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Exogenous plant mir168a specifically targets mammalian ldlrap1: evidence of cross-kingdom regulation by microrna

Cell Research (2012) 22:107-126.
2012 IBCB, SIBS, CAS All rights reserved 1001-0602/12 $ 32.00 Exogenous plant MIR168a specifically targets mammalian
LDLRAP1: evidence of cross-kingdom regulation by
Lin Zhang1, *, Dongxia Hou1, *, Xi Chen1, *, Donghai Li1, *, Lingyun Zhu1, 2, Yujing Zhang1, Jing Li1, Zhen Bian1, Xiangying Liang1, Xing Cai1, Yuan Yin1, Cheng Wang1, Tianfu Zhang1, Dihan Zhu1, Dianmu Zhang1, Jie Xu1, Qun Chen1, Yi Ba3, Jing Liu1, Qiang Wang1, Jianqun Chen1, Jin Wang1, Meng Wang1, Qipeng Zhang1, Junfeng Zhang1, Ke Zen1, Chen-Yu Zhang1 1Jiangsu Engineering Research Center for microRNA Biology and Biotechnology, State Key Laboratory of Pharmaceutical Bio-technology, School of Life Sciences, Nanjing University, 22 Hankou Road, Nanjing, Jiangsu 210093, China; 2Department of Chem-istry and Biology, School of Science, National University of Defense Technology, Changsha, Hunan 410073, China; 3Tianjin Medi-cal University Cancer Institute and Hospital, Huanhuxi Road, Tiyuanbei, Tianjin 300060, China Our previous studies have demonstrated that stable microRNAs (miRNAs) in mammalian serum and plasma are
actively secreted from tissues and cells and can serve as a novel class of biomarkers for diseases, and act as signaling
molecules in intercellular communication. Here, we report the surprising finding that exogenous plant miRNAs are
present in the sera and tissues of various animals and that these exogenous plant miRNAs are primarily acquired
orally, through food intake. MIR168a is abundant in rice and is one of the most highly enriched exogenous plant
miRNAs in the sera of Chinese subjects. Functional studies in vitro and in vivo demonstrated that MIR168a could
bind to the human/mouse low-density lipoprotein receptor adapter protein 1 (LDLRAP1) mRNA, inhibit LDLRAP1
expression in liver, and consequently decrease LDL removal from mouse plasma. These findings demonstrate that ex-
ogenous plant miRNAs in food can regulate the expression of target genes in mammals.
Keywords: microRNA; MIR168a; LDLRAP1; low-density lipoprotein; microvesicle; cross-kingdom
Cell Research (2012) 22:107-126. doi:10.1038/cr.2011.158; published online 20 September 2011
mune response, and the maintenance of cell and tissue identity [1, 2]. Dysregulation of miRNAs has been linked MicroRNAs (miRNAs), a class of 19-24 nucleotide to cancer and other diseases [3, 4]. Recently, we and oth- long non-coding RNAs derived from hairpin precursors, ers found that mammalian miRNAs exist stably in the mediate the post-transcriptional silencing of an estimated sera and plasma of humans and animals [5, 6]. Specific 30% of protein-coding genes in mammals by pairing serum miRNA expression profiles show a great potential with complementary sites in the 3′ untranslated regions to serve as a novel class of biomarkers for the diagnosis (UTRs) of target genes [1, 2]. miRNAs have been widely of cancer and other diseases [5, 6]. Numerous reports shown to modulate various critical biological processes, have subsequently shown that unique expression patterns including differentiation, apoptosis, proliferation, the im- of circulating miRNAs reflect various physiological and pathological conditions [7-12].
We next characterized the possible carrier of circulat- *These four authors contributed equally to this work.
ing miRNAs. Microvesicles (MVs) are small vesicles Correspondence: Chen-Yu Zhanga, Ke Zenb, Junfeng Zhangc that are shed from almost all cell types under both nor- mal and pathological conditions [13, 14]. They bear sur- bE-mail: [email protected] face receptors/ligands of the original cells and have the Received 11 August 2011; revised 23 August 2011; accepted 26 August potential to selectively interact with specific target cells 2011; published online 20 September 2011 and mediate intercellular communication by transporting npg Plant MIR168a downregulates mammalian LDLRAP1108 bioactive lipids, mRNA, or proteins between cells [13, was rice. Plant miRNAs were also detected in the sera 14]. Our recent results demonstrated that MVs from hu- of other animals, such as calves, whose diet was mainly man plasma are a mixture of microparticles, exosomes, green rich fodder (Supplementary information, Table and other vesicular structures and that many types of S2). The significant differences in plant miRNA profiles MVs in human plasma contain miRNAs [15]. These find- between human and calf sera are shown in Supplemen- ings were in agreement with the recent reports by other tary information, Figure S1B, with a Pearson's correla- investigators that exosomes from cultured cells served as tion coefficient (R) of 0.3951. Furthermore, although the physiological carriers of miRNAs [16, 17]. Our further number of plant miRNAs was about 5% of mammalian studies demonstrated that miRNAs could be selectively miRNAs in human and calf sera (Supplementary infor- packaged into MVs and actively delivered into recipient mation, Figure S1C and S1E), there were fewer absolute cells where the exogenous miRNAs can regulate target sequencing reads from the plant miRNAs (Supplementary gene expression and recipient cell function [15]. Thus, information, Figure S1D and S1F). To confirm the Sol- secreted miRNAs can serve as a novel class of signaling exa data, the levels of MIR168a and MIR156a, the two molecules in mediating intercellular communication [15]. plant miRNAs with the highest levels in the sera of Chi- The novel and important functions of the secreted miR- nese subjects, and MIR166a, a plant miRNA with modest NAs were also reported by many other groups [18-21]. level, were assessed by a stem-loop quantitative reverse The identification of circulating miRNAs, mainly deliv- transcription polymerase chain reaction (qRT-PCR) as- ered by cell-secreted MVs, as stable and active signaling say. MIR161, whose expression level was undetectable, molecules opens a new field of research in intercellular served as a negative control. First, the dynamic range and interorganelle signal transduction.
