Dietary plant microRNAs as potential regulators of cellular cholesterol efflux.

AIM: Epidemiological evidence suggests adherence to vegetable-rich diets is associated to atheroprotective effects and bioactive components are most likely to play a relevant role. The notion of inter-kingdom regulation has opened a new research paradigm and perhaps microRNAs (miRNAs) from edible vegetables could influence consumer gene expression and lead to biological effects. We aimed to investigate the potential impact of broccoli-derived miRNAs on cellular cholesterol efflux in vitro. METHODS: Four miRNAs (miR159a, miR159b, miR166a and miR403) from Brassica oleracea var. italica (broccoli), a widely consumed cruciferous vegetable, were selected for further investigation, based on their high abundancy in this vegetable and their presence in other plants. Selected miRNAs were synthesized with a 3'-terminal 2'-O-methylation and their cellular toxicity, in vitro gastrointestinal resistance and cellular uptake were evaluated. Potential target genes within the mammalian transcriptome were assessed in silico following pathway analysis. In vitro cholesterol efflux was assessed in human THP-1-derived macrophages. RESULTS: miRNAs survival to in vitro GI digestion was around 1%, although some variation was seen between the four candidates. Cellular uptake by mammalian cells was confirmed, and an increase in cholesterol efflux was observed. Pathway analysis suggested these miRNAs are involved in biological processes related to phosphorylation, phosphatidylinositol and Wnt signaling, and to the insulin/IGF pathway. CONCLUSIONS: Health-promoting properties attributed to cruciferous vegetables, might be mediated (at least in part) through miRNA-related mechanisms.

Therapeutic potential of broccoli-derived extracellular vesicles as nanocarriers of exogenous miRNAs.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression. The wide-ranging biological activities of microRNAs stimulated research on disease mechanisms and is suggesting appealing therapeutic applications. When unprotected, miRNAs suffer from rapid degradation and appropriate strategies need to be developed to improve their therapeutic potential. Since the first observation of miRNAs being naturally transported by extracellular vesicles (EVs), the latter have been proposed as specific transport means for drug delivery, conferring stability and increasing resistance against RNase degradation. However, a standard, reproducible, and cost-effective protocol for EV isolation is lacking. Here, the use of broccoli-derived EVs as a therapeutic vehicle for extracellular RNA drug delivery was assessed. EVs were isolated from broccoli, combining ultracentrifugation and size exclusion chromatography methodology. Caco-2 cells were exposed to isolated EVs loaded with exogenous miRNAs and cellular viability was tested. The miRNAs were taken up by this intestinal cell line. Our results show that broccoli EVs can be efficiently isolated, characterized, and loaded with exogenous miRNAs, leading to toxicity in caco-2 cells. Because the pharmaceutical industry is searching for novel drug delivery nanovesicles with intrinsic properties such as low immunogenicity, stability to the gastrointestinal tract, ability to overcome biological barriers, large-scale production, cost-effectiveness, etc., broccoli-isolated nanovesicles might be suitable candidates for future pharmacological applications. We propose broccoli as a natural source of EVs, which are capable of transporting exogenous miRNAs with potential therapeutic effects and suggest that appropriate toxicological and randomized controlled trials as well as patent applications are warranted.

Interplay of Walnut Consumption, Changes in Circulating miRNAs and Reduction in LDL-Cholesterol in Elders.

The mechanisms underlying the lipid-lowering effect of nuts remain elusive. This study explores whether one-year supplementation with walnuts decreases LDL-cholesterol (LDL-C) by affecting the expression of circulating microRNAs (c-miRNA). In this sub-study of the Walnuts and Healthy Aging (WAHA) trial, we obtained fasting serum at baseline and at 1 year from 330 free-living participants (63-79 year, 68% women), allocated into a control group (CG, abstinence from walnuts, n = 164) and a walnut group (WG, 15% of daily energy as walnuts, ~30-60 g/d, n = 166). Participants in the WG showed a 1 year decrease in LDL-C (-9.07, (95% confidence interval: -12.87; -5.73) mg/dL; p = 0.010 versus changes in the CG). We conducted a miRNA array in eight randomly selected participants in the WG who decreased in LDL-C. This yielded 53 c-miRNAs with statistically significant changes, 27 of which survived the correction for multiple testing. When validating them in the full population, statistical significance lasted for hsa-miR-551a, being upregulated in the WG. In mediation analysis, the change in hsa-miR-551a was unrelated to LDL-C decrease. Long-term supplementation with walnuts decreased LDL-C independently of the changes in c-miRNA. The hsa-miR-551a upregulation, which has been linked to a reduced cell migration and invasion in several carcinomas, suggests a novel mechanism of walnuts in cancer risk.

