Volume 18 Supplement 2

Selected articles from the 15th Asia Pacific Bioinformatics Conference (APBC 2017)

Open Access

Plant miRNAs found in human circulating system provide evidences of cross kingdom RNAi

BMC Genomics201718(Suppl 2):112

DOI: 10.1186/s12864-017-3502-3

Published: 14 March 2017

Abstract

Background

Emerging evidence indicates that plant miRNAs can present within human circulating system through dietary intake and regulate human gene expression. Hence we deduced that comestible plants miRNAs can be identified in the public available small RNA sequencing data sets.

Results

In this study, we identified abundant plant miRNAs sequences from 410 human plasma small RNA sequencing data sets. One particular plant miRNA miR2910, conserved in fruits and vegetables, was found to present in high relative amount in the plasma samples. This miRNA, with same 6mer and 7mer-A1 target seed sequences as hsa-miR-4259 and hsa-miR-4715-5p, was predicted to target human JAK-STAT signaling pathway gene SPRY4 and transcription regulation genes.

Conclusions

Through analysis of public available plasma small RNA sequencing data, we found the supporting evidence for the plant miRNAs cross kingdom RNAi within human circulating system.

Keywords

Circulating miRNAs Plant miRNAs miR2910 Cross Kingdom RNAi

Background

Micro RNAs (miRNA) are about 22 nucleotides single strand non-coding RNAs, which regulate protein coding genes expression through guiding protein-RNA complex toward mRNAs [1, 2]. Given the conventional recognition that expression of most of the genes are regulated by miRNAs in mammal, research of miRNAs’ roles in pathogenesis in diseases and biological processes is foreseeable [3]. In recent years, stable cell free miRNAs had been found in human circulating system derived samples such as plasma, urine and saliva [4]. Circulating miRNAs are studied to be used as diagnosis biomarkers in many diseases [5].

Plant miRNA miR168a was found to present in the sera of mammals including human and regulate human gene LDLRAP1 [6]. Positive and negative results of experimental test on whether exogenous miRNAs can present in animal circulating system through food ingestion has been published in the following years [710]. Until recently, another indicates that plant miRNAs can present within human circulating system through dietary intake and regulate human gene expression emerged. Broccoli sourced miR159 was found in human sera and proved to inhibit breast cancer growth through targeting the gene TCF7 [11].

Given the assumption that certain comestible plants miRNAs can be absorbed through ingestion and accumulate within human sera, we collected and examined 410 public available small RNA sequencing data sets, detected the existence of plant miRNAs through comparing to genomes of 5 different plant model organism Arabidopsis thaliana, Triticum aestivum, Oryza sativa, Zea mays, and Brachypodium distachyon. In results, we identified abundant plant miRNAs sequences from 410 human plasma small RNA sequencing data sets. Amount of these exogenous miRNA was compared to the ubiquitous human miRNA presented in the samples. One particular plant miRNA miR2910, conserved in fruits and vegetables, was found to present in high relative amount in the plasma samples. This miRNA, with same 6mer and 7mer-A1 target seed sequence as hsa-miR-4259 and hsa-miR-4715-5p, was predicted to target human JAK-STAT signaling pathway gene SPRY4 and transcription regulation genes through miRTar [12].

This discovery not only provides new evidence of cross kingdom RNAi between human and plant, but also expanded the potential of dietary miRNA treatment on a wide range of variable diseases.

Results and discussion

A total 1,301 plant miRNAs, including 654 homologs, with at least one read presented in the sample were identified from the selected transcriptome sequencing runs. Count of the reads within each sample is available in (Additional file 1: Table S1). The relative abundance of these miRNAs were compared to detected human miRNAs. Among them we found a singular case, peu-MIR2910, can be found ubiquitously within the selected samples. In some samples, up to more than one thousand copy of peu-MIR2910 can be found, which is more abundant than most of the human miRNAs detected within the samples. Beside peu-MIR2910, up to hundreds of copies of peu-MIR2916 in 379 samples, peu-MIR2914 in 359 samples, and tae-MIR2018 in 353 samples were also found.

