Written by Kendal Hirschi

“You are what you eat,” as the saying goes, which serves for some as a guide to healthy eating. Even the field of nutrition appreciates the literal nature of this adage: as our bodies renew aging cells, our diets provide the necessary building blocks to repair and maintain them. We need basic nutrients, vitamins, and minerals for proper development and to maintain our health, but what about small RNA molecules?

There is a growing interest in small RNAs because they regulate the expression of genes in living organisms, and are not only present in our food to begin with but are also being tested in genetically engineered crops to create useful traits. While experimental biologists have used dietary small RNAs to exert regulatory changes in worms for decades, few scientists would recognize small RNAs as nutrients. Several recent reports by Baier et al. and Zhou et al. (see references 1, 2 below), suggest that small RNAs from milk and honeysuckle could have a biological impact on people. Many other researchers have failed to show such impacts (3-5), but in light of these two studies, should we reconsider the “pervading negativity” of the field? Has the “worm turned” for small RNAs?

“To whom do lions cast their gentle looks? Not to the beast that would usurp their den. The smallest worm will turn being trodden on, And doves will peck in safeguard of their brood.” – Lord Clifford, Henry VI, Part 3, William Shakespeare.

Our diets contain hundreds of different small RNAs including microRNAs (miRNAs) that are 19-24 nucleotides long (6). Within the plant or animal of origin, these tiny pieces of genetic information attach to specific mRNAs and modify translation. By interrupting the normal flow of information from DNA to mRNA to protein, small RNAs can “silence” genes within the same organism. Several years ago, a report challenged multiple paradigms, suggesting that ingested miRNAs are transferred to blood, accumulate in tissues, and regulate transcripts within the consuming animal (7). These authors had the bravado to suggest food-derived exogenous miRNA may qualify as a novel nutrient. The scientific community sought to authenticate this claim; however, several high-profile studies failed to validate this initial observation (4,5,8). Despite these negative reports, a small dietary RNA hermeneutic circle formed (9-11). Here I review four recent studies that further demonstrate the potential for dietary delivery of small RNAs (1,2).

Milk: an exosomal miRNA transmitter

Mammalian milk appears to serve as an effective vehicle to deliver dietary animal miRNAs since in milk miRNAs are encapsulated in exosomes and maybe more recalcitrant to degradation and receptive for absorption (1). Bovine milk given to volunteers showed a dose dependent response for milk-based miRNAs, while no change was noted in an endogenous miRNA. Mice fed milk diets contained 61% more plasma of a putative bovine miRNA, than mice fed a milk miRNA-depleted diet. Because the content of the nutrients other than miRNAs was identical, the 61% decrease in this plasma miRNA in the depleted group was explained as an insufficient supply of exogenous dietary miRNAs. The compelling message from this milk feeding study is that dietary milk based microsomes appear to provide a mechanism for oral delivery into healthy consumers (1,12).
For those outside the hermeneutic circle, this milk experiment has ambiguities; for instance, assigning point of origins to most cow and human miRNAs obscures matters (13).

Plant Small RNAs to Consumers

Specific diets seem to enable the detection of plant based small RNA (sRNA) in consuming animals. After several days on a honeysuckle (Lonicera japonica)-containing diet, an herb derived small RNA, MIR2911, could be detected (14). This RNA appeared in circulation within days of eating the herbal diets and was no longer detectable after the animals were fed only standard chow. Meanwhile, a chemotherapy drug also facilitated uptake of gavage fed miRNAs independent of the consuming animals dietary history. Interesting, this drug but not the herb appeared to alter gut architecture. This study proposes diets or gastrointestinal injuries favor the delivery of dietary plant-based nucleic acids in consuming populations.
This study has numerous shortcomings, including the fact that it does not address mechanisms of uptake and functionality of MIR2911 in the consumer.