Dietary bovine milk miRNAs transported in extracellular vesicles are partially stable during GI digestion, are bioavailable and reach target tissues but need a minimum dose to impact on gene expression.

PURPOSE: Extracellular RNAs are unstable and rapidly degraded unless protected. Bovine-milk extracellular vesicles (EVs) confer protection to dietary miRNAs, although it remains unclear whether this importantly improves their chances of reaching host target cells to exert biological effects. METHODS: Caco-2, HT-29, Hep-G2 and FHs-74 cell lines were exposed to natural/labelled milk EVs to evaluate cellular uptake. Five frequently reported human milk miRNAs (miR-146b-5p, miR-148a-3p, miR-30a-5p, miR-26a-5p, and miR-22-3p) were loaded into EVs. The intracellular concentration of each miRNA in cells was determined. In addition, an animal study giving an oral dose of loaded EVs in C57BL6/ mice were performed. Gene expression regulation was assessed by microarray analysis. RESULTS: Digestive stability analysis showed high overall degradation of exogenous miRNAs, although EV-protected miRNAs better resisted gastrointestinal digestion compared to free miRNAs (tenfold higher levels). Importantly, orally delivered EV-loaded miRNAs reached host organs, including brain, in mice. However, no biological effect has been identified. CONCLUSION: Milk EVs protect miRNAs from degradation and facilitate cellular uptake. miRNA concentration in EVs from bovine milk might be insufficient to produce gene modulation. Nevertheless, sizable amounts of exogenous miRNAs may be loaded into EVs, and orally delivered EV-loaded miRNAs can reach tissues in vivo, increasing the possibility of exerting biological effects. Further investigation is justified as this could have an impact in the field of nutrition and health (i.e., infant formulas elaboration).

Connection between miRNA Mediation and the Bioactive Effects of Broccoli (Brassica oleracea var. italica): Exogenous miRNA Resistance to Food Processing and GI Digestion.

Broccoli (Brassica oleracea var. italica) and its bioactive compounds are associated with beneficial health effects, which might be enabled, at least in part, through miRNA regulation, despite recent controversial studies suggesting that exogenous dietary miRNAs may reach host circulation and target cells to regulate gene expression. Here, a computational analysis was performed to explore the processes and pathways associated with genes targeted either by (1) host-expressed miRNAs (endogenous) modulated by the bioactive compounds in broccoli or (2) miRNAs derived from broccoli (exogenous). In addition, the stability of exogenous miRNAs from broccoli was assessed after broccoli was subjected to the usual processing methods and in vitro digestion-simulating gastrointestinal (GI) conditions. Overall, bioinformatic results show that the anticarcinogenic and cancer-preventive properties attributed to cruciferous vegetables might be mediated, at least in part, through miRNA-related mechanisms. Moreover, results show that broccoli-derived miRNAs can survive common food-processing conditions and GI digestion.

Comparative Transcriptomes of the Body Wall of Wild and Farmed Sea Cucumber Isostichopus badionotus.

Overfishing of sea cucumber Isostichopus badionotus from Yucatan has led to a major population decline. They are being captured as an alternative to traditional species despite a paucity of information about their health-promoting properties. The transcriptome of the body wall of wild and farmed I. badionotus has now been studied for the first time by an RNA-Seq approach. The functional profile of wild I. badionotus was comparable with data in the literature for other regularly captured species. In contrast, the metabolism of first generation farmed I. badionotus was impaired. This had multiple possible causes including a sub-optimal growth environment and impaired nutrient utilization. Several key metabolic pathways that are important in effective handling and accretion of nutrients and energy, or clearance of harmful cellular metabolites, were disrupted or dysregulated. For instance, collagen mRNAs were greatly reduced and deposition of collagen proteins impaired. Wild I. badionotus is, therefore, a suitable alternative to other widely used species but, at present, the potential of farmed I. badionotus is unclear. The environmental or nutritional factors responsible for their impaired function in culture remain unknown, but the present data gives useful pointers to the underlying problems associated with their aquaculture.

