Effect in Human Gene Regulation of Food-Derived Plant miRNAs

Abstract

MicroRNAs (miRNAs) are a class of non-protein-coding RNA molecules with the ability to regulate gene expression at the posttranscriptional level, abundant in plants and animals, showing a high level of similarity due to their mechanism of biogenesis and action; this led to the discovery of cross-kingdom interactions mediated by exogenous miRNAs, which has been one of the most important scientific advances in recent years. Because plant-derived miRNAs after ingestion can be resistant to diverse conditions such as crossing the gastrointestinal tract in mammals, entering the body fluid and regulating the expression of endogenous mRNAs. Suggesting that food-derived plant miRNAs may control genes in humans through cross-kingdom regulation. More importantly, plant miRNAs may be a new class of molecules with utility in future epigenetic regulatory therapy applications in a wide range of diseases, demonstrating a new and highly specific strategy for the regulation of gene expression.

1. Introduction

In recent years, non-protein-coding RNA transcripts have been associated to regulatory functions in plants and animals, particularly micro RNAs (miRNAs), a class of endogenous single-stranded molecules of ~22 nucleotides, transcribed by polymerase II from MIR genes, involved in negative regulatory activities at the posttranscriptional level in plants and mammals [1, 2, 3]. The sophisticated mechanism used by miRNAs for the regulation of their targets is based on perfect or near-perfect complementarity binding of miRNAs at the ORF, 5’UTR and 3’UTR sites of mRNA, resulting in repression of translation or degradation of the messenger [4]. Since the identification of the first miRNA in Caenorhabditis elegans [5], until now according to the miRBase version 22.1 database (http://www.mirbase.org, accessed April 2021), a total of 38,589 hairpin precursors (pre-miRNAs) from 217 organisms have been recorded. In plants some crucial functions of miRNAs have been established such as response to developmental signals, auxin responsive factors (ARFs), pathogen infection, cell division, metabolism, etc. [6, 7]. In human, these small transcripts have been found to endogenously participate in some diseases such as diabetes, cancer, heart disease, tumors, atherosclerosis, or biomarkers in early stages of particular diseases [8, 9]. Recently, several studies have shown that plant miRNAs can enter the gastrointestinal tract through food in humans, be identified in various tissues and circulatory system, perceiving genes in humans as potential regulatory targets [10]. Because exogenous miRNAs and endogenous miRNAs have no distinguishing characteristics from each other, therefore, it will be recognized as an endogenous miRNA [11]. The cross-kingdom regulation by miRNAs has developed a series of investigations because it is important to determine the effects of miRNAs from plants on gene expression in humans when they enter the human body through food [11, 12, 13].

This chapter will provide a brief overview of the evidence about the impact of exogenous miRNAs and their direct influence on various biological processes in human, a cross-kingdom approach.

2. Biogenesis and mechanisms of miRNAs

miRNAs are single-stranded strands of approximately (18–24) base pairs evolutionarily conserved in diverse species [14], they are transcribed by RNA polymerase II (pol II) giving rise to a miRNA-primary, which possesses a stem-loop structure [3]. The first step occurs in the nucleus and is carried out by cleavage by the RNase III enzyme Drosha and the double-stranded RNA-binding protein (dsRNA) DGCR8, generating a product of approximately 60 nt called pre-miRNA that contains an overhang of 2–3 nt [15]. It is subsequently exported to the cytoplasm by the pre-miRNA/Exportin5/Ran-GTP complex, in the cytoplasm GTP is hydrolyzed to GDP inducing the release of the pre-miRNA [16], then processed by the Dicer RNaseIII protein producing an RNA duplex of approximately 22 nt [17]. The double strand is incorporated into the RNA-induced silencing complex (RISC), a protein nuclease complex, an Argonaute protein (Ago2), and a double-stranded RNA-binding protein, upon incorporation one strand of the duplex is degraded and the other remains as a mature miRNA, with the faculty to regulate the expression of a target mRNA [17, 18]. While, in plants, the pri-miRNA synthesized by pol II in the nucleus is processed by an enzyme of the RNase III family DICER-LIKE1 (DCL1), resulting in an miRNA/miRNA duplex chain. To stabilize and protect from degradation these duplexes are 2′-O-methylated at the 3′-ends by a Hua Enhancer 1 (HEN1) methyltransferase [19]. Finally, one strand of the duplex is incorporated into AGO1 in the cytoplasm to form the RISC complex [20]. One of the determining mechanisms in the interaction of miRNA and its target mRNA is the seed region (nt 2–8, [21]). The seed region appears to be the most important site for miRNA recognition of its target [22]. In addition to taking advantage of its utility to predict regulatory targets, in relation to the characteristics in the mRNA sites necessary for the effective recognition of the miRNA [23].

