miRNA, phytometabolites and disease: Connecting the dots.

miRNAs are tiny noncoding ribonucleotides that function as critical regulators of gene-expression in eukaryotes. A single miRNA may be involved in the regulation of several target mRNAs forming complex cellular networks to regulate diverse aspects of development in an organism. The deregulation of miRNAs has been associated with several human diseases. Therefore, miRNA-based therapeutics is gaining interest in the pharmaceutical industry as the next-generation drugs for the cure of many diseases. Medicinal plants have also been used for the treatment of several human diseases and their curative potential is attributed to their reserve in bioactive metabolites. A role for miRNAs as regulators of the phytometabolic pathways in plants has emerged in the recent past. Experimental studies have also indicated the potential of plant encoded secondary phytometabolites to act as cross-regulators of mammalian miRNAs and transcripts to regulate human diseases (like cancer). The evidence for this cross-kingdom gene regulation through miRNA has gathered considerable enthusiasm in the scientific field, even though there are on-going debates regarding the reproducibility and the effectiveness of these findings. In this review, we provide information to connect the medicinal and gene regulatory properties of secondary phytometabolites, their regulation by miRNAs in plants and their effects on human miRNAs for regulating downstream metabolic or pathological processes. While further extensive research initiatives and good clinical evidence are required to prove or disapprove these findings, understanding of these regulations will have important implications in the potential use of synthetic or artificial miRNAs as effective alternatives for providing health benefits.

Plant-derived exosome-like nanoparticles for microRNA delivery in cancer treatment.

Plant-derived exosome-like nanoparticles (PELNs) are natural nanocarriers and effective delivery systems for plant microRNAs (miRNAs). These PELN-carrying plant miRNAs can regulate mammalian genes across species, thereby increasing the diversity of miRNAs in mammals and exerting multi-target effects that play a crucial role in diseases, particularly cancer. PELNs demonstrate exceptional stability, biocompatibility, and targeting capabilities that protect and facilitate the up-take and cross-kingdom communication of plant miRNAs in mammals. Primarily ingested and absorbed within the gastrointestinal tract of mammals, PELNs preferentially act on the intestine to regulate intestinal homeostasis through functional miRNA activity. The oncogenesis and progression of cancer are closely associated with disruptions in intestinal barriers, ecological imbalances, as well as secondary changes, such as abnormal inflammatory reactions caused by them. Therefore, it is imperative to investigate whether PELNs exert their anticancer effects by regulating mammalian intestinal homeostasis and inflammation. This review aims to elucidate the intrinsic crosstalk relationships and mechanisms of PELNs-mediated miRNAs in maintaining intestinal homeostasis, regulating inflammation and cancer treatment. Furthermore, serving as exceptional drug delivery systems for miRNAs molecules, PELNs offer broad prospects for future applications, including new drug research and development along with drug carrier selection within targeted drug delivery approaches for cancer therapy.

MicroRNAs from edible plants reach the human gastrointestinal tract and may act as potential regulators of gene expression.

MicroRNAs (miRNAs) are small single-stranded non-coding RNA molecules that regulate gene expression at the post-transcriptional level. A cross-kingdom regulatory function has been unveiled for plant miRNAs (xenomiRs), which could shape inter-species interactions of plants with other organisms (bacteria and humans) and thus, be key functional molecules of plant-based food in mammals. However, discrepancies regarding the stability and bioavailability of dietary plant miRNAs on the host cast in doubt whether these molecules could have a significant impact on human physiology. The aim of the present study was to identify miRNAs in edible plants and determine their bioavailability on humans after an acute intake of plant-based products. It was found that plant food, including fruits, vegetables and greens, nuts, legumes, and cereals, contains a wide range of miRNAs. XenomiRs miR156e, miR159 and miR162 were detected in great abundance in edible plants and were present among many plant foods, and thus, they were selected as candidates to analyse their bioavailability in humans. These plant miRNAs resisted cooking processes (heat-treatments) and their relative presence increased in faeces after and acute intake of plant-based foods, although they were not detected in serum. Bioinformatic analysis revealed that these miRNAs could potentially target human and bacterial genes involved in processes such as cell signalling and metabolism. In conclusion, edible plants contain miRNAs, such as miR156e, miR159 and miR162, that could resist degradation during cooking and digestion and reach the distal segments of the gastrointestinal tract. Nevertheless, strategies should be developed to improve their absorption to potentially reach host tissues and organs and modulate human physiology.