The detected plant miRNAs are not human originated or sample contamination

Sequence alignment of the detected plant miRNAs sequence onto human genome was performed with BLAST [13] and resulted in zero alignment hit. Alignment of the conventional adaptor sequences to the plant miRNAs also resulted with zero hits. Through this result we are convinced that these detected plant miRNA sequence were not originated from conserved human sequences or adaptors contamination.

The plant miRNA peu-MIR2910 is conserved within comestible plants

Among the 1,301 plant miRNAs detected in our collected samples, miR2910 presented in all the 404 examined runs in relatively high abundance. Assuming that the source of plant miRNAs present in human plasma originated from food ingestion, then miR2910 must be evolutionally conserved in a wide variable kinds of comestible plants.

From the reports of recent years we found that peu-MIR2910 was reported to be expressed within one of the model organism we used, Zea mays [14]. As one of the main grain consumed all over the world, maize can potentially the source of the miR2910 detected in the data sets. On the other hand, we also found that miR2910 is conserved within fruits and vegetables. Melon [15], Sorghum [16], tomato [17], tea [18] and oil palm [19] are also the potential source of miR2910. All these evidences support the assumption that the peu-MIR2910 present in the human plasma samples originate from food ingestion. On the other hand, peu-MIR2914 and peu-MIR2916 were also found conserved within oil palm [20]. Conserved tae-MIR2018 was found in wheat [21, 22].

Plant miR159 and miR168a were also detected in the examined samples

To verify the analysis result with previous reports, we examined the amount of detected miR159 and miR168a within the examined samples. As listed in the (Additional file 1: Table S1), despite presenting in relatively small amount (less than 4 copies), miR159 can be found in 32 samples while miR168a can be found in 7 samples. The difference might be result from the difference of the diet habit. Most of the examined samples were presumably collected from Wisconsin, USA [23]. The dietary habit of the subjects can be much different from the subjects collect in China [6] and California [11]. On the other hand, given the fact that only roughly 1% of all the adaptor trimmed reads can be aligned onto plant genomes, as well as the generally low concentration of miRNA within plasma samples, detection of the miR159 as well as miR168a in less than 4 copy within one sample can still be considered as supporting evidence of the two previous reports.

Human genes can potentially be targeted by peu-MIR2910 and other plant miRNAs

In the previous reports [6, 11], plant miRNAs present in human circulating system were proven to be able to target human genes through similar AGO protein correlated mechanism. Given the assumption that miRNAs with similar or same target seed sequences can target same sets of genes, we tested the similarity of plant miRNAs and human miRNAs in target seed regions.

Through the result of the target seed analysis, we found that despite the whole sequence, including the hairpin, of peu-MIR2910 cannot be aligned onto human genome, the 6mer and 7mer-A1 target seed sequences of miR2910, CCAACT and CCAACTA, is the same as hsa-miR-4259 and hsa-miR-4715-5p. Given the previously reported case of miR168a [6] and miR159 [11], we hypothesis that peu-MIR2910 within the circulating system can target human genes as well. From the 51 target genes of hsa-miR-4259 and hsa-miR-4715-5p reported in miRTarBase [24], and the mature sequence of miR2910, we found 10 possible target genes predicted through miRTar [12], as illustrated in Fig. 1. We found that miR2910 can potentially target on 5′ UTR of LIMA1, and the CDS of CTNND1, FOLR1, LBX1 STK38, FAM127B, PHF19, ZNF295, SPRY4 and MRRF.
Fig. 1

Predicted target human genes of abundant plant miRNAs. The predicted miR2910, miR2916 and miR2018 targets through miRTar [12] are illustrated in this figure. The pink diamond shape nodes represent plant miRNAs while the round blue nodes represent human genes

Interestingly, we also found that miR2916 has exact same 8mer target seed sequence, AGTCCCCA, with human miRNA hsa-miR-4652-5p. From the 40 target genes of hsa-miR-4652-5p reported in miRTarBase [24], and the mature sequence of miR2916, we found other 20 possible target genes predicted through miRTar [12]. Same analysis process was conducted on miR2018, and three potential human gene targets were found. These findings were also illustrated in Fig. 1.