3. Endogenous function by plant and animal miRNAS

In recent years, a series of research studies have provided important advances in plant molecular biology by discovering that plants can regulate the expression of some target genes [11, 24, 25]. The first example was discovered in the nematode C. elegans by studying the lin-4 gene in 1993, which transcribes for a small RNA complementary to some segments of the 3’untranslated region of an mRNA encoding the LIN-14 protein, required for passage to the late larval stage [26]. However, up until 2000, it was reported that these small transcripts, due to their size, had gone unnoticed. But they were the perpetrators of gene silencing. Over time it has been evidenced that animals and plants produce a large amount of miRNAs [27]. In plants miRNAs generally participate in the process of growth, disease resistance, morphogenesis, leaf and fruit size, development of healthy plant characteristics, and the process of flowering regulation [6]. Some miRNA targets have been found to be involved in processes such as metabolism, transport, cell signaling, stress response [28]. In humans, it is estimated that approximately 60% of all protein-coding genes are miRNA targets, which could practically affect most physiological processes in the body [29]. The miRNAs are, therefore, important regulatory molecules of gene expression in different processes, such as neuronal development, differentiation, proliferation, and cell survival [2]. There is evidence that miRNAs offer potential targets for the diagnosis, prognosis, and treatment of a wide variety of diseases [21]. More importantly, miRNA profiles, especially in serum, plasma, and urine, have been reported to be closely related to various diseases and disease states, including cancer, diabetes, inflammation, infections, and tissue injury [8, 14, 30].

4. Resistance and stability of plant miRNAs to harsh conditions

In 2017, Luo et al. [31] revealed the abundance of miRNAs in Zea mays (maize), obtaining 18 highly represented miRNAs, fresh maize samples were subjected to different treatments (elevated temperature and pressure, starch dextrinization, and protein denaturation), observing that all miRNAs resisted the treatments with only decrease in their concentrations. Subsequently, the resistance of miRNAs from maize was analyzed in pigs, due to their attractive biomedical model and organ size similar to humans, they were fed for 7 days with fresh maize-based diet, and evaluated the presence of the 18 previously selected miRNAs, finding 16 in tissue and serum. They verified the crossing of the gastrointestinal barrier of synthetic miRNAs and evaluated by bioinformatic analysis the possible regulation of pig genes by miRNAs from maize, obtaining as a result that MIR164a-5p has CSPG4, OTX1, and PLAGL2 genes as a potential target, with a reduction in the gene activity compared with control. This suggests the likelihood that exogenous miRNAs regulate gene expression in endogenous mRNAs in a similar way to mammalian miRNAs.

4.1 Mechanisms of transport and absorption

Exogenous miRNAs are selectively packaged into small microvesicles (MVs). MVs are a mixture of microparticles, exosomes, and other vesicular structures found in human plasma [12]. They are shed from epithelial cells under normal or pathological conditions and can enter the circulatory system to be transported. They contain receptors and ligands on the surfaces of the cells of origin, giving them the ability to interact with target cells and mediate intercellular communication. Exogenous miRNAs take advantage of this ability to regulate host cell gene expression. MVs serve as stable signaling molecules, they protect exogenous miRNAs against serum RNAases and facilitate their transport to target genes [12, 32, 33, 34]. They are classified according to their origin, size, and formulation mechanism: exosomes, microvesicles, and apoptotic bodies [35].

5. Regulation in human given by exogenous miRNAs from plant cross-kingdoms

5.1 MIR168a of plants first evidence of cross-kingdom regulation

The paradigm that miRNAs originated exclusively for endogenous assimilation was changed in 2012 by Zhang et al. [12], when the first evidence of cross-kingdom interactions was demonstrated by sequencing serum from men and women in China and finding present human and other animal miRNAs from dietary plant miRNAs, identifying MIR168a from Oriza sativa (rice), analyzed through bioinformatics, finding as a regulatory target the gene encoding for the low-density lipoprotein receptor adapter protein 1 (LDLRAP1). Confirming their various hypotheses on the regulation of the LDLRAP1 gene, predicted target of exogenous MIR168a, through an in vivo assay where serum and tissues were collected from mice previously fed with rice and in vitro transfecting HepG2 cells with MIR168a MVs, finding a decrease in the levels of the protein in vitro and in vivo, thus interfering with the cholesterol transport mechanism.