and sensitivity of the qRT-PCR assay for measuring plant In the present study, we were surprised to detect ex- miRNAs in serum were evaluated. A series of synthetic ogenous plant miRNAs in the serum and plasma of hu- plant miRNA oligonucleotides with known concentra- man and animals. Over half of plant miRNAs detected tions were reverse transcribed and amplified to generate in serum and plasma are present in MVs. Further in vitro a standard curve. As shown in Supplementary informa- and in vivo analysis demonstrated for the first time that tion, Figure S1G, these plant miRNAs had a linear semi- food-derived exogenous plant MIR168a can pass through logarithmic plot in a range from 1 fM to 104 fM. In other the mouse gastrointestinal (GI) track and enter the cir- words, plant miRNAs at a level as low as 1 fM can be culation and various organs especially the liver where efficiently examined by this assay. The absolute concen- it cross-kingdomly regulates mouse LDLRAP1 protein tration of each miRNA was then calculated according expression and physiological condition.
to the standard curve. As can be seen in Figure 1B, the tested plant miRNAs were clearly present in sera from humans, mice, and calves. Moreover, when compared to the endogenous mammalian miRNAs known to be Plant miRNAs are present in human and animal sera and stably present in animal serum [5, 15], these plant miR- NAs were relatively lower, but in a similar concentration Upon investigation of the global miRNA expres- range. The levels of plant miRNAs in various serum sion profile in human serum, we found that exogenous samples were about one-tenth of that of endogenous plant miRNAs were consistently present in the serum of miR-16 (Figure 1B, insert), one of the major endogenous healthy Chinese men and women. As shown in Figure miRNAs in animal serum [5, 6]. Furthermore, the pres- 1A and Supplementary information, Table S1, Solexa se- ence of plant miRNAs in human and animal serum was quencing revealed 30 known plant miRNAs in Chinese confirmed by semi-quantitative RT-PCR and northern healthy donors, among which MIR156a and MIR168a blotting (Figure 1C and 1D). To determine whether the showed considerable levels of expression (the mean Sol- MIR156a, MIR168a, and MIR166a identified in serum exa reads/total mammalian miRNAs > 0.005). A compar- are genuine plant miRNAs, we treated the total small ison of the Solexa reads of plant miRNAs between male RNA isolated from human serum with sodium perio- and female sera revealed no significant difference for the date (oxidizing agent). Plant miRNAs are 2′-O-methyl majority of plant miRNAs. As shown in the Pearson's modified on their terminal nucleotide, which renders correlation scatter plot comparing plant miRNAs in male them resistant to periodate [22]. In contrast, mammalian and female sera, the Pearson's correlation coefficient miRNAs with free 2′ and 3′ hydroxyls are sensitive to (R) was close to 1 (R = 0.9002; Supplementary informa- periodate. Since oxidized terminal nucleotides can not tion, Figure S1A). These healthy Chinese women and be ligated to the cloning adapter, a comparison of deep men had no metabolic dysfunctions, and their main diet sequencing libraries before and after oxidation allows Cell Research Vol 22 No 1 January 2012 Lin Zhang et al. npg www.cell-research.com Cell Research npg Plant MIR168a downregulates mammalian LDLRAP1110 Figure 1 Plant miRNAs are present in human and animal sera and organs. (A) The levels (Solexa reads) of 10 plant miRNAs
detected by Solexa sequencing in sera from healthy Chinese men and women, and eight pooled samples (each pooled from 10 healthy Chinese subjects). For normalization, the sequencing frequency of each plant miRNA was normalized to the total amount of mammalian miRNAs. (B) The absolute levels of plant miRNAs in the sera of various mammals detected by qRT-
PCR (n = 6). Endogenous animal miRNAs, miR-16, and miR-25 serve as controls (insert). (C) Semi-quantitative RT-PCR
analysis of the indicated miRNAs in the serum from human, mouse, rat, calf, horse, and sheep. Accurate amplification of each miRNA was confirmed by Sanger-based method to sequence the PCR products. (D) Northern blotting analysis of the
expression levels of MIR168a, MIR156a, and miR-16 in human serum (100 ml) and calf serum (40 ml). Synthetic MIR168a, MIR156a, and miR-16 (1 pmol) served as positive controls. (E) Equal amount of total small RNAs (each from 100 ml of hu-
man serum) were treated with/without sodium periodate. After the reactions, the RNAs were purified and then subjected to Solexa sequencing. Solexa reads of the plant miRNAs in oxidized and unoxidized groups were compared. Total and indi- vidual mammalian miRNAs were compared to serve as controls (insert). The absolute Solexa reads of miRNAs are indicated. (F) Equal amount of synthetic plant miRNAs (without 2′-O-methylated 3′ ends) and total small RNAs isolated from rice and
human serum were treated with/without sodium periodate. After the reactions, the RNAs were subjected to qRT-PCR with the miScript PCR system. Synthetic miR-16 and total small RNAs isolated from mouse liver and human serum were treated as above. MiR-16 expression levels with/without oxidation were compared to serve as controls (insert). (G) The levels of
plant miRNAs detected by qRT-PCR in MVs isolated from C57BL/6J mouse plasma (n = 4). (H) The levels of plant miRNAs
detected by Solexa sequencing in various organs of C57BL/6J mice. (I) The levels of plant miRNAs detected by qRT-PCR
in various organs of C57BL/6J mice (normalized to U6; n = 6). As before, miR-16 and miR-25 serve as controls (insert). (J,
K) The levels of plant miRNAs detected by Solexa sequencing (J) and qRT-PCR (K) in mouse liver after oxidation. Similarly,
mammalian miRNAs serve as controls (insert). The absolute Solexa reads of miRNAs are indicated.