From the gene sets enrichment, we found that LBX1, PHF19, STK36, ZNF295 and CTNND1 are associated with regulation of transcription, while SPRY 4 belongs to human JAK-STAT signaling pathway, as summarized in Table 1. CTNND1 is also associated with Adherens junctions and endocytosis [25]. We also found that three genes MEN1, LDB1 and TLX1, predicted to be targeted by miR2916 were associated with Transcriptional misregulation in cancer, as summarized in Table 2.
Table 1

Gene set enrichment of miR2910 target genes

Category

Term

P Value

Genes

GOTERM_CC_FAT

GO:0042995 ~ cell projection

0.026595

FOLR1, CTNND1, SPRY4

SP_PIR_KEYWORDS

developmental protein

0.048812

LBX1, STK36, SPRY4

GOTERM_BP_FAT

GO:0045449 ~ regulation of transcription

0.049408

LBX1, PHF19, STK36, ZNF295, CTNND1

INTERPRO

IPR011989: Armadillo-like helical

0.059853

STK36, CTNND1

SP_PIR_KEYWORDS

transcription regulation

0.060318

LBX1, PHF19, ZNF295, CTNND1

SP_PIR_KEYWORDS

Transcription

0.06372

LBX1, PHF19, ZNF295, CTNND1

This table summarized the gene set enrichment of miR2910 targets. The human genes listed here were predicted to be targeted by plant miR2910 through sequence analysis. The terms were originated from the web based tool DAVID [33]

Table 2

Gene set enrichment of miR2916 target genes

Category

Term

P Value

Genes

KEGG_PATHWAY

ptr05202: Transcriptional misregulation in cancer

0.011556

MEN1, LDB1, TLX1

GOTERM_BP_DIRECT

GO:0043484 ~ regulation of RNA splicing

0.02339

SON

INTERPRO

IPR000467:G-patch domain

0.025805

SON

UP_KEYWORDS

DNA-binding

0.028196

TBX2, PPP1R10, H2AFX, TLX1

INTERPRO

IPR014720: Double-stranded RNA-binding-like domain

0.029124

SON

GOTERM_CC_DIRECT

GO:0071011 ~ precatalytic spliceosome

0.033471

SON

SMART

SM00443:G_patch

0.036062

SON

GOTERM_BP_DIRECT

GO:0000910 ~ cytokinesis

0.036161

SON

GOTERM_MF_DIRECT

GO:0003723 ~ RNA binding

0.037823

SON, TDRKH

GOTERM_CC_DIRECT

GO:0000781 ~ chromosome, telomeric region

0.046328

PPP1R10, H2AFX

GOTERM_BP_DIRECT

GO:0006397 ~ mRNA processing

0.053776

SON

GOTERM_BP_DIRECT

GO:0000226 ~ microtubule cytoskeleton organization

0.058753

SON

GOTERM_BP_DIRECT

GO:0032092 ~ positive regulation of protein binding

0.063706

MEN1, PLXND1

GOTERM_CC_DIRECT

GO:0000785 ~ chromatin

0.073822

MEN1, PPP1R10

UP_KEYWORDS

Nucleus

0.081889

TBX2, PPP1R10, H2AFX, TLX1

GOTERM_CC_DIRECT

GO:0005634 ~ nucleus

0.08342

MEN1, TBX2, LDB1, MKNK2, H2AFX, MID1IP1, TLX1

GOTERM_BP_DIRECT

GO:0051726 ~ regulation of cell cycle

0.091709

SON

This table summarized the gene set enrichment of miR2916 targets. The human genes listed here were predicted to be targeted by plant miR2916 through sequence analysis. The terms were originated from the web based tool DAVID [33]

Methods

The data analysis process in this research is summarized in Fig. 2. Small RNA sequencing data sets of human plasma was dowloaded from NCBI SRA [26]. The adaptors within the data set were trimmed with the tool Skewer [27]. Quality control of the remaining reads was further conducted through NGSQCToolkit [28]. The miRNA detection was performed through miRDeep 2 [29]. To detect the plant miRNAs within the samples, we collected the genomes of 5 different plant model organism Arabidopsis thaliana, Triticum aestivum, Oryza sativa, Zea mays, and Brachypodium distachyon. The assembly version used were GCA_000001735.1, GCA_000005505.2, GCA_001305255.1, GCA_000210335.1 and GCA_000210335.1, which were downloaded from NCBI assembly. The plant miRNAs used for detection were collected from the database PMRD [30].
Fig. 2