Atypical Plant miRNA cal-miR2911: Robust Stability against Food Digestion and Specific Promoting Effect on Bifidobacterium in Mice.

Previous studies showed that cal-miR2911, featuring an atypical biogenesis, could target genes of virus and in turn inhibit virus replication. Given its especial sequence motif and cross-kingdom potential, the stability of miR2911 under digestive environment and its impact on intestinal microbes in mice were examined. The results showed that miR2911 was of considerable stability during oral, gastric, and intestinal digestion. The coingested food matrix enhanced its stability in the gastric phase, contributing to the existence of miR2911 in mouse intestines. The survival miR2911 promoted the growth of Bifidobacterium in mice and maintained the overall composition and diversity of the gut microbiota. miR2911 specifically entered the cells of Bifidobacterium adolescentis and potentially modulated the gene expression as evidenced by the dual-luciferase assay. The current study provided evidence on the cross-kingdom communication between dietary miRNAs and gut microbes, suggesting that modulating target bacteria using miRNAs for nutritional and therapeutic ends is promising.

Plant miR8126-3p and miR8126-5p Decrease Lipid Accumulation through Modulation of Metabolic Genes in a Human Hepatocyte Model That Mimics Steatosis.

Plant-based food interventions are promising therapeutic approaches for non-alcoholic fatty liver disease (NAFLD) treatment, and microRNAs (miRNAs) have emerged as functional bioactive components of dietary plants involved in cross-kingdom communication. Deeper investigations are needed to determine the potential impact of plant miRNAs in NAFLD. This study aimed to identify plant miRNAs that could eventually modulate the expression of human metabolic genes and protect against the progression of hepatic steatosis. Plant miRNAs from the miRBase were used to predict human target genes, and miR8126-3p and miR8126-5p were selected as candidates for their potential role in inhibiting glucose and lipid metabolism-related genes. Human HepG2 cells were transfected with plant miRNA mimics and then exposed to a mixture of oleic and palmitic acids to mimic steatosis. miR8126-3p and miR8126-5p transfections inhibited the expression of the putative target genes QKI and MAPKAPK2, respectively, and had an impact on the expression profile of key metabolic genes, including PPARA and SREBF1. Quantification of intrahepatic triglycerides revealed that miR8126-3p and miR8126-5p attenuated lipid accumulation. These findings suggest that plant miR8126-3p and miR8126-5p would induce metabolic changes in human hepatocytes eventually protecting against lipid accumulation, and thus, they could be potential therapeutic tools for preventing and alleviating lipid accumulation.

microRNAs: Key Regulators in Plant Responses to Abiotic and Biotic Stresses via Endogenous and Cross-Kingdom Mechanisms.

Dramatic shifts in global climate have intensified abiotic and biotic stress faced by plants. Plant microRNAs (miRNAs)-20-24 nucleotide non-coding RNA molecules-form a key regulatory system of plant gene expression; playing crucial roles in plant growth; development; and defense against abiotic and biotic stress. Moreover, they participate in cross-kingdom communication. This communication encompasses interactions with other plants, microorganisms, and insect species, collectively exerting a profound influence on the agronomic traits of crops. This article comprehensively reviews the biosynthesis of plant miRNAs and explores their impact on plant growth, development, and stress resistance through endogenous, non-transboundary mechanisms. Furthermore, this review delves into the cross-kingdom regulatory effects of plant miRNAs on plants, microorganisms, and pests. It proceeds to specifically discuss the design and modification strategies for artificial miRNAs (amiRNAs), as well as the protection and transport of miRNAs by exosome-like nanovesicles (ELNVs), expanding the potential applications of plant miRNAs in crop breeding. Finally, the current limitations associated with harnessing plant miRNAs are addressed, and the utilization of synthetic biology is proposed to facilitate the heterologous expression and large-scale production of miRNAs. This novel approach suggests a plant-based solution to address future biosafety concerns in agriculture.

Cross-kingdom regulation by plant-derived miRNAs in mammalian systems.