assessing whether the small RNAs tested bear 2′-O- In contrast, MIR156a, MIR168a, and MIR166a in human methylated 3′ ends [23, 24]. Indeed, as shown in Figure serum remained unchanged with this treatment and were 1E, most mammalian miRNAs in human serum, such sequenced by Solexa, suggesting that they bear 2′-O- as miR-423-5p, miR-320a, miR-483-5p, miR-16, and methylated 3′ ends and are therefore genuine plant miR- miR-221, had an unmodified 2′, 3′ hydroxyls and were NAs. Moreover, the methylation status of small RNAs therefore oxidized and failed to be sequenced by Solexa. can be evaluated by qRT-PCR with miScript PCR system Cell Research Vol 22 No 1 January 2012 Lin Zhang et al. npg after treating the small RNAs with sodium periodate. and 0.66 fmol/g (miRNA level/weight of chow diet), This method blocks ligation of a poly(A) tail to small respectively. However, the levels of these three miRNAs RNAs bearing 2′,3′ hydroxy termini, preventing them were 6-, 10- and 3-fold higher, respectively, in fresh from being reverse transcribed and amplified. Consistent rice than in chow diet (Figure 2A). In contrast, mammal with the results of oxidized deep sequencing, MIR156a miRNAs were expressed in chow diet but were undetect- and MIR168a in rice and serum showed high levels of able in fresh rice (Figure 2A, insert). It is worth noting resistance to periodate and could be amplified, whereas that these three plant miRNAs, MIR168a, MIR156a, and miR-16 in liver and serum was oxidized and unable to be MIR166a, were detected in other foods, including Chi- detected (Figure 1F). We next determined the portion of nese cabbage (Brassica rapa pekinensis), wheat (Triticum circulating plant miRNAs in MVs compared to MV-free aestivum), and potato (Solanum tuberosum) (Supplemen- plasma. Similar to most endogenous miRNAs, such as tary information, Table S3). Interestingly, plant miRNAs miR-16, miR-21, and miR-150 (Figure 1G, insert), plant were stable in cooked foods (Supplementary information, MIR168a and MIR156a were primarily detected in MVs Table S3). The levels of MIR168a and MIR156a were in C57BL/6J mouse plasma (Figure 1G). Further study further assessed in the sera and tissues of mice fed with revealed that plant MIR168a and MIR156a were detected either a chow diet or fresh rice following 12 h of fasting. in various mouse tissues, including liver, small intestine, As shown in Figure 2B and 2C and Supplementary in- and lung (Figure 1H and 1I). Interestingly, MIR166a was formation, Figure S2C, the levels of plant MIR168a and nearly undetectable in various mouse tissues, although it MIR156a were not elevated in the serum or liver at 0.5, 3, was present in the serum. In these studies, the levels of and 6 h after chow diet feeding. In contrast, the levels of miRNAs were normalized to U6 snRNA and other small these two miRNAs were significantly increased in both RNAs such as snoRNA146 and snoRNA251. As shown the sera and the livers of mice fed with fresh rice for 6 h in Supplementary information, Figure S2A, the expres- (Figure 2B and 2C and Supplementary information, Fig- sion levels of U6 and small RNAs were approximately ure S2C). We further compared the levels of MIR168a equivalent between different mouse tissues. Moreover, in perfused and non-perfused organs after rice feeding no plant pre-miRNA was detected by semi-quantitative and found no difference (Supplementary information, RT-PCR in human and animal sera and tissues (Supple- Figure S2D), which indicates that the elevation of plant mentary information, Figure S2B). The presence of miRNAs in mouse organs was not due to contamination genuine plant miRNAs in mouse tissues was further by blood cells. In agreement with the hypothesis that demonstrated by oxidized deep sequencing and validated exogenous plant miRNAs are derived from food intake, by qRT-PCR with miScript PCR system. As shown in the levels of MIR168a were increased in the stomach and Figure 1J and 1K, mammalian miRNAs in mouse liver small intestine, but not in the kidney, of mice following were oxidized and undetectable by both Solexa sequenc- feeding with a chow diet or fresh rice (Supplementary ing and qRT-PCR assay with miScript PCR system. In information, Figure S2E-S2G). Furthermore, when mice contrast, MIR168a and MIR156a in mouse liver were were gavage fed with total RNA extracted from fresh resistant to oxidation by periodate.