Plant small RNAs as antivirals

The most elaborate evidence of the efficacy of dietary sRNAs in therapeutics comes from a recent study out of Zhang’s group in Nanjing, the same lab that published the initial study on cross-kingdom dietary gene regulation (2). When animals were fed honeysuckle tea, the MIR2911 from the tea survives the preparation process, withstands digestion and is delivered to the sera and lungs of the consuming animals. Furthermore, this small RNA is biologically active as it suppresses the influenza A virus.

One may envision that boiling honeysuckle would degrade MIR2911; however, this particular small RNA appears resilient to degradation (2). This resiliency and the biological effects forebodes MIR2911 as a ‘virological penicillin’ to treat numerous viruses.

The studies out of Nanjing are breathtaking. However, as the gulf between their results and other findings widen, vigilant focus on unearthing the differences among labs must persist. From my interactions, the group from Nanjing appears receptive to collaborations and it has been suggested that a consortium approach be used to address this scientific divide (3).

Chemopreventive plant-based miRNAs

A Chemopreventive role for dietary miRNAs was established in a small mouse feeding study (15). Using a colon cancer mouse model, three groups of seven animals were treated with one of the follow dietary supplements: water, an array of plant based miRNAs, or 3 tumor suppressor miRNA that were synthesized to mimic miRNAs of plant origin. The animals were gavaged for 28 days (400 pm of each tumor suppressor miRNA in the cocktail) and tumor burden was measured. In the tumor suppressor group, 6/7 had tumor values lower than the lowest water control fed animals.
These dietary miRNAs may trigger a general immune response that minimizes tumor burden. This immune response should be similar in the animals fed the plant based miRNAs or the tumor suppressor cocktail. However, there was only a slight tumor reduction feeding the non-specific plant RNAs compared to the water controls, suggesting the tumor suppressing specificity of the dietary miRNA cocktail.

The presence of the suppressing miRNAs in the colon could only be verified for one of the miRNAs. Do these miRNAs have different half-lives in the colon?

Despite the small sample size, this study is groundbreaking in that it helps alter our concept of the relationship between health and nutrition and potentially opens up new vistas for cancer therapy.
Several themes can now be drawn from these four studies:

Packaging Packs a Punch

The bovine study demonstrates that the microsomal fractions from the milk can provide a conduit for safe passage through the digestive system. Can some plants and gut pathogens also bundle their RNAs for regulatory roles in the host (16)?

Bulking Up

High dosages of dietary RNAs are needed to facilitate transmission. We speculate that in healthy consumers dietary uptake of RNAs will be effective through prolonged exposure to high levels of specific small RNAs.

Health Impacts Delivery

The circulation or tissue level of a nutrient depends in part on degradation and processing by the GI tract. Can gut injuries potentiate dietary RNA uptake? Drug treatments and pathologies may produce gut injuries that form a conduit for small RNA uptake.

Closing Remarks

Several themes can be drawn from recent studies (See Figure). It is worth noting that transgenic expression of small RNAs is the vanguard approach in agbiotechnology for the global enhancement of foods. Should we as consumers now be alarmed about the potential side-effects of these transgenic foods on our own gene expression?

Work in my lab suggests gastrointestinal uptake of these plant-based miRNAs is NOT a general means of RNA transfer and subsequent gene regulation. However, we propose that specific diets and pathological conditions favor the detection of dietary-based nucleic acids in specific consuming populations. We are examining how particular dietary regimes, pathologies, drugs, and stresses alter the uptake, retention and excretion of dietary nucleic acids in the sera, tissues and urine of consuming animals. I believe that dietary small RNA delivery could transform our concepts about diet and health. This could modify our view of plant bioactive compounds and establish new potential delivery systems for gene therapy. In the future, a doctor may prescribe specific plant-based dietary therapies tailored to delivery medicinal small RNAs to children and adults identified as at-risk for a particular metabolic or genetic disorder. This approach would be affordable, non-invasive and have high-compliance – creating a dream scenario for both doctor and patient!