5.2 Stability and survival of plant miRNAs to the gastrointestinal tract in mammals

In a second approach to cross-kingdom approach, as far as was known, there was no major evidence demonstrating the survival of exogenous miRNAs to the gastrointestinal (GI) system, blood, or organs in mammals. Plant-derived MIR172 was detected in a range of 2–72 hours and remained stable in organs, tissues, feces, and blood in mice after being fed total RNA from Brassica oleracea in amounts between 10 and 50 μg, administered orally, suggesting the survival of exogenous miRNAs to the GI system, organs, and blood in mammals [36]. Subsequently, in another study in humans [10], 16 miRNAs from plants were evaluated and detected after ingestion (ath-MIR156a, ath-MIR157a, ath-MIR162a, ath-MIR167a, ath-MIR168a, ath-MIR172a, ath-MIR172a, ath-MIR390a, osa-MIR528, ppt-MIR894, ath-MIR166a, ath-MIR158a, ath-MIR159a, ath-MIR160a, ath-MIR163a, ath-MIR169a, ath-MIR824). The concentrations of plant miRNAs were measured by qRT-PCR, in nine volunteers administered orally with 2.5 L of watermelon juice and 2.5 kg total mixture of other fruits, finding the presence of exogenous miRNAs (MIR156a, MIR157a, MIR162a, MIR167a, MIR168a, MIR172a, MIR390a, MIR528, MIR894, and MIR166a) in serum for up to 9 hours, demonstrating that a variety of exogenous plant miRNAs can be found in human plasma following ingestion.

5.3 Antiviral activity of miR2911 from plants

According to the authors Zhou et al. [37], the repercussions, given by exogenous miRNAs from plants, together with the mechanisms involved in absorption and transfer “remain largely unknown”. In their study they sequenced the plant honeysuckle (HS, Lonicera japonica) used for thousands of years to treat influenza infection and were able to obtain a total of 148 miRNAs in their reads. Additionally, the plant was subjected to a decoction process, finding only the MIR2911 stable after treatment, remaining in high concentrations (0.2 g HS/ml), was 0.06 pmol/ml suggesting its stability due to its sequence and high GC content. Subsequently, mice administered with the 500 μl decoction product of the plant reached a peak in the concentrations of MIR2911 in plasma and lung 6 h post-administration and a decrease to a basal level after 12 h. A series of potential target genes for MIR2911 of different viruses such as H1N1, H3N2, H5N1, and H7N9 were found through bioinformatic analysis. In cell lines infected with H1N1 and transfected with synthetic MIR2911, they achieved a significant decrease in viral count due to the fact that the target genes, PB2 and NS1, are essential for its replication. The in vivo study demonstrated that miR2911 inhibited the replication of several IAV (influenza A viruses) including H1N1, H5N1, and H7N9, decreasing mortality in mice, being the first evidence of a natural product that directly targets and suppresses IAV. In addition, the authors in 2020 conducted an emerging investigation of MIR2911, now against the SARS-CoV-2 virus, the cause of COVID-19, severe acute respiratory syndrome, which spread rapidly around the world, causing an unprecedented pandemic. Among the results, the researchers were able to inhibit viral replication in vitro, using exosomes from serum of donors who consumed the plant decoction and exosomes of synthetic MIR2911, showing a very high antiviral activity of plant MIR2911 on SARS-CoV-2 virus replication, since bioinformatics results showed at least 179 putative binding sites for the miRNA on the SARS-CoV-2 genome, with 28 binding sites subsequently confirmed, indicating that the plant miRNA could inhibit the translation of almost all SARS-CoV-2 proteins [38].