rice, the levels of MIR168a in mouse serum and liver were elevated 3 h after feeding (Figure 2D and 2E). Ad- The exogenous mature plant miRNAs in food can pass ditionally, mice were gavage fed with synthetic MIR168a through mouse GI tract and enter the sera and organs (single-stranded mature MIR168a) or synthetic methy- Next, we sought to identify the source of these exog- lated MIR168a because it has been reported that plant enous plant miRNAs in the sera and organs of mammals. miRNAs are methylated [27]. The levels of MIR168a in Given that plant MIR168a and MIR156a have been mouse serum and liver were elevated 3 h after feeding reported to be enriched in various plants, including rice (Figure 2F and 2G). To identify the form in which MI- (Oryza sativa) and crucifers (Brassicaceae) [25, 26], R168a in a food such as rice was taken up by C57BL/6J and that rice is the main stable food in China, we hy- mice, mice were gavage fed with various forms of MI- pothesized that plant miRNAs in the sera and tissues of R168a, including double-stranded miRNA (dsMIR168a), humans and animals are primarily a result of food intake. precursor miRNA (pre-MIR168a), single-stranded DNA To test this hypothesis, the expression levels and stabil- miRNA (ssDNA-MIR168a), and DNA precursor miRNA ity of plant miRNAs in various foods were investigated. (pre-DNA-MIR168a), and the level of each form of MI- As shown in Supplementary information, Table S3, R168a in mouse serum was assessed after 6 h. Interest- plant MIR168a, MIR156a, and MIR166a were detected ingly, we only detected single-stranded mature MIR168a in chow diet (a mouse diet with a relatively low fat and by qRT-PCR (Supplementary information, Figure S2H- cholesterol content) in the concentrations of 0.43, 0.54 S2K), strongly suggesting that food-derived MIR168a is www.cell-research.com Cell Research npg Plant MIR168a downregulates mammalian LDLRAP1112 Figure 2 The exogenous mature plant miRNAs in food can pass through gastrointestinal (GI) tract and enter the sera and or-
gans. (A) The levels of MIR168a, MIR156a, and MIR166a detected by qRT-PCR in fresh rice and chow diet (n = 6). Two en-
dogenous animal miRNAs, miR-16, and miR-150, served as controls (insert). UD, undetectable. (B, C) The levels of MIR168a
in mouse serum (B) and liver (C) after feeding with fresh rice or chow diet for 0.5 h, 3 h, or 6 h (n = 8). The control group (named
0 h) was euthanized after a 12-h of fasting. (D, E) The levels of MIR168a in mouse serum (D) and liver (E) following gavage
feeding with RNA extracted from fresh rice (n = 8). After 0.5 h, 3 h, or 6 h, MIR168a levels were detected by qRT-PCR. The control group (0 h) was euthanized after a 12-h of fasting. (F, G) The levels of MIR168a in mouse serum (F) and liver (G) fol-
lowing gavage feeding with synthetic MIR168a and synthetic methylated MIR168a (n = 8). The control group was gavage fed with ncRNA. *P < 0.05; **P < 0.01.
taken up in its mature form. To mimic GI tract environ- 4.0-fold induction of secreted endogenous monocytic ment, the effect of acidification on the stability of plant miR-150 in the plasma of patients with atherosclerosis miRNAs and mammalian miRNAs was examined. Total could sufficiently decrease the protein level of its target RNA isolated from rice or mouse liver was adjusted to gene, c-Myb, and increase cell migration in recipient en- pH 2.0 and kept at 37 °C for several hours. As shown in dothelial cells [15]. Given that structurally functional ex- Supplementary information, Figure S2L, acidification did ogenous plant miRNAs can enter human and animal sera not significantly affect the yield and quality of miRNAs. and tissues through food intake and can be maintained The majority of plant miRNAs and mammalian miRNAs at elevated levels, we hypothesized that they may play a can survive under acidic condition for at least 6 h. Inter- role in regulating the functions of mammalian cells and estingly, the degradation rate of mammalian miRNAs un- organs. Because most plant miRNAs can act like RNA der acidic condition was similar to that of their synthetic interference (RNAi), which requires a high degree of form (without 2′-O-methylated 3′ ends), whereas plant complementarity between miRNAs and target RNAs [28], miRNAs had a much slower degradation rate compared we performed bioinformatic analysis to identify any se- with their synthetic form (without 2′-O-methylated 3′ quences in the human, mouse, or rat genome with perfect ends), suggesting that methylation had a protective effect or near-perfect match to MIR168a. Approximately 50 on the stability of plant miRNAs.
putative target genes were identified as the target genes of MIR168a (Supplementary information, Table S4). Plant MIR168a binds to exon 4 of mammalian LDLRAP1 The most highly conserved sequence of a putative bind- and decreases LDLRAP1 protein level in vitro ing site among various species is located in exon 4 of In a previous study, it was demonstrated that a 2.5- the low-density lipoprotein receptor adapter protein 1 Cell Research Vol 22 No 1 January 2012 Lin Zhang et al. npg (LDLRAP1) (Figure 3A). We calculated the minimum phenomenon, the LDLRAP1 ORF was GFP tagged at free energy of binding to be −29.2 kcal/mol. LDLRAP1 its carboxyl terminus (Figure 3L). Compared to cells is a liver-enriched gene that plays a critical role in facili- transfected with ncRNA, the number of cells expressing tating the removal of LDL from the circulatory system GFP-tagged LDLRAP1 was drastically decreased in cells [29]. As shown in Figure 3B, transfection of HepG2 transfected with pre-MIR168a (Figure 3M and 3N).
