Abstract

   Gastrodia elata Blume (G. elata) is a valuable traditional Chinese
   medicine with neuroprotection, anti-inflammatory, and immune regulatory
   functions. MicroRNAs (miRNA) is a kind of endogenous noncoding small
   RNAs that plays distinctly important roles for gene regulation of
   organisms. So far, the research on G. elata is mainly focused on the
   pharmacological functions of the natural chemical ingredients, and the
   function of G. elata miRNA remains unknown. In this study, 5,718 known
   miRNAs and 38 novel miRNAs were identified by high-throughput
   sequencing from G. elata. Based on GO and KEGG analysis, we found that
   the human genes possibly regulated by G. elata miRNAs were related to
   the cell cycle, immune regulation, intercellular communication, etc.
   Furthermore, two novel miRNAs as Gas-miR01 and Gas-miR02 have stable
   and high expression in the medicinal tissues of G. elata. Further
   bioinformatics prediction showed that both Gas-miR01 and Gas-miR02
   could target Homo sapiens A20 gene, furthermore, the dual-luciferase
   reporter gene assay and Western Blotting verified the interaction of
   Gas-miR01 or Gas-miR02 with A20. These evidences suggested that G.
   elata-unique miRNAs might be involved in certain physiological
   processes. The animal experiment showed that Gas-miR01 and Gas-miR02
   could be detected in some tissues of mice by intragastric
   administration; meanwhile, the A20 expression in some tissues of mice
   was downregulated. These results supported for the functional study of
   G. elata miRNAs.

   Keywords: Gastrodia elata Blume, microRNA, high-throughput sequencing,
   expression profiles, A20, functional study

Introduction

   Gastrodia elata (G. elata) Blume is a precious Chinese herbal medicine
   in China ([37]Yuan et al., 2018). Many kinds of active ingredients in
   G. elata play an important role in the treatment of diseases. Since the
   1950s, 81 compounds have been isolated from G. elata, including
   phenols, polysaccharides, and various sterols ([38]Duan et al., 2013).
   Gastrodin is the main biologically active ingredient of G. elata
   ([39]Liu et al., 2018).

   In ancient China, G. elata was widely used to treat headache,
   dizziness, paralysis, epilepsy, joint pain, and numbness of limbs
   ([40]Zhan et al., 2016). Some studies on its pharmacological properties
   have shown that G. elata has neuroprotective and antioxidant functions
   and could be used as an anti-inflammatory and antipsychotic drug
   ([41]Kumar et al., 2013; [42]Hu et al., 2014; [43]Du et al., 2016;
   [44]Wang et al., 2016). The pharmacological mechanisms of potential
   anticonvulsant activity of arrow G. elata have been extensively
   studied, which is similar to the mechanisms of known antiepileptic
   drugs ([45]Wu et al., 2017). G. elata has also been found to treat
   anxiety, regulate the circulatory system, and improve memory ([46]Niu
   et al., 2004). Additionally, G. elata could achieve its anti-aging
   effect by regulating certain signaling pathways ([47]Song et al.,
   2016). Except for these, some chemical constituents such as anti-fungal
   protein GAFP- 1 in G. elata also play an important role. Certain
   phenols in G. elata can regulate the apoptosis-related signaling
   pathway by upregulating Bcl-2 and protect the nerves of mice ([48]Wu
   et al., 1996). Recent studies have shown that phenolic glycoside
   gastrodin, the main component of G. elata, showed potential
   neuroprotective effects by inhibiting neurotoxic proinflammatory
   mediators ([49]Li et al., 2018). G. elata has a high potential
   medicinal value. However, research on G. elata in China and abroad
   mainly focuses on the pharmacological functions and product development
   of natural chemical constituents of G. elata. To date, there is no
   research report on the miRNA and its function in G. elata.