Summary of the data analysis process. The data analysis pipeline designed to detect the comestible plants miRNAs in human plasma samples is summarized in this figure. The collected human circulating small RNA-seq reads were align to the plant genome. The existence of plant miRNAs was examined through the algorithm of miRDeep2 package [29]

The human plasma small RNA sequencing reads were aligned onto each of the plant genomes through Bowtie [31] and the miRDeep2 package [29]. Whether the plant miRNAs presented in the sample was determined by the copy of read aligned on the genomes, precursor and mature sequences of miRNAs. On the other hand, the HG19 human genome was used to detect the human miRNA. The target seed sequences [3] of the detected plant miRNAs were compared to the human mature miRNA sequence downloaded from miRBase [32]. Target genes of human miRNAs with close target seed sequences were assumed to be potentially targeted by the plant miRNAs. The target gene of these human miRNAs were acquired from miRTarBase [24]. Finally these target genes and the sequence of the interested plants miRNAs were used as candidates for miRNA target prediction. The target prediction was conducted through miRTar [12]. The target gene prediction of miRNAs was conducted on 3′UTR, CDS and 5′UTR of the genes, with the threshold of MFE −14 and score 140. The gene set enrichment was performed with DAVID [33].

Conclusions

Through analysis of public available plasma small RNA sequencing data, we found the supporting evidence for the plant miRNAs cross kingdom RNAi within human circulating system.

Singular amount of plant miRNA peu-MIR2910, conserved in fruits and vegetables, was found in the plasma samples presumably collected from the population of Wisconsin, USA. This miRNA, with same 6mer and 7mer-A1 target seed sequences as hsa-miR-4259 and hsa-miR-4715-5p, CCAACT and CCAACTA, was predicted to target human JAK-STAT signaling pathway gene SPRY4 and transcription regulation genes. Whether the detected miR2910 can be absorbed into circulating system through food ingestion should be further tested through xenograft experiments.

Declarations

Acknowledgements

The authors would like to thank the Ministry of Science and Technology and the National Chiao Tung University and Ministry of Education, Taiwan, R.O.C. Veterans General Hospitals and University System of Taiwan (VGHUST), for financially supporting this research.

About this supplement

This article has been published as part of BMC Genomics Volume 18 Supplement 2, 2017. Selected articles from the 15th Asia Pacific Bioinformatics Conference (APBC 2017): genomics. The full contents of the supplement are available online http://bmcgenomics.biomedcentral.com/articles/supplements/volume-18-supplement-2

Funding

This work is supported by multiple programs from the Ministry of Science and Technology, Taiwan [MOST 105-2627-M-009-007-, MOST 103-2628-B-009-001-MY3, MOST 105-2319-B-400-002-, MOST 104-2911-I-009-509, and MOST 105-2633-B-009-003-, MOST 104-2314-B-195-014]. This study was also supported by research grants from Health and welfare surcharge of tobacco products, Ministry of Health and Welfare, Taiwan [MOHW105-TDU-B-212-134002]. Finally, this work is particularly supported by “Aiming for the Top University Program” of the National Chiao Tung University and Ministry of Education, Taiwan, R.O.C. Veterans General Hospitals and University System of Taiwan (VGHUST) Joint Research Program [VGHUST105-G1-4-2]. The funding includes the cost of labor force, publication costs of the manuscript and maintenance fee of the research facilities as well as the computational devices.

Availability of data and materials

Supplemental table 1 is available in the Additional file 1.

Authors’ contributions

Y.C. L. proposed and conducted this study. H.D.H. supervised the progress of the study, provided guidance in its design and coordination. W.L.C, W.H. K. and H.D.H. helped to draft and revise the manuscript. All read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

Ethics approval and consent to participate

Not applicable.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Institute of Bioinformatics and Systems Biology, National Chiao Tung University
(2)
Department of Biological Science and Technology, National Chiao Tung University
(3)
Center for Bioinformatics Research, National Chiao Tung University
(4)
Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University

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Copyright

© The Author(s). 2017

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