cells (a hepatocyte carcinoma cell line) with MIR168a Although overexpression of pre-miRNA is the con- precursor (pre-MIR168a) resulted in a 1 000-fold eleva- ventional method to assess the function of a miRNA, we tion in MIR168a, suggesting that plant pre-MIR168a determined the direct effect of a single-stranded mature can be properly processed in mammalian HepG2 cells. plant MIR168a on LDLRAP1 expression in HepG2 cells Additionally, the LDLRAP1 protein level in these cells for two reasons. First, neither plant pri-miRNA nor plant was significantly reduced (Figure 3C and 3D), whereas pre-miRNA has been detected in the sera and tissues the LDLRAP1 mRNA level was not affected (Figure of human and animals. Second, and more importantly, 3E and 3F). A luciferase reporter assay was employed single-stranded mature MIR168a is likely to be the form to demonstrate the direct binding of MIR168a to exon 4 taken up by animals via the GI tract. By transfecting of LDLRAP1. The same strategy has been used by oth- HepG2 cells with equivalent amounts of single-stranded ers in studying miRNA targeting in coding regions [30- mature plant MIR168a, we increased the level of mature 32]. In this experiment, the wild-type (WT) or mutant MIR168a in HepG2 cells by 10 000-fold (Supplemen- (MUT) MIR168a complementary site (CS), WT or MUT tary information, Figure 3C) and observed a concomitant LDLRAP1 binding site (BS), the human LDLRAP1 reduction in LDLRAP1 (Supplementary information, exon 4, and human LDLRAP1 coding sequence (CDS) Figure S3D and S3E). Similar to the results from trans- were cloned into a luciferase reporter plasmid (Figure fection with pre-MIR168a, the overexpression of single- 3G) and transfected into HepG2 cells combined with stranded mature MIR168a had no effect on LDLRAP1 pre-MIR168a. As expected, luciferase reporter activity mRNA levels (Supplementary information, Figure S3F for WT was significantly reduced following transfection and S3G). A luciferase reporter assay also demonstrated with pre-MIR168a, whereas the MUT luciferase reporter that high levels of mature MIR168a decreased luciferase was unaffected by transfection with pre-MIR168a (Fig- activity (Supplementary information, Figure S3H), in ure 3H). Because the predicted MIR168a binding site is agreement with the observation that mature MIR168a not in the 3′ UTR but in the open reading frame (ORF) could affect LDLRAP1 expression despite a relatively of LDLRAP1, we further compared the efficiency of our low affinity for the putative binding site of LDLRAP1.
luciferase reporter assay with those of typical reporters in which the endogenous miRNAs bind to the 3′ UTR AGO2-associated mature MIR168a in Caco-2 cel -derived of target genes. We found that the rate of the inhibition MVs sufficiently reduces mammalian LDLRAP1 protein of luciferase activity by MIR168a was dependent on level in recipient HepG2 cells the ratio of pre-MIR168a to BS-containing luciferase In this study, we hypothesized that epithelial cells, par- reporter (Supplementary information, Figure S3A) and ticularly in the small intestine, might take up plant miR- that the efficiency of MIR168a inhibition of LDLRAP1 NAs in food, then package them into MVs and release expression was similar to the inhibition of target genes them into the circulatory system. The secreted MVs from by endogenous miRNAs (miR-16, miR-21, and miR- small intestinal epithelial cells could then deliver exog- 150; Supplementary information, Figure S3B). To deter- enous plant miRNA to target cells of other organs and mine whether MIR168a could directly target LDLRAP1 regulate recipient cell function. To test this hypothesis, within its CDS, we followed the strategy proposed by human intestinal epithelial Caco-2 cells were transfected others [30-32] and created a construct that expressed the with single-stranded mature MIR168a. As indicated by full-length ORF of LDLRAP1 (Figure 3I). Furthermore, Figure 4A, the MVs released by the Caco-2 cells were we used site-directed mutagenesis to create a variant of collected and used to treat HepG2 cells. The LDLRAP1 the LDLRAP1 construct in which the MIR168a target protein level in HepG2 cells was then assessed. As site was mutated while leaving the LDLRAP1 amino- shown in Figure 4B, the levels of mature MIR168a were acid sequence intact (Figure 3I). These constructs were elevated by 200-fold in MVs released by Caco-2 cells co-transfected with pre-MIR168a or pre-ncRNA into transfected with MIR168a and by 100-fold in Caco-2 293T cells. WT LDLRAP1 was strongly downregulated MV-treated HepG2 cells. Plant MIR168a delivered into by MIR168a, whereas MIR168a had no effect on the HepG2 cells via Caco-2 MVs significantly decreased expression of LDLRAP1 when the MIR168a target site the LDLRAP1 protein level in the recipient HepG2 was mutated (Figure 3J and 3K). To further explore this cells (Figure 4C and 4D), while it had no effect on LD- www.cell-research.com Cell Research npg Plant MIR168a downregulates mammalian LDLRAP1114 Cell Research Vol 22 No 1 January 2012 Lin Zhang et al. npg Figure 3 Plant MIR168a binds to exon 4 of mammalian LDLRAP1 and decreases LDLRAP1 protein level in vitro. (A) Sche-