Considering the impact that dietary delivery of small RNAs could have on nutrition, disease and agriculture (17) – we should not disparage the concept based on the failure to replicate initial findings (5).

1. Baier, S.R., et al., MicroRNAs Are Absorbed in Biologically Meaningful Amounts from Nutritionally Relevant Doses of Cow Milk and Affect Gene Expression in Peripheral Blood Mononuclear Cells, HEK-293 Kidney Cell Cultures, and Mouse Livers. J Nutr, 2014.
2. Zhou, Z., et al., Honeysuckle-encoded atypical microRNA2911 directly targets influenza A viruses. Cell Res, 2014.
3. Witwer, K.W. and K.D. Hirschi, Transfer and functional consequences of dietary microRNAs in vertebrates: concepts in search of corroboration: negative results challenge the hypothesis that dietary xenomiRs cross the gut and regulate genes in ingesting vertebrates, but important questions persist. Bioessays, 2014. 36(4): p. 394-406.
4. Snow, J.W., et al., Ineffective delivery of diet-derived microRNAs to recipient animal organisms. RNA Biol, 2013. 10(6).
5. Dickinson, B., et al., Lack of detectable oral bioavailability of plant microRNAs after feeding in mice. Nat Biotechnol, 2013. 31(11): p. 965-7.
6. Bartel, D.P., MicroRNAs: Target Recognition and Regulatory Functions. Cell, 2009. 136(2): p. 215-233.
7. Zhang, L., et al., Exogenous plant MIR168a specifically targets mammalian LDLRAP1: evidence of cross-kingdom regulation by microRNA. Cell Res, 2012. 22(1): p. 107-26.
8. Witwer, K.W., et al., Real-time quantitative PCR and droplet digital PCR for plant miRNAs in mammalian blood provide little evidence for general uptake of dietary miRNAs: limited evidence for general uptake of dietary plant xenomiRs. RNA Biol, 2013. 10(7): p. 1080-6.
9. Wang, K., et al., The complex exogenous RNA spectra in human plasma: an interface with human gut biota? PLoS One, 2012. 7(12): p. e51009.
10. Beatty, M., et al., Small RNAs from plants, bacteria and fungi within the order Hypocreales are ubiquitous in human plasma. BMC Genomics, 2014. 15: p. 933.
11. Liang, G., et al., Assessing the survival of exogenous plant microRNA in rice. Food Science and Nutrition, 2014. 2(4): p. 8.
12. Melnik, B.C., S.M. John, and G. Schmitz, Milk: an exosomal microRNA transmitter promoting thymic regulatory T cell maturation preventing the development of atopy? J Transl Med, 2014. 12: p. 43.
13. Witwer, K.W., Diet-responsive mammalian miRNAs are likely endogenous. J Nutr, 2014. 144(11): p. 1880-1.
14. Yang, J., et al., Detection of dietary plant-based small RNAs in animals. Cell Res, 2015.
15. Mlotshwa, S., et al., A novel chemopreventive strategy based on therapeutic microRNAs produced in plants. Cell Res, 2015.
16. Bayer-Santos, E., et al., Characterization of the small RNA content of Trypanosoma cruzi extracellular vesicles. Mol Biochem Parasitol, 2014. 193(2): p. 71-4.
17. Yang, J., K.D. Hirschi, and L.M. Farmer, Dietary RNAs: New Stories Regarding Oral Delivery. Nutrients, 2015. 7(5): p. 3184-3199.

Written by Guest Expert

Kendal Hirschi works at the Children’s Nutrition Research Center at Baylor College of Medicine and is Associate Director of Research at the Vegetable and Fruit Improvement Center at Texas A&M. His research program centers on many biomedical issues and has published papers using bacteria, yeast, crops, zebrafish, mice, and human subjects. His research goal is to increase the nutritional content of crops, in collaboration with clinical faculty at Baylor College of Medicine. His long-term goal is to bridge the chasm between plant biology and nutritional sciences.