   MicroRNAs (miRNAs) are conserved small RNA molecules, which can
   negatively regulate target genes at the transcriptional and
   posttranscriptional level through near-perfect complementarity to
   target mRNA ([50]Nam et al., 2011; [51]Razaviyan et al., 2018). Over
   the past decade, more and more reports have paid attention to their
   cross-kingdom regulation ([52]Wang et al., 2014; [53]Oghbaei et al.,
   2017; [54]Zhou et al., 2017). The regulation of human target genes by
   rice MIR168a was first discovered in 2012 ([55]Zhang et al., 2012). In
   2013, Yang et al. demonstrated that plant-derived small RNAs are
   present in animal serum and urine through two independent studies
   ([56]Yang et al., 2015a; [57]Yang et al., 2015b). In 2014, Zhang et al.
   found that honeysuckle had little degradation of miR2911 after the
   decoction, and further experiments in vitro and in vivo showed that
   honeysuckle could target influenza A virus (IAV), thereby inhibiting
   its replication and reducing mouse mortality ([58]Zhou et al., 2015).
   Furthermore, these studies demonstrate that storage, processing, and
   cooking do not eliminate plant miRNAs in food sources and that
   plant-derived miRNAs could survive in the simulated digestive system
   for at least 75 min ([59]Philip et al., 2015). Additionally, miRNAs
   detected in human plasma were found to resist to ribonuclease A,
   suggesting that miRNA molecules may be accompanied by certain proteins,
   lipids, or other particles that protect them from degradation ([60]Wang
   et al., 2012). To further explore the properties of small RNA through
   the placenta, Li et al. found that the level of small interfering RNA
   (siRNA) in mouse embryos increased significantly after feeding siRNA,
   and the level of alpha-fetoprotein (AFP) targeted by siRNA was
   downregulated ([61]Wong et al., 2015). In addition, the factors like
   ultrasonic treatment, extreme heat, especially RNase treatment that
   could cause miRNA degradation, while being associated with plant
   molecules such as proteins and exosomes might be an effective way to
   protect miRNAs from degradation ([62]Xie and Melzig, 2018). These
   studies confirmed the stability of plant-derived miRNAs during herb
   preparations, suggesting the possibility of medicinal plant miRNAs in
   mammals with complete function. The therapeutic potential for plant
   miRNAs has been confirmed by two different laboratories. A study
   reported that a mixture of three tumor suppressor miRNAs (simulated
   plant miRNAs) reduced colon tumor burden by oral administration in a
   mouse model of colon cancer ([63]Mlotshwa et al., 2015). Another study
   found that plant miR159 was present in female serum, and its level was
   inversely correlated with breast cancer incidence and progression
   ([64]Chin et al., 2016). Most of the identified miR159 are abundant in
   extracellular vesicles (EV), and in vitro experiments have shown that
   synthetic miR159 could reduce the proliferation of breast cancer cells
   ([65]Chin et al., 2016). Furthermore, oral administration of the miR159
   mimics significantly inhibited the growth of venous angiographic breast
   tumors in mice ([66]Chin et al., 2016). Currently, many studies have
   provided evidence that exogenous miRNAs can play an important
   biological role in animals through oral administration.

   In this study, using high-throughput sequencing, we comprehensively
   identified 5718 known miRNAs and 38 novel miRNAs from G. elata for the
   first time. The relative levels of these miRNAs in various stages of G.
   elata were further verified by qRT-PCR. The bioinformatics prediction
   showed these miRNAs could target the Homo sapiens genes, the
   dual-luciferase reporter gene assay and Western blotting indicated that
   both Gas-miR01 and Gas-miR02 could interact with the A20 gene in 293T
   cell. As a zinc finger protein in the NF-κB signaling pathway, A20 is
   involved in cytokine-mediated immune and inflammatory responses, which
   has been proved to play a complex role impacting tumor development and
   therapeutic response according to some reports ([67]Ma and Malynn,
   2012; [68]Santolla et al., 2018). Then, animal experiments also
   confirmed that Gas-miR01 and Gas-miR02 could be detected in some
   tissues of mice by intragastric administration with fresh G. elata
   total RNAs, Gastrodia decoction, and Gastrodia powder. Moreover, the
   A20 expression in some tissues of mice was downregulated in some
   tissues of mice. Our research provides a theoretical basis for the
   functional study of G. elata.

Materials and Methods

Sample Collection

   The life cycle of G. elata includes G. elata seeds, protocorms, white
   G. elata, arrow G. elata, and bolted G. elata, which is shown as
   [69]Figure 1 . The sample of fresh arrow G. elata, numbered as
   CL20151008, which was authenticated by Professor Zongsuo Liang in
   Zhejiang Provincial Key Laboratory of Plant Secondary Metabolism and
   Regulation, was collected at coordinate 115°93′12.5″E 31°27′50.0″N in
   Dabie Mountain, Anhui Province in China and stored at -80°C until total
   RNA was extracted. The voucher specimen is deposited at Herbarium of
   Northwest A&F University (WUK). After collecting the sample, G. elata
   seeds from fresh arrow G. elata were cultured. Protocorms, white
   G. elata and bolted G. elata were collected separately according to
   their growing stages.