MicroRNAs (miRNAs) are small noncoding RNA molecules ubiquitously distributed across diverse organisms, serving as pivotal regulators of genetic expression. Notably, plant-derived miRNAs have been demonstrated to have unique bioactivity and certain stability in mammalian systems, thereby facilitating their capacity for cross-kingdom modulation of gene expression. While there is substantial evidence supporting the regulation of mammalian cells by plant-derived miRNAs, several questions remain unanswered. Specifically, a comprehensive investigation of the mechanisms underlying the stability and transport of plant miRNAs and their cross-kingdom regulation of gene expression in mammals remains to be done. In this review, we summarized the origin, processing, and functional mechanisms of plant miRNAs in mammalian tissues and circulation, emphasizing their greater resistance to mammalian digestion and circulation systems compared to animal miRNAs. Additionally, we introduce four well-known plant miRNAs that have been extensively studied for their functions and mechanisms in mammalian systems. By delving into these aspects, we aim to offer a fundamental understanding of this intriguing field and shed light on the complex interactions between plant miRNAs and mammalian biology.

miR482f and miR482c-5p from edible plant-derived foods inhibit the expression of pro-inflammatory genes in human THP-1 macrophages.

Background: Edible plants can exert anti-inflammatory activities in humans, being potentially useful in the treatment of inflammatory diseases. Plant-derived microRNAs have emerged as cross-kingdom gene expression regulators and could act as bioactive molecules involved in the beneficial effects of some edible plants. We investigated the role of edible plant-derived microRNAs in the modulation of pro-inflammatory human genes. Methods: MicroRNAs from plant-derived foods were identified by next-generation sequencing. MicroRNAs with inflammatory putative targets were selected, after performing in silico analyses. The expression of candidate plant-derived miRNAs was analyzed by qPCR in edible plant-derived foods and their effects were evaluated in THP-1 monocytes differentiated to macrophages. The bioavailability of candidate plant miRNAs in humans was evaluated in feces and serum samples by qPCR. Results: miR482f and miR482c-5p are present in several edible plant-derived foods, such as fruits, vegetables, and cooked legumes and cereals, and fats and oils. Transfections with miR482f and miR482c-5p mimics decreased the gene expression of CLEC7A and NFAM1, and TRL6, respectively, in human THP-1 monocytes differentiated to macrophages, which had an impact on gene expression profile of inflammatory biomarkers. Both microRNAs (miR482f and miR482c-5p) resisted degradation during digestion and were detected in human feces, although not in serum. Conclusion: Our findings suggest that miR482f and miR482c-5p can promote an anti-inflammatory gene expression profile in human macrophages in vitro and their bioavailability in humans can be achieved through diet, but eventually restricted at the gut level.

Progress and challenges of plant-derived nucleic acids as therapeutics in macrophage-mediated RNA therapy.

Plant-derived nucleic acids, especially small RNAs have been proved by increasing evidence in the pharmacological activities and disease treatment values in macrophage meditated anti-tumor performance, immune regulating functions and antiviral activities. But the uptake, application and delivery strategies of RNAs as biodrugs are different from the small molecules and recombinant protein drugs. This article summarizes the reported evidence for cross-kingdom regulation by plant derived functional mRNAs and miRNAs. Based on that, their involvement and potentials in macrophage-mediated anti-tumor/inflammatory therapies are mainly discussed, as well as the load prospect of plant RNAs in viruses and natural exosome vehicles, and their delivery to mammalian cells through macrophage were also summarized. This review is to provide evidence and views for the plant derived RNAs as next generation of drugs with application potential in nucleic acid-based bio-therapy.

A Relationship Prediction Method for Magnaporthe oryzae-Rice Multi-Omics Data Based on WGCNA and Graph Autoencoder.

Magnaporthe oryzae Oryzae (MoO) pathotype is a devastating fungal pathogen of rice; however, its pathogenic mechanism remains poorly understood. The current research is primarily focused on single-omics data, which is insufficient to capture the complex cross-kingdom regulatory interactions between MoO and rice. To address this limitation, we proposed a novel method called Weighted Gene Autoencoder Multi-Omics Relationship Prediction (WGAEMRP), which combines weighted gene co-expression network analysis (WGCNA) and graph autoencoder to predict the relationship between MoO-rice multi-omics data. We applied WGAEMRP to construct a MoO-rice multi-omics heterogeneous interaction network, which identified 18 MoO small RNAs (sRNAs), 17 rice genes, 26 rice mRNAs, and 28 rice proteins among the key biomolecules. Most of the mined functional modules and enriched pathways were related to gene expression, protein composition, transportation, and metabolic processes, reflecting the infection mechanism of MoO. Compared to previous studies, WGAEMRP significantly improves the efficiency and accuracy of multi-omics data integration and analysis. This approach lays out a solid data foundation for studying the biological process of MoO infecting rice, refining the regulatory network of pathogenic markers, and providing new insights for developing disease-resistant rice varieties.