One-year dietary supplementation with walnuts modifies exosomal miRNA in elderly subjects.

PURPOSE: Epidemiological studies and clinical trials support the association of nut consumption with a lower risk of prevalent non-communicable diseases, particularly cardiovascular disease. However, the molecular mechanisms underlying nut benefits remain to be fully described. MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression and play a pivotal role in health and disease. Exosomes are extracellular vesicles released from cells and mediate intercellular communication. Whether nut consumption modulates circulating miRNAs (c-miRNAs) transported in exosomes is poorly described. METHODS: Cognitively healthy elderly subjects were randomized to either control (n = 110, abstaining from walnuts) or daily supplementation with walnuts (15% of their total energy, ≈30-60 g/day, n = 101) for 1-year. C-miRNAs were screened in exosomes isolated from 10 samples, before and after supplementation, and identified c-miRNA candidates were validated in the whole cohort. In addition, nanoparticle tracking analysis and lipidomics were assessed in pooled exosomes from the whole cohort. RESULTS: Exosomal hsa-miR-32-5p and hsa-miR-29b-3p were consistently induced by walnut consumption. No major changes in exosomal lipids, nanoparticle concentration or size were found. CONCLUSION: Our results provide novel evidence that certain c-miRNAs transported in exosomes are modulated by walnut consumption. The extent to which this finding contributes to the benefits of walnuts deserves further research.

Intestinal miRNAs regulated in response to dietary lipids.

The role of miRNAs in intestinal lipid metabolism is poorly described. The small intestine is constantly exposed to high amounts of dietary lipids, and it is under conditions of stress that the functions of miRNAs become especially pronounced. Approaches consisting in either a chronic exposure to cholesterol and triglyceride rich diets (for several days or weeks) or an acute lipid challenge were employed in the search for intestinal miRNAs with a potential role in lipid metabolism regulation. According to our results, changes in miRNA expression in response to fat ingestion are dependent on factors such as time upon exposure, gender and small intestine section. Classic and recent intestinal in vitro models (i.e. differentiated Caco-2 cells and murine organoids) partially mirror miRNA modulation in response to lipid challenges in vivo. Moreover, intestinal miRNAs might play a role in triglyceride absorption and produce changes in lipid accumulation in intestinal tissues as seen in a generated intestinal Dicer1-deletion murine model. Overall, despite some variability between the different experimental cohorts and in vitro models, results show that some miRNAs analysed here are modulated in response to dietary lipids, hence likely to participate in the regulation of lipid metabolism, and call for further research.

Intestinal Lipid Metabolism Genes Regulated by miRNAs.

MicroRNAs (miRNAs) crucial roles in translation repression and post-transcriptional adjustments contribute to regulate intestinal lipid metabolism. Even though their actions in different metabolic tissues have been elucidated, their intestinal activity is yet unclear. We aimed to investigate intestinal miRNA-regulated lipid metabolism-related genes, by creating an intestinal-specific Dicer1 knockout (Int-Dicer1 KO) mouse model, with a depletion of microRNAs in enterocytes. The levels of 83 cholesterol and lipoprotein metabolism-related genes were assessed in the intestinal mucosa of Int-Dicer1 KO and Wild Type C57BL/6 (WT) littermates mice at baseline and 2 h after an oral lipid challenge. Among the 18 genes selected for further validation, Hmgcs2, Acat1 and Olr1 were found to be strong candidates to be modulated by miRNAs in enterocytes and intestinal organoids. Moreover, we report that intestinal miRNAs contribute to the regulation of intestinal epithelial differentiation. Twenty-nine common miRNAs found in the intestines were analyzed for their potential to target any of the three candidate genes found and validated by miRNA-transfection assays in Caco-2 cells. MiR-31-5p, miR-99b-5p, miR-200a-5p, miR-200b-5p and miR-425-5p are major regulators of these lipid metabolism-related genes. Our data provide new evidence on the potential of intestinal miRNAs as therapeutic targets in lipid metabolism-associated pathologies.