Figure 1.

   [70]Figure 1
   [71]Open in a new tab

   The life cycle of G. elata (trace diagram). The life cycle of G. elata
   has five stages, including G. elata seeds, protocorms, white G. elata,
   arrow G. elata, and bolted G. elata.

   In animal experiment, total RNAs of fresh G. elata, Gastrodia decoction
   and Gastrodia powder were used. Gastrodia superfine powder purchased
   from Yunnan Panlong Yunhai Ltd. Total RNA was extracted from fresh
   arrow G. elata. Gastrodia decoction was made by putting 150 mg of fresh
   arrow G. elata in 90 mL ddH[2]O, with cutting into pieces, soaking at
   room temperature for 30 min, simmering over low heat for 20 min to
   simulate traditional Chinese herbal decoction, finally getting 30 mL of
   Gastrodia decoction. Gastrodia powder was made as the follows: put 30
   mg (the moisture content of arrow G. elata is 80%) of Gastrodia
   superfine powder in 30 mL ddH[2]O, and then blend to get a mixture
   called Gastrodia powder.

RNA Extraction and qRT-PCR

   Total RNAs extracted from different developmental stages from G. elata
   were reverse transcribed into the cDNAs using the Transcriptor
   First-Strand cDNA Synthesis Kit (Roche). The following program was used
   for the qRT-PCR using Fast Start Universal SYBR Green Master (Rox)
   (Roche): 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and
   60°C for 1 min, 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s.
   Likewise, stem-loop qRT-PCR was used to detect miRNA (Specific
   RT-primer) using 5.8S rRNA as a normal control ([72]Li et al., 2016).
   Likewise, total RNAs of mouse tissues in animal experiments were
   extracted. Then, the expression of Gas-miR01, Gas-miR02, and A20 were
   also detected by qRT-PCR using Fast Start Universal SYBR Green Master
   (Rox) (Roche). Primers for the quantitative real-time PCR and reverse
   transcription ([73] Supplementary materials Table 1 ) were synthesized
   by Sangon Biotech (Shanghai). The data analysis was performed using ABI
   Prism 7500 SDS Software (Applied Biosystems, USA).

Bioinformatics Analysis

   We used the two-phase model (TPM) of Gastrodia to normalize the
   expression level. Normalized expression = (read count ×
   1,000,000)/libsize, where libsize refers to the number of reads on a
   miRNA compared to a sample ([74]Zhou et al., 2010). We use DEGSeq for
   differential analysis ([75]Wang et al., 2010) and count its Q-value (or
   adjust p-value) ([76]Pounds and Cheng, 2004). The predicted target
   genes were subjected aligned against to GO and KEGG database, and the
   specific processes and pathways were emphatically analyzed ([77]Nie
   et al., 2018). The GO enrichment analysis method refers to GOseq, and
   we counted the number of genes belonging to a certain GO number in the
   whole genome background and differentially expressed genes. Then,
   according to Fisher’s test, chi-squared test, and binominal test, the
   enrichment of GO entries in differentially expressed genes was
   determined relative to the background. RNAhybrid and miRanda softwares
   were used to predict differential miRNA target genes.

Transfection With miRNA Mimics

   Gas-miR01, Gas-miR02, and negative control were synthetized by Gene
   Pharma (Shanghai, China). 293T cells were cultured in six-well plates
   overnight in 37°C at 5% CO[2], and Gas-miR01 and Gas-miR02 were
   overexpressed in 293T cells by transfection of the mimics for 20 pmol/1
   × 10^5 cells. The NC mimics was used as the negative control. Cells
   were collected 48 h post transfection for real-time qPCR analysis.

Dual Luciferase Reporter Assay

   The 293 T cells were cultured in a 24-well plate. And after 24 hours,
   the cells were transfected with pGL3-basic-A20 vector and
   co-transfected with Gas-miR01/Gas-miR02 mimics or negative control by
   Lipofectamine 3000TM transfection reagent (Thermo Scientific, MA, USA).
   The luciferase vectors were constructed by General Biol (Anhui, China).
   The sequence of Gas-miR01 and Gas-miR02 were as follows: Gas-miR01:
   5’-GUUCAGGAAUGCUGUGGGAAG-3’; Gas-miR02: 5’-UUCAAUAAAGCUGUGGGAAA-3’.
   After 48 hours of transfection, the luciferase activity was detected by
   Dual-Luciferase Reporter Assay System (Promega, USA, E1910) according
   to the manufacturer’s protocol.