Plant miRNA bol-miR159 Regulates Gut Microbiota Composition in Mice: In Vivo Evidence of the Crosstalk between Plant miRNAs and Intestinal Microbes.

New evidence reveals that bol-miR159, an miRNA rich in fruits and vegetables, cross-kingdomly functions in mammalian bodies. However, whether the miRNA could regulate gut microbiota remains unclear. Here, the effect of miR159 on mouse intestinal microbes was comprehensively examined. The results showed that supplementation of miR159 to the chow diet significantly enhanced the diversity of mouse gut microbiota without causing pathological lesions or inflammatory responses on the intestines. At the phylum level, miR159 increased the abundance of Proteobacteria and decreased the Firmicute-to-Bacteroidetes (F/B) ratio. miR159 had prebiotic-like effects on mouse gut microbiota, as it promoted the growth of the bacteria that is beneficial for maintaining gut health. The miRNA can target bacteria genes and get into the bacteria cells. The data provide direct in vivo evidence on the crosstalk between plant miRNAs and intestinal microbes, highlighting the potential for miRNA-based strategies that modulate gut microbes to improve host health.

Profile and potential bioactivity of the miRNome and metabolome expressed in Malva sylvestris L. leaf and flower.

Malva sylvestris L. (common mallow) is a plant species widely used in phytotherapy and ethnobotanical practices since time immemorial. Characterizing the components of this herb might promote a better comprehension of its biological effects on the human body but also favour the identification of the molecular processes that occur in the plant tissues. Thus, in the present contribution, the scientific knowledge about the metabolomic profile of the common mallow was expanded. In particular, the phytocomplex of leaves and flowers from this botanical species and the extraction capacity of different concentrations of ethanol (i.e., 95%, 70%, 50%, and 0%; v/v in ddH2O) for it were investigated by spectrophotometric and chromatographic approaches. In detail, 95% ethanol extracts showed the worst capacity in isolating total phenols and flavonoids, while all the hydroalcoholic samples revealed a specific ability in purifying the anthocyanins. HPLC-DAD system detected and quantified 20 phenolic secondary metabolites, whose concentration in the several extracts depended on their own chemical nature and the percentage of ethanol used in the preparation. In addition, the stability of the purified phytochemicals after resuspension in pure ddH2O was also proved, considering a potential employment of them in biological/medical studies which include in vitro and in vivo experiments on mammalian models. Here, for the first time, the expressed miRNome in M. sylvestris was also defined by Next Generation Sequencing, revealing the presence of 33 microRNAs (miRNAs), 10 typical for leaves and 2 for flowers. Then, both plant and human putative mRNA targets for the detected miRNAs were predicted by bioinformatics analyses, with the aim to clarify the possible role of these small nucleic acids in the common mallow plant tissues and to try to understand if they could exert a potential cross-kingdom regulatory activity on the human health. Surprisingly, our investigations revealed that 19 miRNAs out of 33 were putatively able to modulate, in the plant cells, the expression of various chromosome scaffold proteins. In parallel, we found, in the human transcriptome, a total of 383 mRNAs involved in 5 fundamental mammalian cellular processes (i.e., apoptosis, senescence, cell-cycle, oxidative stress, and invasiveness) that theoretically could be bound and regulated by M. sylvestris miRNAs. The evidence collected in this work would suggest that the beneficial properties of the use of M. sylvestris, documented by the folk medicine, are probably linked to their content of miRNAs and not only to the action of phytochemicals (e.g., anthocyanins). This would open new perspectives about the possibility to develop gene therapies based on miRNAs isolated from medicinal plants, including M. sylvestris.

A comprehensive analysis of the Bencao (herbal) small RNA Atlas reveals novel RNA therapeutics for treating human diseases.

Cross-kingdom herbal miRNA was first reported in 2012. Using a modified herbal extraction protocol, we obtained 73,677,287 sequences by RNA-seq from 245 traditional Chinese Medicine (TCM), of which 20,758,257 were unique sequences. We constructed a Bencao (herbal) small RNA (sRNA) Atlas ( http://bencao.bmicc.cn ), annotated the sequences by sequence-based clustering, and created a nomenclature system for Bencao sRNAs. The profiles of 21,757 miRNAs in the Atlas were highly consistent with those of plant miRNAs in miRBase. Using software tools, our results demonstrated that all human genes might be regulated by sRNAs from the Bencao sRNA Atlas, part of the predicted human target genes were experimentally validated, suggesting that Bencao sRNAs might be one of the main bioactive components of herbal medicines. We established roadmaps for oligonucleotide drugs development and optimization of TCM prescriptions. Moreover, the decoctosome, a lipo-nano particle consisting of 0.5%-2.5% of the decoction, demonstrated potent medical effects. We propose a Bencao (herbal) Index, including small-molecule compounds (SM), protein peptides (P), nucleic acid (N), non-nucleic and non-proteinogenic large-molecule compounds (LM) and elements from Mendeleev's periodic table (E), to quantitatively measure the medical effects of botanic medicine. The Bencao sRNA Atlas is a resource for developing gene-targeting oligonucleotide drugs and optimizing botanical medicine, and may provide potential remedies for the theory and practice of one medicine.

