Detection of an Abundant Plant-Based Small RNA in Healthy Consumers

Abstract

The mechanisms of delivery of plant small RNAs to consumers must be investigated in order to harness this technology to positively impact biotechnology. Two groups have used honeysuckle (Lonicera japonica) feeding regimes to detect a plant-based small RNA, termed MIR2911, in sera. Meanwhile, numerous groups have failed to detect dietary plant-based small RNAs in consumers. Here we catalog levels of MIR2911 in different herbs, and suggest that in particular herb MIR2911 levels are elevated. Feeding these different herb-based diets to mice, we found MIR2911 levels in the sera and urine were associated with dietary intake levels. Abundance was not the sole determinate of apparent RNA bioavailability, as gavage-feeding large-doses of synthetic MIR2911 permitted only small transient increases in serum levels. Dietary MIR2911 were not modified in circulation by association with the host's RNA-induced silencing complex, as the RNA did not co-immunoprecipitate with AGO2. The stability of dietary MIR2911 in circulation differed from synthesized small RNAs, as tail vein administration of various synthetic plant-based small RNAs resulted in rapid clearance. However, synthetic MIR2911 appeared to be more stable than the other plant miRNAs tested. Notably, this uptake of dietary MIR2911 was not related to perturbations in the host's microbiome or gut permeability. We suggest dietary uptake of MIR2911 is commonplace in healthy consumers, and reproducible detection of plant-based small RNAs in consumers depends on dietary abundance, RNA stability and digestion from within the food-matrix.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1. Serum and urine MIR2911 levels in mice fed various herb- or flower-containing diets.

(A) Detection of MIR2911 in sera from mice fed various herb and flower diets. (B) Detection of MIR2911 in urine from mice fed various herb and flower diets. For (A) and (B), mice were fed diets for 7 days before RNA was isolated form serum and urine samples and analyzed. N = 5. Asterisk: p

Fig 2. Time course analysis of absorption of gavage fed synthetic MIR2911 Time course analysis of serum MIR2911 levels in mice gavage fed 400 pmols synthetic MIR2911 at 0.5 hour, 1 hour, 3 hours after gavage feeding.

Mice were pre-fed chow diet. N = 5. Experiment replicated three times.

Fig 3. Serum MIR2911 is not associated primarily with AGO2.

(A) Quantification of MIR2911 in AGO2-associated immunoprecipitates and unbound fractions of serum from animals fed an herb-based diet or a standard chow diet. (B) miR-16 immunoprecipitation was analyzed as an endogenous control for AGO2 precipitation. Levels of MIR2911 and miR-16 were each normalized to spiked-in exogenous MIR161. This experiment is representative of more than five different experiments done with herbal-fed mice (honeysuckle, chamomile).

Fig 4. Stability of various synthetic plant microRNAs injected into mouse circulation.

Time course analysis of serum microRNA levels after the injection of microRNA cocktail (5 pmol each) via lateral tail vein. 1X PBS was injected as a control. Experiment replicated more than three times.

Fig 5. Antibiotic treatments do not appear to impact MIR2911 serum detection in honeysuckle fed mice.

Analysis of serum MIR2911 levels in mice fed honeysuckle (HS), mice fed honeysuckle and treated with antibiotics (HS+Ab), and mice fed chow. Mice were fed the diets for 7 days before analysis of serum MIR2911 levels. N = 5. Experiment replicated more than three times with each diet and condition.

Fig 6. Gut permeability is not altered in honeysuckle fed mice.

Quantification of FITC-Dextran 4000 translocation into the circulation of mice fed chow, honeysuckle diet, or mice fed chow diet and treated with cisplatin. N = 5. p (a/b)