Western Blotting

   The 293T cells transfected with Gas-miR01/Gas-miR02/NC mimics were
   collected, and then the total proteins were extracted using the Cell
   Lysis Reagent (Roche). Total proteins were separated by 12% SDS-PAGE
   and transferred onto a polyvinylidene difluoride (PVDF) membrane
   (Millipore, USA) by electroblotting. After blocking with 5% skim milk
   in tris-buffered saline with Tween (TBST) (pH 7.4) for 2 h at room
   temperature, the protein was detected with an anti-A20 antibody (Cell
   Signaling Technology, USA) overnight at 4°C. The membrane was washed
   with TBST, and HRP-labeled (horseradish peroxidase-labeled) mouse
   anti-rabbit antibody was used as the secondary antibody (Cell Signaling
   Technology, USA). The signals were detected using the ECL Detection Kit
   (Advansta).

Animal Experiments

   Twenty-four female ICR mice were purchased from a pathogen-free animal
   facility at Zhejiang Chinese Medical University. The Institutional
   Review Board of Zhejiang Chinese Medical University approved all
   housing and surgical procedures. Animals were housed in cages (three to
   five mice per cage) and given free access to rodent chow and water ad
   libitum in a room maintained at 24°C and a 12-h/12-h light-dark cycle.
   At 7 weeks of age, each ICR mouse was fed with fresh G. elata total
   RNAs (30 μg/kg/2 h), Gastrodia decoction (3 mg/kg/2 h), Gastrodia
   powder (3 mg/kg/2 h), or double distilled H[2]O by gavage after fasting
   overnight. The mice were treated with total RNAs of fresh G. elata (30
   μg/kg), Gastrodia decoction (3 mg/kg) and Gastrodia powder (3 mg/kg) by
   feeding a time per 2 h with six times during 12 h. After the sixth
   gavage for 0.5 h, tissues were collected, and total RNAs were
   extracted. The flow chart of animal experiments is shown in [78]Figure
   6A . All procedures used in this study were approved by the Zhejiang
   Sci-Tech University Animal Experimental Ethics Committee.

Figure 6.

   Figure 6
   [79]Open in a new tab

   Gas-miR01 and Gas-miR02 were detected in some tissues of mice by
   intragastric administration. (A) Flow chart of animal experiments. The
   levels of Gas-miR01 (B) and Gas-miR02 (C) in fresh G. elata tuber,
   Gastrodia decoction and Gastrodia powder. The fresh G. elata total
   RNAs, Gastrodia decoction and Gastrodia powder were gavaged and the
   levels of Gas-miR01 and Gas-miR02 in various tissues of mice were
   detected by qRT-PCR. The levels of Gas-miR01 and Gas-miR02 in mouse
   tissues. (D) cerebrum; (E) cerebellum; (F) hypothalamus; (G) heart; (H)
   kidney; (I) spleen. *p < 0.05; **p < 0.01; ***p < 0.001 compared with
   the control group, n = 6. Values are the mean ± SEM.

Statistical Analysis

   All data are the average of three separate experiments presented as the
   mean ± SEM. Data were analyzed by one-way ANOVA and LSD test with SPSS
   statistical analysis software.

