Research Article
Stability and absorption mechanism of typical plant miRNAs in an in vitro gastrointestinal environment: basis for their cross-kingdom nutritional effects

https://doi.org/10.1016/j.jnutbio.2020.108376Get rights and content

Abstract

Plant miRNAs, a group of 19–24 nt noncoding RNAs from plant foods, were recently found to have immunomodulatory and nutritional effects on mammalian and human bodies. However, how the miRNAs survive gastrointestinal (GI) environment and how the stable miRNAs are absorbed, which serve the basis for their biological functions, were not unraveled. Here, we investigated the stabilities of six typical plant miRNAs in simulated gastric and intestinal environments, and the absorption mechanisms by Caco-2 cells. The results showed that the miRNAs can survive the environment with certain concentrations. The mixture of food ingredients enhanced the stabilities of the plant miRNAs in the gastric conditions, while 2′-O-methyl modification protects the miRNAs in intestinal juice. The stabilities of the miRNAs vary significantly in the environment and are related to their secondary structures. The stable plant miRNAs can be absorbed by Caco-2 cells via clathrin- and caveolin-mediated endocytosis. Uptake of the miRNAs was sequence dependent, facilitated by NACh and TLR9, two typical receptors on cell membrane. The results suggest that some of plant miRNAs are stable in the mimic GI environment and can be absorbed by Caco-2 cells, underlying the potential of their cross-kingdom regulation effects.

Introduction

Nucleic acids, including deoxyribonucleic acids (DNAs) and ribonucleic acids (RNAs), are long-chain biomacromolecules composed of nucleotides and account for 15% of the dry weight of food ingredients, second only to carbohydrate and protein [1]. Along with the daily diet, large amount of nucleic acids is simultaneously assimilated by human body. The conventional opinion holds that nucleic acids from food are completely degraded into small molecules in the gastrointestinal (GI) tract and absorbed in the form of monomer nucleotides, which have been demonstrated to be capable of improving infants' ability to absorb nutrients, promoting regeneration and repair of damaged cells, as well as maintaining immunity homeostasis of the elderly [2], [3]. Therefore, it is generally believed that food nucleic acids exert their biological functions by nucleotides, their GI metabolites, while the long-chain nucleic acids themselves do not have nutritional effects on human body [4].

Nonetheless, the traditional view was shaken by a recent work reported by Zhang et al. that an intact single-stranded RNA molecule from food has nutritional utility. They found that miR168a (a plant-specific miRNA) can transport from rice to mammalian circulating system, in turn down-regulating gene expression by binding the mRNA dealing with lipid metabolism [5]. The presence of plant miRNAs can be found nearly in all bodily fluids, but any potential biological function following ingestion would likely rely on the miRNAs remaining intact rather than on degraded nucleotides. As soon as this study was reported, it attracted worldwide attention and triggered a boom in investigating food-source miRNAs [6]. In 2015, Zhou et al. reported the related investigation demonstrating that mice immunity could be enhanced by administration of honeysuckle-derived miR2911 [7]. The follow-up studies were performed by Yang et al., who validated the presence of miR2911 in mice bodily fluids using digital-droplet polymerase chain reaction (PCR) and elucidated that its biogenesis was related to the integrity of plant 26S rRNA [8], [9]. In 2016, two independent research groups almost simultaneously discovered that food-derived microRNAs were capable of binding receptors on animal cells and producing the biological effects via the elucidated signaling pathways [10], [11]. The study on miR2911 by Yang et al. demonstrated that some dietary microRNAs may be more digestively stable if the miRNAs were encapsulated in liposome and exosome or other delivery vehicles [12]. These researches suggest that miRNAs from plant food may play important roles in regulating nutrient metabolism and physiological processes of animal body. Both mRNAs and receptors can serve as targets for actions of the plant miRNAs, which are very similar to protein-mediated nutritional regulations [13], [14].

Although the exciting discoveries open up a new field for exogenous miRNAs investigation, whether the plant miRNAs have cross-kingdom regulatory effects is still on debate on account of the reports that deny the existence of plant miRNAs in animal bodies. Dickinson et al. designed a validation study examining the miR168a content in mice after being fed with rice, and the results showed that accumulation of the plant miRNA was negligible [15]. Witwer et al. measured five plant miRNAs in the plasma of pigtailed macaques fed with the plant miRNA-enriched food and failed to observe any specific amplification results of the plant miRNAs [16]. Serial investigations conducted by Chan's group showed that accumulation of the fruit-specific miRNAs in the plasma of healthy subjects was insignificant after intake of the corresponding fruits [17], [18]. Kang et al. analyzed more than 800 samples of body fluids and tissues and concluded that the detection of plant miRNA in human bodies was due to crossover contamination or even artifacts [19]. Apart from that, Denzler et al. and Howard et al. independently published research papers indicating lack of evidence that plant miRNAs can enter into mammal circulatory system, arguing that no pathway was elucidated for the miRNAs to regulate the nutritional metabolic process of animal bodies [20], [21].