A Glycosaminoglycan-Rich Fraction from Sea Cucumber Isostichopus badionotus Has Potent Anti-Inflammatory Properties In Vitro and In Vivo.

Sea cucumber body wall contains several naturally occurring bioactive components that possess health-promoting properties. Isostichopus badionotus from Yucatan, Mexico is heavily fished, but little is known about its bioactive constituents. We previously established that I. badionotus meal had potent anti-inflammatory properties in vivo. We have now screened some of its constituents for anti-inflammatory activity in vitro. Glycosaminoglycan and soluble protein preparations reduced 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammatory responses in HaCaT cells while an ethanol extract had a limited effect. The primary glycosaminoglycan (fucosylated chondroitin sulfate; FCS) was purified and tested for anti-inflammatory activity in vivo. FCS modulated the expression of critical genes, including NF-ĸB, TNFα, iNOS, and COX-2, and attenuated inflammation and tissue damage caused by TPA in a mouse ear inflammation model. It also mitigated colonic colitis caused in mice by dextran sodium sulfate. FCS from I. badionotus of the Yucatan Peninsula thus had strong anti-inflammatory properties in vivo.

Arthropod-Borne Bacteria Cause Nonmalarial Fever in Rural Ethiopia: A Cross-Sectional Study in 394 Patients.

Bacterial arthropod-borne pathogens are a common cause of fever in Africa, but their precise impact is unknown and usually underdiagnosed in the basic rural laboratories of low-resourced African countries. Our aim was to determine the prevalence of arthropod-borne bacterial diseases causing fever among malaria smear-negative patients in a rural hospital located in Ethiopia. The study population included patients aged 2 years or older; referred to Gambo Rural General Hospital (West Arsi, Ethiopia), between July and November 2013, for fever or report of fever in the previous 48 h; attending the outpatient department; and testing negative for malaria by Giemsa-stained thin blood smears. We extracted DNA from 394 whole blood samples, using reverse line blot assays of amplicons to look for bacteria from the genera: Anaplasma, Bartonella, Borrelia, Coxiella, Ehrlichia, Francisella, and Rickettsia. Thirteen patients showed presence of DNA for these pathogens: three each by Borrelia spp., the Francisella group (F. tularensis tularensis, F. tularensis holartica, and F. novicia), Rickettsia bellii, and Rickettsia Felis, and one by Bartonella rochalimae. Thus, in this rural area of Africa, febrile symptoms could be due to bacteria transmitted by arthropods. Further studies are needed to evaluate the pathogenic role of R. bellii.

Postprandial Circulating miRNAs in Response to a Dietary Fat Challenge.

Postprandial lipemia has many physiopathological effects, some of which increase the risk of cardiovascular disease. MicroRNAs (miRNAs) can be found in almost all biological fluids, but their postprandial kinetics are poorly described. We aimed to profile circulating miRNAs in response to a fat challenge. In total, 641 circulating miRNAs were assessed by real-time PCR in plasmas from mice two hours after lipid gavage. Mice with intestine-specific loss of Dicer were screened to identify potential miRNAs released by the intestine. A total of 68 miRNAs were selected for further validation. Ten circulating miRNAs were finally validated as responsive to postprandial lipemia, including miR-206-3p, miR-543-3p, miR-466c-5p, miR-27b-5p, miR-409-3p, miR-340-3p, miR-1941-3p, miR-10a-3p, miR-125a-3p, and miR-468-3p. Analysis of their possible tissues of origin/target showed an enrichment of selected miRNAs in liver, intestine, brain, or skeletal muscle. miR-206, miR-27b-5p, and miR-409-3p were validated in healthy humans. Analysis of their predicted target genes revealed their potential involvement in insulin/insulin like growth factor (insulin/IGF), angiogenesis, cholecystokinin B receptor signaling pathway (CCKR), inflammation or Wnt pathways for mice, and in platelet derived growth factor (PDGF) and CCKR signaling pathways for humans. Therefore, the current study shows that certain miRNAs are released in the circulation in response to fatty meals, proposing them as potential novel therapeutic targets of lipid metabolism.

