B1 in Caco-2 Cells.

Interaction of foods with intestinal transporters has generally been ascribed to small molecules, but recently, edible-plant-derived nanoparticles (NPs) have been suggested to affect intestinal function. Here, we examined the effects of NPs contained in edible fruits on intestinal transporters. Apple-derived NPs (APNPs) were isolated by ultracentrifugation and characterized by measurement of particle size distribution and electron microscopy. Human epithelial colorectal adenocarcinoma (Caco-2) cells internalized fluorescently labeled APNPs, suggesting that fruit-derived NPs would be internalized into intestinal epithelial cells in vivo. We found that the mRNA expression levels of several transporters, including organic-anion-transporting polypeptide (OATP) 2B1, were changed in APNP-treated Caco-2 cells. The protein expression and activity of OATP2B1 were also decreased by APNP exposure, as determined by Western blotting and measurements of [3H]estrone-3-sulfate uptake by Caco-2 cells, respectively. These actions required intact APNPs, because sonication or boiling abrogated the effects. Since the content of apple-derived small molecules in APNPs was negligible, the observed decrease of OATP2B1 expression appears to be mediated by large molecules in the APNPs. We further found that the 3'-untranslated region of the OATP2B1 gene was required for the response to APNPs, suggesting that microRNA in the APNPs might be involved. These results propose a novel mechanism, in which large molecules such as microRNA in food could affect intestinal transporters through food-derived NPs, which also demonstrates that food-derived NPs should be useful for delivery of biologically active large molecules to intestinal tissues.

Association of miR-145 With Statin-Induced Skeletal Muscle Toxicity in Human Rhabdomyosarcoma RD Cells.

Skeletal muscle toxicity including rhabdomyolysis in severe case is a major side effect of low-density lipoprotein cholesterol-lowering statin drugs. We, therefore, aimed at exploring microRNA (miRNA) expression to understand molecular mechanism of statin-induced toxicity. miRNA expression profiling assay for cerivastatin (1 µM for 48 h)-treated RD cells showed more than 2-fold decrease in 26 miRNA expressions with miR-145 being downregulated prominently. When RD cells were treated with cerivastatin at 10 µM for 36 h, mitochondrial dysfunction was observed in 49.6% of the population without causing apoptosis, whereas 82% underwent apoptosis when treated at 10 µM for 48 h. In RD cells treated under the same condition (10 µM for 48 h), miR-145 expression and mRNA expressions of proapoptotic APAF1 and CASP10 genes, potential targets of miR-145, significantly decreased and increased, respectively. Moreover, enforced expression of miR-145 reduced apoptotic cell population of cerivastatin-treated RD cells (10 µM for 36 h). Because miR-145 increased in extracellular medium from cerivastatin-treated RD cells, miR-145 was suggested to be secreted in response to statin-induced toxicity. These results provide a new rationale for statin's toxicity that statin-induced apoptosis is caused by enhanced expression of proapoptotic genes mediated by decreased intracellular miR-145 due to statin-induced mitochondrial dysfunction.