matic description of the hypothesized duplexes formed by interactions between the exon 4 of LDLRAP1 and MIR168a. Paired bases are indicated by a black oval and G:U pairs are indicated by two dots. The predicted free energy of the hybrid is indi- cated. Note that the potential binding site of MIR168a to LDLRAP1 mRNA is highly conserved across species. (B) qRT-PCR
analysis of MIR168a levels in pre-MIR168a-transfected HepG2 cells (n = 9). (C) Western blot analysis of LDLRAP1 protein
levels in pre-MIR168a-transfected HepG2 cells. (D) The quantification of the LDLRAP1 protein expression in C (n = 9). (E, F)
Semi-quantitative RT-PCR (E) and qRT-PCR (F) analysis of LDLRAP1 mRNA levels in pre-MIR168a-transfected HepG2 cells (n
= 5). (G) Diagram of the luciferase reporter plasmid carrying the firefly luciferase-coding sequence attached with the wild-type
(WT) or mutant (MUT) MIR168a complementary site (CS), WT, or MUT LDLRAP1 BS, LDLRAP1 exon 4, and the LDLRAP1 CDS. (H) Luciferase activities in HepG2 cells co-transfected with luciferase reporters described in G and pre-MIR168a or pre-
ncRNA (n = 9). (I) Constructs for the expression of the WT or MUT ORFs of LDLRAP1. (J) LDLRAP1 level in 293T cells co-
transfected with WT or MUT LDLRAP1 ORFs and pre-MIR168a or pre-ncRNA. (K) The quantification of the LDLRAP1 protein
expression in J (n = 3). (L) The LDLRAP1 ORF tagged with GFP at its carboxyl terminus. (M) 293T cells were co-transfected
with the GFP-tagged LDLRAP1 ORF (top) and pre-MIR168a or pre-ncRNA. GFP-positive cells were analyzed by fluores- cence microscopy at 24 h of post-transfection. (N) The percentage of GFP-positive cells in M (n = 6). *P < 0.05; **P < 0.01.
LRAP1 mRNA levels (Figure 4E and 4F). Interestingly, (Figure 4M and 4N). The association of MIR168a with repression of LDLRAP1 protein levels in HepG2 cells AGO2 was additionally detected in HepG2 cells directly by Caco-2 MVs was dose dependent, as Caco-2 MVs transfected with pre-MIR168a or mature MIR168a (Sup- containing more MIR168a decreased the expression of plementary information, Figure S3I and S3J). In separate LDLRAP1 more effectively (Figure 4G-4I). A luciferase experiments, we tested whether plant miRNAs can be assay further demonstrated that plant MIR168a secreted processed and packaged into secreted MVs by cell types by Caco-2 cells could effectively enter HepG2 cells other than colonic epithelial Caco-2 cells. As can be and recognize exon 4 of the LDLRAP1 transcripts. WT seen in Supplementary information, Figure S4, similar to luciferase reporter activity was significantly reduced fol- Caco-2 cells, 293T cells were able to package MIR168a lowing incubation with MVs derived from Caco-2 cells into MVs and then deliver MIR168a into HepG2 cells.
transfected with mature MIR168a, whereas the MUT luciferase reporter was unaffected (Figure 4J). In 293T Anti-MIR168a ASO reverses rice feeding-induced reduc- cells transfected with WT LDLRAP1 ORF, treatment tion of mouse liver LDLRAP1 protein at 6 h feeding with MIR168a-containing Caco-2 MVs but not ncRNA- In order to determine the specificity of the inhibitory containing Caco-2 MVs significantly reduced the level of effect of MIR168a on LDLRAP1 and exclude the possi- LDLRAP1 expression (Figure 4K and 4L). In contrast, bility that the reduction of LDLRAP1 protein was caused MIR168a-containing Caco-2 MVs had no effect on the by factors other than MIR168a, an anti-MIR168a anti- expression of the MUT LDLRAP1 ORF (Figure 4K and sense oligonucleotide (ASO) was injected intravenously 4L). These results clearly indicate a possible mechanism into mice during rice feeding. The injection of anti- for the action of exogenous plant miRNAs in an animal MIR168a ASO not only significantly reduced MIR168a model. Moreover, because Argonaute2 (AGO2) is present levels in the livers of fresh rice-fed mice (Figure 5A), in MVs and facilitates miRNA binding to its target gene but also remarkably reversed the reduction of mouse via the RNA-induced silencing complex (RISC) [33], we liver LDLRAP1 by fresh rice-derived MIR168a (Figure performed an immunoprecipitation experiment to assess 5B and 5C). Taken together, these results indicate that whether plant MIR168a was associated with mammalian exogenous plant miRNAs are able to enter the serum AGO2 and LDLRAP1. As can be seen in Figure 4M-4O, and organs of mammals via food intake, and that plant both MIR168a (Figure 4N) and LDLRAP1 mRNA (Fig- MIR168a can bind to the nucleotide sequence located in ure 4O) were detected in the product precipitated by an exon 4 of mammalian LDLRAP1, leading to the inhibi- anti-AGO2 antibody from the HepG2 cells treated with tion of LDLRAP1 expression in vivo.