Results

Length Distribution of G. elata Small RNA and Expression Profiles of miRNAs

   In the clean total reads, the length distribution show higher peaks at
   24 nt and 35 nt; in the clean unique reads, the highest peak of the
   length distribution is at 24 nt ([80] Figures 2A, B ). The length of
   miRNA and siRNA is approximately 24 nt, suggesting that the majority of
   G. elata small RNAs (sRNAs) were miRNAs or siRNAs, which may play an
   important role in regulating gene expression. Then, the expression of
   miRNAs from different G. elata tissues was explored. We firstly drew a
   hierarchical clustering map of 38 miRNAs with high expression and good
   reproducibility. The results are shown in [81]Figure 2C . The arrow G.
   elata bud and bloted G. elata tuber showed a relative higher expression
   level of miRNAs. Eight known miRNAs (Gas-miR159, Gas-miR6478,
   Gas-miR148a-3p, Gas-miR99, Gas-miR143-3p, Gas-miR319f, Gas-miR396e, and
   Gas-miR26a) and two new miRNAs (Gas-miR01 and Gas-miR02) were selected
   to detect for expression levels ([82] Supplementary materials Table 2
   ). In traditional medicinal tissue of G. elata (arrow G. elata tuber),
   Gas-miR6478, Gas-miR148a-3p, Gas-miR99, Gas-miR143-3p, and Gas-miR26a
   had higher expression. Furthermore, the expression levels of 10 G.
   elata miRNAs were verified by qRT-PCR, and the qRT-PCR results were
   generally consistent with the sequencing results. It is worth
   mentioning that the two G. elata-unique miRNAs (Gas-miR01 and
   Gas-miR02) had relatively high expression levels in G. elata tissues
   ([83] Figure 2D ).

Figure 2.

   [84]Figure 2
   [85]Open in a new tab

   Length distribution of G. elata sRNA and expression profiles of 10 G.
   elata miRNAs in different tissues. Length distributions of clean total
   reads (A) and clean unique reads (B). (C) Clustering map of difference
   miRNAs. (D) Relative expression levels of 10 G. elata miRNAs in
   different tissues.

   The relative G. elata miRNA expression levels in different growth
   stages were also determined by qRT-PCR. The results showed that all the
   detected 10 miRNAs showed a different expression levels in different
   growth stages ([86] Figure 3 ). On the whole, the Gas-miR01,
   Gas-miR319f and Gas-miR396e had relative high expression levels in
   certain growth stages. The G. elata-unique Gas-miR01 has lower
   expression levels in protocorms and white G. elata but higher
   expression levels in G. elata seeds and arrow G. elata. Compared with
   G. elata seeds, white G. elata and arrow G. elata, Gas-miR02 had the
   highest expression level in protocorms ([87] Figure 3 ).

Figure 3.

   [88]Figure 3
   [89]Open in a new tab

   Expression profiles of 10 G. elata miRNAs in different growth stages.
   The relative G. elata miRNA expression levels in different growth
   stages were determined by qRT-PCR. The vertical coordinate means fold
   change.

GO and KEGG Enrichment Analysis of Differential miRNA Target Genes

   To explore the potential functions of some specific miRNAs in G. elata,
   the potential Homo sapiens target genes of G. elata miRNAs were
   obtained by prediction using RNAhybrid and miRanda softwares. The
   candidate target genes were further analyzed by Gene Ontology analysis.
   In the medicinal tissue of G. elata (arrow G. elata tuber), the
   enrichment GO annotation of miRNA target genes involves biological
   regulation, immune system regulation, intercellular communication, and
   nucleic acid binding in three aspects: biological processes, cellular
   components, and molecular functions ([90] Figure 4A ). The KEGG pathway
   enrichment analysis indicated that the target genes annotation of the
   G. elata miRNAs is mainly enriched in the cell cycle, intestinal immune
   network, tumor transcriptional regulation, and olfactory signal
   transduction pathway ([91] Figure 4B ).

Figure 4.

   Figure 4
   [92]Open in a new tab

   GO and KEGG enrichment analysis of differential miRNA target genes. (A)
   The target genes of miRNA involve BP (biological processes), CC
   (cellular components), and MF (molecular functions). (B) The closer the
   Q-value is to zero, the more significant the particular GO term
   associated with the group of genes; the closer the Q-value is to one,
   the opposite. The closer the Q-value is to 0, the more significant the
   particular GO term associated with the group of genes; the closer the
   Q-value is to one, the opposite.

The G. elata-Unique Gas-miR01 and Gas-miR02 Targeted the Homo sapiens A20
Gene

   The bioinformatics analysis found that the seed sequences of the 5’ end
   of G. elata-unique miRNA Gas-miR01 and Gas-miR02 could perfectly match
   the targeted sites in the 3’ UTR region of the Homo sapiens A20 gene
   ([93] Figure 5A ). Subsequently, the relative luciferase activity of
   pGL3-basic-A20 vector was significantly reduced when transfected with
   Gas-miR01 mimic or Gas-miR02 mimic, and this indicated that Gas-miR01
   and Gas-miR02 could suppress the translation of A20 by interacting with
   the A20 gene ([94] Figures 5B, C ). The Gas-miR01 and Gas-miR02 mimics
   were then transfected into 293T cells successfully ([95] Figure 5D ),
   and the down-regulated expression level of A20 protein was detected by
   Western blotting ([96] Figure 5E ). The result showed that both
   Gas-miR01 and Gas-miR02 could significantly downregulate the expression
   of the protein A20 in vitro.