Looking for Plant microRNAs in Human Blood Samples: Bioinformatics Evidence and Perspectives.

Literature has proposed the existence of a cross kingdom regulation (CRK) between human and plants. In this context, microRNAs present in edible plants would be acquired through diet by the consumer's organism and transported via bloodstream to tissues, where they would modulate gene expression. However, the validity of this phenomenon is strongly debated; indeed, some scholars have discussed both the methodologies and the results obtained in previous works. To date, only one study has performed a bioinformatics analysis on small RNA-sequencing data for checking the presence of plant miRNAs (pmiRNAs) in human plasma. For that investigation, the lack of reliable controls, which led to the misidentification of human RNAs as pmiRNAs, has been deeply criticized. Thus, in the present contribution, we aim to demonstrate the existence of pmiRNAs in human blood, adopting a bioinformatics approach characterized by more stringent conditions and filtering. The information obtained from 380 experiments produced in 5 different next generation sequencing (NGS) projects was examined, revealing the presence of 350 circulating pmiRNAs across the analysed data set. Although one of the NGS projects shows results likely to be attributed to sample contamination, the others appear to provide reliable support for the acquisition of pmiRNAs through diet. To infer the potential biological activity of the identified pmiRNAs, we predicted their putative human mRNA targets, finding with great surprise that they appear to be mainly involved in neurogenesis and nervous system development. Unfortunately, no consensus was identified within the sequences of detected pmiRNAs, in order to justify their stability or capability to be preserved in human plasma. We believe that the issue regarding CKR still needs further clarifications, even if the present findings would offer a solid support that this hypothesis is not impossible.

Cross-Kingdom Regulation of Plant-Derived miRNAs in Modulating Insect Development.

MicroRNAs (miRNAs), a class of non-coding small RNAs, are crucial regulatory factors in plants and animals at the post-transcriptional level. These tiny molecules suppress gene expression by complementary oligonucleotide binding to sites in the target messenger. Recently, the discovery of plant-derived miRNAs with cross-kingdom abilities to regulate gene expression in insects has promoted exciting discussion, although some controversies exist regarding the modulation of insect development by plant-derived miRNAs. Here, we review current knowledge about the mechanisms of miRNA biogenesis, the roles of miRNAs in coevolution between insects and plants, the regulation of insect development by plant-derived miRNAs, the cross-kingdom transport mechanisms of plant-derived miRNAs, and cross-kingdom regulation. In addition, the controversy regarding the modulation of insect development by plant-derived miRNAs also was discussed. Our review provides new insights for understanding complex plant-insect interactions and discovering new strategies for pest management and even crop genetic improvement.

Circulating exosome miRNA, is it the novel nutrient molecule through cross-kingdom regulation mediated by food chain transmission from microalgae to bivalve?

MicroRNAs (miRNAs) can efficiently regulate gene expression at intracellular and extracellular levels. Plant-derived miRNAs are highly enriched in animal haemolymph and regulate mammalian gene expression. However, evidence for food-derived miRNAs in Mollusca species is lacking. In this study, we fed the microalga Nannochloropsis oculata to the pearl oyster Pinctada fucata martensii and detected dietary miRNAs in exosomes isolated from the haemolymph by RNA-seq. In total, 273 endogenous miRNAs were identified in all biological replicates. We identified 23 microalgae-derived miRNAs in the exosomes of pearl oyster haemolymph. Most microalgae-derived miRNAs showed high expression levels in both exosomes and microalgae and exhibited apparent variation among individuals. These food-derived miRNAs were predicted to participate in endocytosis, apoptosis, signal transduction, energy metabolism, and biomineralization by targeting multiple genes. These findings demonstrated the cross-kingdom transport of miRNAs from microalgae to bivalves and provide insights into novel nutrient transmission through the food chain.

[Advances of miRNA-mediated regulatory roles in plant-microbe interaction].