Caco-2 MVs, while only MIR168a (Figure 4M) but not LDLRAP1 mRNA (Figure 4O) was detected in the anti- Exogenous MIR168a inhibits mouse liver LDLRAP1 ex- AGO2 antibody-precipitated product from the Caco-2 pression and elevates plasma LDL-cholesterol level at 3 MVs. As a control, endogenous mammalian miR-16 was days after food intake detected with the anti-AGO2 antibody-precipitated prod- LDL is the major cholesterol-carrying lipoprotein of uct from both Caco-2 MVs and MV-treated HepG2 cells, human plasma and plays an essential role in the patho- but its level was not affected by MIR168a transfection genesis of atherosclerosis [34]. Downregulation of www.cell-research.com Cell Research npg Plant MIR168a downregulates mammalian LDLRAP1116 Cell Research Vol 22 No 1 January 2012 Lin Zhang et al. npg Figure 4 AGO2-associated mature MIR168a in Caco-2 cell-derived MVs sufficiently reduces mammalian LDLRAP1 protein
level in recipient HepG2 cells. (A) A flow chart of the experimental design. (B) The elevation of MIR168a in Caco-2 MVs after
transfection with mature MIR168a (left) and in HepG2 cells after treatment with Caco-2 MVs (right; n = 9). Caco-2 MVs were harvested after transfecting the cells with mature MIR168a or ncRNA. (C) Western blot analysis of LDLRAP1 protein lev-
els in HepG2 cells treated with or without Caco-2 MVs. Caco-2 MVs were harvested after transfecting the cells with mature MIR168a or ncRNA. (D) The quantification of the LDLRAP1 protein expression in C (n = 6). (E, F) Semi-quantitative RT-PCR
(E) and qRT-PCR (F) analysis of the LDLRAP1 mRNA levels in HepG2 cells treated with or without Caco-2 MVs (n = 5). (G,
H) The levels of MIR168a (G) and LDLRAP1 protein (H) in HepG2 cells treated with Caco-2 MVs derived from Caco-2 cells
transfected with different doses (10, 50, or 100 pmol/105 cells) of mature MIR168a or ncRNA. (I) The quantification of the
LDLRAP1 level in H (n = 3). (J) The luciferase activities in luciferase reporter-transfected HepG2 cells treated with or without
Caco-2 MVs (n = 9). (K) LDLRAP1 protein level in 293T cells transfected with WT or MUT LDLRAP1 ORF and then treated
with Caco-2 MVs. (L) The quantification of the LDLRAP1 protein expression in K (n = 3). (M, N) The association of MIR168a
with AGO2 in Caco-2 MVs (M) and HepG2 cells treated with Caco-2 MVs (N). The levels of MIR168a and miR-16 (control) in
anti-AGO2 immunoprecipitated products detected by qRT-PCR (n = 9). (O) The association of LDLRAP1 mRNA with AGO2
in Caco-2 MV-treated HepG2 cells or Caco-2 MVs. The LDLRAP1 mRNA in anti-AGO2 immunoprecipitated products from HepG2 cells (lanes 1 and 2) and Caco-2 MVs (lanes 3 and 4) was detected by semi-quantitative RT-PCR with 25-30 cycles. *P < 0.05; **P < 0.01.
LDLRAP1 in the liver causes decreased endocytosis of the chow diet group (Figure 6C). Likewise, levels of LDL by liver cells and impairs the removal of LDL from MIR168a in the liver were increased in fresh rice-fed plasma [35, 36]. To assess the physiological function mice compared to chow diet-fed mice after 1 day (Figure of food-enriched MIR168a in mammals, mice were fed 6D). Supplementary information, Figure S5A to S5C with fresh rice and chow diets for 7 days after a 12-h of shows the level of MIR168a in other mouse organs after fasting. Body weight was not different between the two a long period of feeding. Although miR168a was ini- groups of mice during the experimental period (Figure tially increased in the stomach of mice fed with rice, no 6A), although the fresh rice-fed group had an increased significant difference in MIR168a levels in the stomach, food intake (Figure 6B). In addition, the serum MIR168a intestine, or kidney were found between chow diet- and levels of the chow diet-fed group were not significantly rice-fed mice. Concomitant with a significant elevation in altered during the experimental period (Figure 6C). MIR168a levels in the livers of mice after 1 day of fresh In contrast, there was a significant induction of serum rice feeding (Figure 6D), LDLRAP1 expression dramati- MIR168a levels in mice after 1 day of feeding with fresh cally decreased in the group of fresh rice-fed mice (Figure rice, and these levels remained elevated over those of 6E and 6F). In these experiments, LDLRAP1 levels in Figure 5 Anti-MIR168a ASO reverses rice feeding-induced reduction of mouse liver LDLRAP1 protein at 6 h feeding. (A, B)
The levels of MIR168a (A) and LDLRAP1 protein (B) in mouse liver after feeding with chow diet, fresh rice, or fresh rice ac-
companying an intravenous injection of anti-MIR168a ASO or anti-ncRNA for 6 h (n = 8). (C) The quantification of the LDL-
RAP1 level in B (n = 8). *P < 0.05; **P < 0.01.
www.cell-research.com Cell Research npg Plant MIR168a downregulates mammalian LDLRAP1118 Cell Research Vol 22 No 1 January 2012 Lin Zhang et al. npg Figure 6 Exogenous MIR168a inhibits mouse liver LDLRAP1 expression and elevates plasma LDL-cholesterol level at 3
days after food intake. (A) The weight changes of mice fed with chow diet or fresh rice (n = 8). (B) The food intake of chow
diet or fresh rice (n = 8). (C, D) The levels of MIR168a in mouse sera (C) and livers (D) after chow diet or fresh rice feeding (n
= 8). (E) The LDLRAP1 protein levels in mouse livers after chow diet or fresh rice feeding. (F) The quantification of LDLRAP1
protein expression in E (n = 8). (G) The levels of LDL-cholesterol in mouse plasma after chow diet or fresh rice feeding (n = 8).