Olive Oil, Palm Oil, and Hybrid Palm Oil Distinctly Modulate Liver Transcriptome and Induce NAFLD in Mice Fed a High-Fat Diet.

Nonalcoholic fatty liver disease (NAFLD) is highly prevalent worldwide. The most severe form is nonalcoholic steatohepatitis (NASH). Among risk factors for the development of NAFLD is excessive lipid intake. Since palm (P) oil is the most consumed oil in the world, we aimed to investigate the effects of high-fat diets made with P oil, hybrid palm (HP) oil, or olive (O) oil in liver. Twenty-four male mice (C57Bl/6J) were fed a high-fat diet (41% fat) containing P, HP, or O oils for 8 weeks and compared to a control (C) group fed a chow diet. Adiposity was measured with computed tomography. Body, adipose tissue, and liver weights, as well as liver fat (Bligh⁻Dyer), blood lipid profile, glucose, and liver enzymes were measured. Liver histology (hematoxylin⁻eosin) and transcriptome (microarray-based) were performed. ANOVA tests with Newman⁻Keuls were used. Body weight was increased in the P group (p < 0.001) and body fat in the O group (C vs. O p ≤ 0.01, P vs. O p ≤ 0.05, HP vs. O p ≤ 0.05). All high-fat diets disturbed the blood lipid profile and glucose, with marked effects of HP on very low-density lipoprotein cholesterol (VLDL), triglycerides, and alkaline phosphatase (p ≤ 0.001). HP had the highest liver fat (42.76 ± 1.58), followed by P (33.94 ± 1.13). O had a fat amount comparable to C (16.46 ± 0.34, 14.71 ± 0.70, respectively). P and HP oils induced hepatocyte ballooning. Transcriptome alterations of the O group were related to amino acid metabolism and fatty acid (FA) metabolism, the P group to calcium ion homeostasis, and HP oil to protein localization. Both P and HP oils induced NASH in mice via disturbed hepatocyte transcription. This raises concerns about the content of these oils in several industrialized foods.

Dietary supplementation with hybrid palm oil alters liver function in the common Marmoset.

Hybrid palm oil, which contains higher levels of oleic acid and lower saturated fatty acids in comparison with African palm oil, has been proposed to be somehow equivalent to extra virgin olive oil. However, the biological effects of its consumption are poorly described. Here we have explored the effects of its overconsumption on lipid metabolism in a non-human primate model, the common marmoset. Dietary supplementation of marmoset with hyperlipidic diet containing hybrid palm oil for 3 months did not modify plasma lipids levels, but increased glucose levels as compared to the supplementation with African palm oil. Liver volume was unexpectedly found to be more increased in marmosets consuming hybrid palm oil than in those consuming African palm oil. Hepatic total lipid content and circulating transaminases were dramatically increased in animals consuming hybrid palm oil, as well as an increased degree of fibrosis. Analysis of liver miRNAs showed a selective modulation of certain miRNAs by hybrid palm oil, some of which were predicted to target genes involved in cell adhesion molecules and peroxisomal pathways. Our data suggest that consumption of hybrid palm oil should be monitored carefully, as its overconsumption compared to that of African palm oil could involve important alterations to hepatic metabolism.

Hydroxytyrosol supplementation modulates the expression of miRNAs in rodents and in humans.

Dietary microRNAs (miRNAs) modulation could be important for health and wellbeing. Part of the healthful activities of polyphenols might be due to a modulation of miRNAs' expression. Among the most biologically active polyphenols, hydroxytyrosol (HT) has never been studied for its actions on miRNAs. We investigated whether HT could modulate the expression of miRNAs in vivo. We performed an unbiased intestinal miRNA screening in mice supplemented (for 8 weeks) with nutritionally relevant amounts of HT. HT modulated the expression of several miRNAs. Analysis of other tissues revealed consistent HT-induced modulation of only few miRNAs. Also, HT administration increased triglycerides levels. Acute treatment with HT and in vitro experiments provided mechanistic insights. The HT-induced expression of one miRNA was confirmed in healthy volunteers supplemented with HT in a randomized, double-blind and placebo-controlled trial. HT consumption affects specific miRNAs' expression in rodents and humans. Our findings suggest that the modulation of miRNAs' action through HT consumption might partially explain its healthful activities and might be pharmanutritionally exploited in current therapies targeting endogenous miRNAs. However, the effects of HT on triglycerides warrant further investigations.