There are many bidirectional communication and crosstalk between microbes and host plants. The plant-pathogen interaction directly affects the survival of host plants, while the interaction between plants and their probiotics benefits both. Plant miRNA responds quickly to pathogenic or beneficial microbes when they enter the plant tissues, while microbes also produce miRNA-like RNA (milRNA) to affect plant health. These means miRNA or milRNA is an important fast-responding molecular mediator in plant-microbe interactions, and these internal mechanisms have been better understood in recent years. This review summarized the regulatory roles of miRNA in plant-pathogens and plant-probiotics interaction. The regulatory role of miRNA in disease resistance of host plants during plant-pathogens interaction, and the regulatory role of miRNA in promoting host growth and development during plant-probiotics interaction, as well as the cross-kingdom regulatory role of milRNA in host plants, were discussed in-depth.

Digestion of Plant Dietary miRNAs Starts in the Mouth under the Protection of Coingested Food Components and Plant-Derived Exosome-like Nanoparticles.

The regulatory functions of plant miRNAs on mammalian bodies are controversial, mainly because stability of the miRNAs in the digestive tract, as the prerequisite for their cross-kingdom effects, has somehow been overlooked. Hence, as the first stage of food ingestion, stability of plant miRNAs in human saliva has been investigated. The results show that plant miRNAs are of considerable resistance against salivary digestion, as surviving miRNAs more than 20 fM are detected. The stability varies dramatically, which can be explained by the difference in tertiary structure, governing their affinities to RNase. Surprisingly, miRNAs of low initial concentrations can end up with high survival rates after digestion. Plant miRNAs can be loaded into exosome-like nanoparticles (ELNs) and microcapsules formed by food components, both of which protect the miRNAs from being degraded in human saliva. Overall, plant miRNAs can apply certain strategies to maintain constant concentrations, paving the way for their potential cross-kingdom effects.

Advances of miRNA-mediated regulatory roles in plant-microbe interaction / 生物工程学报

There are many bidirectional communication and crosstalk between microbes and host plants. The plant-pathogen interaction directly affects the survival of host plants, while the interaction between plants and their probiotics benefits both. Plant miRNA responds quickly to pathogenic or beneficial microbes when they enter the plant tissues, while microbes also produce miRNA-like RNA (milRNA) to affect plant health. These means miRNA or milRNA is an important fast-responding molecular mediator in plant-microbe interactions, and these internal mechanisms have been better understood in recent years. This review summarized the regulatory roles of miRNA in plant-pathogens and plant-probiotics interaction. The regulatory role of miRNA in disease resistance of host plants during plant-pathogens interaction, and the regulatory role of miRNA in promoting host growth and development during plant-probiotics interaction, as well as the cross-kingdom regulatory role of milRNA in host plants, were discussed in-depth.

Exogenous Plant gma-miR-159a, Identified by miRNA Library Functional Screening, Ameliorated Hepatic Stellate Cell Activation and Inflammation via Inhibiting GSK-3ß-Mediated Pathways.

PURPOSE: Plant-derived exogenous microRNAs (miRNAs) regulate human physiological functions by blocking the translation of target mRNAs. Although several computational approaches have been developed to elucidate the interactions of cross-species miRNAs and their targets in mammals, the number of verified plant miRNAs is still limited, and the biological roles of most exogenous plant miRNAs remain unknown. METHODS: A miRNA mimic library-based phenotypic screening, which contained 8394 plant mature miRNAs published in the official database miRbase, was performed to identify more novel bioactive plant miRNAs for the prevention of hepatic fibrosis. Inhibition of candidates for the activation of hepatic stellate cells (HSCs) and the underlying mechanisms were evaluated in TGF-ß1- and PDGF-exposed HSC models. The protective effects of the candidates against CCl4-induced liver fibrosis were evaluated in a mouse model. RESULTS: Among the 8394 plant mature miRNAs reported in the official database miRBase, five candidates were found to effectively inhibit the differentiation of HSCs. gma-miR-159a (miR159a) exerted the strongest inhibitory activities on both TGF-ß1- and PDGF-induced HSC activation and proliferation by inhibiting the GSK-3ß-mediated NF-κB and TGF-ß1 pathways. Moreover, miR159a was mainly accumulated in the liver after intravenous injection, and it reduced CCl4-induced hepatic fibrosis and inflammation in mice. CONCLUSION: Results indicated that miR159a has the therapeutic potential for preventing hepatic fibrosis. This study provides a novel strategy for achieving natural nucleic acid drugs.