Figure 5.

   Figure 5
   [97]Open in a new tab

   The down-regulation of A20 by Gas-miR01 and Gas-miR02. (A) Matching
   between Gas-miR01, Gas-miR02 and their target sites in A20 gene. (B)
   The relative luciferase activity down-regulated by Gas-miR01. (C) The
   relative luciferase activity down-regulated by Gas-miR02. (D) Levels of
   Gas-miR01 and Gas-miR02 in 293T cells after transfected with
   corresponding mimics. (E) The expression level of A20 protein after
   transfected with Gas-miR01 and Gas-miR02 mimics. *p < 0.05; **p < 0.01;
   ***p < 0.001. Values are the mean ± SEM.

Gas-miR01 and Gas-miR02 Were Detected in Some Tissues of Mice by Intragastric
Administration

   The total RNAs were extracted from fresh G. elata tuber, Gastrodia
   decoction, and Gastrodia powder, respectively, and both Gas-miR01 and
   Gas-miR02 were all detected in them by using qPCR ([98] Figures 6B, C
   ). To further determine the cross-kingdom regulation of G. elata
   miRNAs, the fresh G. elata total RNAs, Gastrodia decoction Gastrodia
   powder, and double distilled H[2]O were gavaged. The levels of
   Gas-miR01 and Gas-miR02 in various tissues of mice were detected by
   qRT-PCR ([99] Figures 6D–I ). Compared with the control, the levels of
   Gas-miR01 and Gas-miR02 in the brain, cerebellum, hypothalamus, heart,
   kidney, and spleen of mice were significantly increased after total
   RNAs were gavaged, especially in the kidneys. After the Gastrodia
   powder and Gastrodia decoction were gavaged, the levels of Gas-miR01
   and Gas-miR02 in the brain, cerebellum, and spleen tissues of mice also
   increased significantly. These results indicated that Gas-miR01 and
   Gas-miR02 could be into the mouse internal environment through the
   gastrointestinal tract and could be detected in some tissues of mice.
   Furthermore, qRT-PCR showed the A20 expression was also downregulated
   in some tissues of mice ([100] Figures 7A–F ).

Figure 7.

   [101]Figure 7
   [102]Open in a new tab

   The A20 expression was downregulated in many tissues of mouse. The
   result of qRT-PCR showed the mRNA level of A20 in various tissues of
   mice. (A) Cerebrum. (B) Cerebellum. (C) Hypothalamus. (D) Heart. (E)
   Kidney. (F) Spleen. *p < 0.05; **p < 0.01; ***p < 0.001 compared with
   the control group, n = 6. Values are the mean ± SEM.

Discussion

   miRNA, a small endogenous RNA, plays an important role by targeting
   specific mRNAs in plants, animals and humans ([103]Sala-Cirtog et al.,
   2015). Studies have shown that many plant miRNAs are indispensable in
   cell proliferation and the secondary process of plant secondary
   metabolism. miRNA detection analysis displayed that Gas-miR319f had
   slightly different expression levels in G. elata seeds, protocorms, and
   white G. elata, but it would rise sharply 20 times to the original in
   arrow G. elata, which is the main medicinal tissue of G. elata.
   Therefore, we inferred that the sharp increase in the level of
   Gas-miR319f during this stage might be related to the large amount of
   synthetic medicinal components, such as gastrodin.

   In China, the traditional medicinal herbs have been used for thousands
   of years and are usually decocted and orally administrated. A growing
   body of research suggests that the molecular mechanisms of medicinal
   herbs have been revealed. Since the discovery of plant miRNAs in human
   tissues and serum after ingestion, the relationship between the two
   kingdoms has been presented in a new perspective ([104]Han and Luan,
   2015). For example, miRNA therapy might be the next best step in
   determining new treatment options for medicinal plants ([105]Thakur
   et al., 2014; [106]Bai et al., 2018). Plant miRNAs could be detected in
   the sera and tissues of various animals, and these exogenous miRNAs
   were primarily acquired orally through food intake ([107]Zhang et al.,
   2012). In addition to the findings on transfer of miRNAs and their
   therapeutic applications, similar studies could provide evidences for
   further research that might influence modern definitions of herbs. A
   recent study showed that synthetic MIR2911, extracted RNA from
   honeysuckle decoction, and honeysuckle decoction all significantly
   inhibited H1N1 viral replication and rescued viral infection-induced
   mouse weight loss. The results indicated that MIR2911 is an active
   component identified in traditional Chinese medicine to directly target
   various IAVs and may represent a novel type of natural product that
   effectively suppresses viral infection ([108]Zhou et al., 2015).