Docosahexaenoic acid modulates the enterocyte Caco-2 cell expression of microRNAs involved in lipid metabolism.

Consumption of the long-chain ω-3 (n-3) polyunsaturated fatty acid docosahexaenoic acid (DHA) is associated with a reduced risk of cardiovascular disease and greater chemoprevention. However, the mechanisms underlying the biologic effects of DHA remain unknown. It is well known that microRNAs (miRNAs) are versatile regulators of gene expression. Therefore, we aimed to determine if the beneficial effects of DHA may be modulated in part through miRNAs. Loss of dicer 1 ribonuclease type III (DICER) in enterocyte Caco-2 cells supplemented with DHA suggested that several lipid metabolism genes are modulated by miRNAs. Analysis of miRNAs predicted to target these genes revealed several miRNA candidates that are differentially modulated by fatty acids. Among the miRNAs modulated by DHA were miR-192 and miR-30c. Overexpression of either miR-192 or miR-30c in enterocyte and hepatocyte cells suggested an effect on the expression of genes related to lipid metabolism, some of which were confirmed by endogenous inhibition of these miRNAs. Our results show in enterocytes that DHA exerts its biologic effect in part by regulating genes involved in lipid metabolism and cancer. Moreover, this response is mediated through miRNA activity. We validate novel targets of miR-30c and miR-192 related to lipid metabolism and cancer including nuclear receptor corepressor 2, isocitrate dehydrogenase 1, DICER, caveolin 1, ATP-binding cassette subfamily G (white) member 4, retinoic acid receptor ß, and others. We also present evidence that in enterocytes DHA modulates the expression of regulatory factor X6 through these miRNAs. Alteration of miRNA levels by dietary components in support of their pharmacologic modulation might be valuable in adjunct therapy for dyslipidemia and other related diseases.

One-year supplementation with a grape extract containing resveratrol modulates inflammatory-related microRNAs and cytokines expression in peripheral blood mononuclear cells of type 2 diabetes and hypertensive patients with coronary artery disease.

Numerous studies have shown that resveratrol (RES) exerts anti-inflammatory effects but human trials evidencing these effects in vivo are limited. Furthermore, the molecular mechanisms triggered in humans following the oral intake of RES are not yet understood. Therefore, the purpose of this study was to investigate the molecular changes in peripheral blood mononuclear cells (PBMCs) associated to the one-year daily intake of a RES enriched (8 mg) grape extract (GE-RES) in hypertensive male patients with type 2 diabetes mellitus (T2DM). We used microarrays and RT-PCR to analyze expression changes in genes and microRNAs (miRs) involved in the inflammatory response modulated by the consumption of GE-RES in comparison to a placebo and GE lacking RES. We also examined the changes in several serobiochemical variables, inflammatory and fibrinolytic markers. Our results showed that supplementation with GE or GE-RES did not affect body weight, blood pressure, glucose, HbA1c or lipids, beyond the values regulated by gold standard medication in these patients. We did not find either any significant change on serum inflammatory markers except for a significant reduction of ALP and IL-6 levels. The expression of the pro-inflammatory cytokines CCL3, IL-1ß and TNF-α was significantly reduced and that of the transcriptional repressor LRRFIP-1 increased in PBMCs from patients taking the GE-RES extract. Also, a group of miRs involved in the regulation of the inflammatory response: miR-21, miR-181b, miR-663, miR-30c2, miR-155 and miR-34a were found to be highly correlated and altered in the group consuming the GE-RES for 12 months. Our results provide preliminary evidence that long-term supplementation with a grape extract containing RES downregulates the expression of key pro-inflammatory cytokines with the involvement of inflammation-related miRs in circulating immune cells of T2DM hypertensive medicated patients and support a beneficial immunomodulatory effect in these patients.