To date, whether the food-source miRNAs have regulatory effects on mammals is still on debate. The previous studies mainly focused on the presence of plant miRNAs in circulatory system of animal or human body and their potential regulatory effects on nutritional metabolism processes [22]. However, due to the complexity of mammalian bodily fluids, detection of the exogenous plant miRNAs would be seriously interfered by endogenous miRNAs [23]. More importantly, whether plant miRNAs from the daily diet can survive the unfavorable conditions in the GI tract and how the stable exogenous miRNAs are absorbed by the body are the two key points which serve as the basis for the cross-kingdom bioactivity of plant miRNAs; however, these were ignored in the former investigations. Therefore, it is imperative to study the stability of plant miRNAs under GI tract environment and the mechanism by which the miRNAs are taken into intestinal epithelial cells. A pioneering study on miRNAs from soybean and rice demonstrated that the miRNAs are of robustness during food processing and of significant bioavailability in a simulated digestion system [24]. Further investigations are necessary to reveal metabolism pathway and absorption mechanism of plant miRNAs and to clarify the long-debated issue concerning the bioactivity of the exogenous miRNAs on the molecular level.

In this study, we evaluated the stability of plant miRNAs under GI tract conditions and investigated absorption mechanism of the stable miRNAs in vitro. Six plant miRNAs, miR157a (Peanut), miR172a (Tomato), miR894 (Sorghum), miR159 (Cabbage), miR160 (Soybean) and miR168a (Rice), were investigated. All of them were reported to be abundant in daily vegetable diets, and some of the sequences (miR159, miR160, miR168a) were observed with potential cross-kingdom biological effects on mammals. The results showed that, different from traditional belief, some plant miRNAs can tolerate the extreme pH of gastric juice and resist the digestion in small intestine. Starch, protein and lipid failed to protect the miRNAs when added separately, but a significant protective effect was observed when they were mixed together with plant miRNAs as daily diet intake. The 2′-O-methylation at the 3′ end of the plant miRNAs is conducive to the stability in simulated intestinal environment, as evidenced previously [25]. Furthermore, we found that some stable plant miRNAs were selectively absorbed by Caco-2 cells (human intestinal epithelial cells) via endocytosis and receptor protein-mediated internalization. The experimental data here provide primary evidence laying the foundation for plant miRNAs to exert biological functions in a cross-kingdom fashion and guidance to exploring nutritional effects of other miRNAs from plant food.

Section snippets

Reagents

All oligonucleotides used in this study including miRNAs, 2′-O-methyl miRNAs and FITC-modified miRNAs were synthesized and purified by Genscript Biotech (Piscataway, NJ, USA) under RNase-free conditions. Sequences of plant miRNAs were listed in Table 1. The RNase-free water was provided by Thermo Fisher Scientific (Pittsburgh, PA, USA). Single-stranded binding protein (SSB) was obtained from New England Biolabs Inc. (Ipswich, MA, USA). SYBR Green II was provided by Life Technologies (Carlsbad,

Stability of plant miRNAs in human gastric juice

The study carried out by Liu et al. demonstrated that RNA can be digested by pepsin [26]. Therefore, we first studied the stability of those plant miRNAs against digestion in human gastric juice, a property that could enable them to survive the extreme gastric environ and enter into the small intestine. The fluorescently labeled miRNA sequences were applied because some of miRNA sequences are hard to be dyed even by SYBR Green II [27]. The half-life was estimated based on the information

Concluding remarks

In conclusion, this study demonstrated that plant miRNAs, which were previously considered to be unstable, could be of robustness in simulated gastric environment, and the mixture of food ingredients from daily diet enhances their tolerance to the extreme gastric conditions. Plant miRNAs are degraded to certain degrees by nucleases in intestinal tract, yet their final concentrations are higher than those needed to exert biological functions. 2′-O-methylation effectively protects the plant

Acknowledgment

This work was supported by National Natural Science Foundation for Young Scholar of China (31901653 and 81601877), Key R&D Program of Shaanxi Province (2018NY-095) and Fundamental Research Funds for the Central Universities (GK202003087).

Declarations of interest

The authors declare no conflict of interest.

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