   In our research, the results suggested that the human genes related to
   immune system regulation could be regulated by G. elata miRNAs, and in
   vitro experiment evidenced that both Gas-miR01 and Gas-miR02 mimics
   could downregulate the expression of the Homo sapiens A20, the key
   protein in the NF-κB signaling pathway. NF-κB is present in many
   eukaryotic cells, widely involved in many physiological processes in
   humans, and related to many diseases such as Parkinson’s disease,
   Alzheimer’s disease and fatty liver disease ([109]Lee and Ambros, 2001;
   [110]Gommans and Berezikov, 2012; [111]Ge et al., 2016;
   [112]Bazazzadegan et al., 2017). And according to some reports, A20
   expression is increased in a number of solid human tumors and cancers,
   which likely contributes to both carcinogenesis and response to
   chemotherapy. These evidences uncovered the complexities of the
   mechanisms involved in A20’s impact on tumor development and response
   to treatment ([113]Hjelmeland et al., 2010; [114]Bellail et al., 2012;
   [115]Dong et al., 2012; [116]Qiao et al., 2015). Animal experiments
   showed that the G. elata-unique miRNAs as Gas-miR01 and Gas-miR02 could
   be detected in both Gastrodia decoction and Gastrodia powder,
   suggesting these two G. elata-unique miRNAs could still remain stable
   after decoction and long-time storage. Furthermore, Gas-miR01 and
   Gas-miR02 could be detected in some tissues of mice by intragastric
   administration of the total RNAs of fresh G. elata tuber, Gastrodia
   decoction, and Gastrodia powder; meanwhile, the A20 expression in some
   tissues of mice was also downregulated. These evidences suggest that G.
   elata miRNAs could be into the mouse internal environment and
   conductcross-kingdom regulation. The above results provided a new
   theoretical basis for the further study on the cross-kingdom regulation
   of G. elata miRNAs. Our findings may provide data support for the
   functional study of miRNAs and the medicinal ingredients in G. elata in
   the future.

Conclusion

   In this study, we firstly obtained 5,718 known miRNAs and 38 G.
   elata–specific miRNAs by high-throughput sequencing. The expression
   levels of the miRNAs in various stages and tissues of G. elata were
   different by qRT-PCR, which was found to be high expression in the
   medicinal tissue of G. elata (arrow G. elata tuber). Bioinfomatics
   analysis indicated that G. elata miRNAs might be involved in the
   cross-kingdom regulation of Homo sapiens genes. It was confirmed for
   the first time that the two G. elata-unique miRNAs Gas-miR01 and
   Gas-miR02 could downregulate the expression of the Homo sapiens A20
   gene in vitro. Furthermore, G. elata-unique miRNAs Gas-miR01 and
   Gas-miR02 could be detected in some tissues of mice through the
   gastrointestinal tract, and A20 expression was downregulated. These
   findings lay a foundation for the study of the cross-kingdom regulation
   of G. elata miRNAs.

Data Availability Statement

   All datasets generated for this study are included in the
   article/supplementary material.

Ethics Statement

   The animal study was reviewed and approved by Zhejiang Sci-Tech
   University Animal Experimental Ethics Committee.

Author Contributions

   QS designed the project and instructed all the process. CX and HZ
   designed and performed the main experiments and wrote the article under
   the guidance of QS. XX and TJ undertook part of the experiments and
   analyzed the data. SL participated in the research and helped to write
   the article. DW and ZN helped to design the project and revise
   articles.

Funding

   This work was supported by grants from Project of Public Welfare
   Technology Research in Zhejiang Province (grant numbers LGF18H250004
   and LGF19H250002).

Conflict of Interest

   The authors declare that the research was conducted in the absence of
   any commercial or financial relationships that could be construed as a
   potential conflict of interest

Acknowledgments