5.4 Potential role of plant miRNAs in breast cancer

According to the potential function of plant miRNAs, the authors sought to clarify the influence of exogenous molecules on tissues outside the GI tract. By sequencing serum and tumor tissue samples from stage II and III breast cancer patients, plant miR159 was detected up to six times more frequently than plant miRNAs. The results showed a higher abundance of miRNA in healthy patients than in breast cancer (BC) patients and patients with metastasis. Therefore, it was proposed that miR159 might influence breast cancer progression. By transfecting cells containing miR159 isolated from patient serum, they had the ability to reduce proliferation in BC cells. To identify miR159 target genes they used three independent methods: computational prediction, RNA-induced silencing, and sequencing. All three methods identified three potential target genes: transcription factor 7 (TCF7), nuclear receptor coactivator 6 (NCOA6), and engrailed homeobox 2 (EN2). Of which the TCF7 gene belongs to a family of transcription factors of the Wnt signaling pathway, which is overexpressed in breast cancer. Finally, the authors report that mice fed with MIR159 showed a significant reduction of TCF7 and MYC expression in their tumors with decreased tumor cell proliferation, reduced tumor growth, and increased apoptosis, providing important information on kingdom interactions for the prevention and treatment of various human diseases [39].

5.5 Plant MIR167e-5p suppresses intestinal cell proliferation

A recent study has found plant-derived MIR167e-5p to regulate enterocyte proliferation in vitro in cancer cell lines. Plant MIR167e-5p decreased cell proliferation following treatment with 20 pmol of MIR167e-5p over a 72 h period. The tests included viability assays determined by MTT at different times. In this study, a bioinformatic program was employed to identify plant miRNA targets due to their involvement in auxin response factor processes in plants, resulting in a conserved putative site in β-catenin gene in human, key in the Wnt/ β-catenin pathway related to cell proliferation, differentiation, and maintenance. Demonstrating that the plant MIR167e-5p could decrease protein levels and inhibit cell proliferation [40].

6. Future approaches and applications of miRNAs

Identifying gene targets in human in a timely manner using plant miRNAs offers a reliable, effective, and economical way to determine the possible effects of interactions given by exogenous molecules, regardless of their origin, in human mRNA, studies have focused on providing information of importance for future projects or research, as in 2017, Kumar et al. [41] presented a cross-kingdom bioinformatics analysis, showing the importance of some plant miRNAs, coming from the plant Camptotheca acuminata commonly known as Happy Tree, with anticancer attributions, showing in their results, a strong association with several cancer pathways in human, and a possible important role in the regulation of complex disease networks for humans. Similarly, another plant Ocimum bascilicum, used for its therapeutic properties, underwent a bioinformatics analysis, by Patel et al. in 2019 [42], finding a close relationship of plant miRNAs with various genes involved in signaling pathways, various functional processes, and several organs in human. Not only have predictive studies been performed in plants that are used for their medicinal properties, there are also analyses where foods of daily consumption have shown interesting results as in the year 2020, the authors Rakhmetullina et al. [43] searched for target genes in human for 227 miRNAs of the Oryza sativa (rice) plant, finding 942 possible genes, which represent 5.4% of the total number of human genes studied, being this of major importance, due to the fact that four miRNAs present (osa-miR2102-5p, osa-miR5075-3p, osa-miR2097-5p, and osa-miR2919) were associated with a greater number of target genes, and since miRNAs can reach some tissues, and the circulatory system through ingestion, some targets were related to some biological processes involved in the development of cardiovascular and neurodegenerative diseases, the authors emphasize the importance of continuing to study them. Likewise, in 2022, Sánchez-Romo et al. [44] determined through bioinformatics analysis the possible mRNA targets in humans, resulting in 787 different genes for 84 miRNAs of the plant Triticum aestivum (wheat), showing some genes involved in cancer processes, risk of dementia and schizophrenia. In addition, functional enrichment analysis highlighted some pathways such as the Fanconi anemia pathway, circadian rhythm, and the dopaminergic synapse pathway, related to attention deficit hyperactivity disorder ADHD.

7. Conclusion

The impact of endogenous and exogenous miRNAs that directly influence various biological processes is an important current topic, as it opens the horizons of understanding gene regulation in organisms. Considering that plants and humans are different species, plant miRNAs and their interactions have been actively studied, which has contributed to the knowledge of the mechanisms of coevolution between plant miRNAs and human mRNAs. The information gathered in these articles could facilitate the approach and could open a number of opportunities for the development of new studies and therapies for various diseases based on miRNAs, as well as extensive molecular validation of predictions.