(H, I) The levels of MIR168a (H) and LDLRAP1 protein (I) in mouse livers after 3 days of feeding with chow diet, fresh rice,
or fresh rice accompanying an injection of anti-MIR168a ASO or anti-ncRNA (n = 8). (J) The quantification of LDLRAP1 pro-
tein expression in I (n = 8). (K) The levels of LDL-cholesterol in mouse plasma after 3 days of feeding with chow diet, fresh
rice, or fresh rice accompanying an injection of anti-MIR168a ASO or anti-ncRNA (n = 8). (L) The association of MIR168a
with AGO2 in mouse livers after 3 days of feeding with chow diet, fresh rice, or fresh rice plus injection of anti-MIR168a ASO or anti-ncRNA. The levels of MIR168a and miR-16 (control) in anti-AGO2 immunoprecipitated products were detected by qRT-PCR. (M) The association of LDLRAP1 mRNA with AGO2 in mouse livers after 3-day feeding. The LDLRAP1 mRNA in
anti-AGO2 immunoprecipitated products was detected by semi-quantitative RT-PCR. (N, O) The levels of MIR168a (N) and
LDLRAP1 protein (O) in mouse livers after 3 days of feeding with chow diet and mature MIR168a or ncRNA (n = 8). (P) The
quantification of LDLRAP1 protein expression in O (n = 8). (Q) The levels of LDL-cholesterol in mouse plasma after 3 days of
feeding with chow diet and mature MIR168a or ncRNA (n = 8). *P < 0.05; **P < 0.01.
the control group (0 d) were obtained from mice that had creased compared to those in chow diet-fed mice and this undergone a 12-h period of fasting. It has been found rice feeding-induced elevation of liver AGO2-associated that fasting decreases LDLRAP1 expression in mouse MIR168a and LDLRAP1 mRNA could be blocked by livers, and re-feeding was sufficient to rescue this reduc- administration of anti-MIR168a ASO. Furthermore, by tion (Supplementary information, Figure S5D and S5E). adding mature MIR168a into chow diet and feeding In agreement with the reduction of LDLRAP1 in the mice, we found that the MIR168a levels in chow diet mouse liver, LDL levels in mouse plasma were signifi- (Supplementary information, Figure S5F), sera (Supple- cantly elevated on days 3 and 7 after fresh rice feeding mentary information, Figure S5G) and mouse liver (Fig- (Figure 6G). The rice-induced elevation of mouse liver ure 6N) were increased but the LDLRAP1 protein level MIR168a (Figure 6H) and, more importantly, MIR168a- in the mouse liver (Figure 6O and 6P) was decreased. mediated reduction of liver LDLRAP1 (Figure 6I and In contrast, there is no difference of MIR156a levels 6J) was largely blocked by the administration of anti- between the mice after chow diet feeding with addition MIR168a ASO but not negative control oligonucleotides. of MIR168a or ncRNA (Supplementary information, Consistently, the rice-induced elevation of LDL levels in Figure S5G). Additionally, decreased liver LDLRAP1 mouse plasma was blocked by the anti-MIR168a ASO levels led to an elevation in the mouse plasma LDL level (Figure 6K). Furthermore, the association of MIR168a (Figure 6Q). In separate experiments, we tested the link with LDLRAP1 mRNA through AGO2 in mouse liver between the downregulation of mouse liver LDLRAP1 was tested. For this experiment, AGO2 protein in mouse and the elevation of mouse plasma LDL level by directly liver following different treatment was immunoprecipi- decreasing liver LDLRAP1 by administration of LDL- tated by anti-AGO2 antibody and the levels of MIR168a RAP1 siRNA. Downregulation of liver LDLRAP1 via (Figure 6L) and LDLRAP1 mRNA (Figure 6M) in anti- LDLRAP1 siRNA (Supplementary information, Figure AGO2 antibody IP product were assessed. As can be S6A and S6B) resulted in the elevation of plasma LDL seen, in rice-fed mice, AGO2-associated MIR168a and level (Supplementary information, Figure S6C), sug- LDLRAP1 mRNA in mouse livers were significantly in- gesting that LDLRAP1 is responsible for plasma LDL www.cell-research.com Cell Research npg Plant MIR168a downregulates mammalian LDLRAP1120 clearance. However, the level of liver LDLRAP1 was not did not significantly affect the stability of plant miR- related to the levels of plasma cholesterol or triglycer- NAs, and that methylation may have a protective effect ides (Supplementary information, Figure S6D and S6E). on plant miRNAs (Supplementary information, Figure Decreased levels of plasma cholesterol (Supplementary S2L). Interestingly, pre-MIR168a and pre-MIR156a can information, Figure S7A) but unchanged plasma ApoA be detected in fresh rice but not in either cooked rice (Supplementary information, Figure S7B) and triglycer- or mouse serum (Supplementary information, Figure ide levels (Supplementary information, Figure S7C) fur- S2B), implicating that these pre-miRNAs may not be ther supported the hypothesis that the elevation of fresh stable and able to pass through GI tract. In support of rice-derived MIR168a in the mouse liver specifically this, the gavage feeding experiment using equal amount decreased liver LDLRAP1 expression and thus caused of single-stranded miRNA (ssRNA), double-stranded an elevated LDL level in mouse plasma. Interestingly, miRNA (dsRNA), precursor miRNA (preRNA), single- no significant differences in the levels of plasma cho- stranded DNA form of miRNA (ssDNA), and DNA form lesterol (Supplementary information, Figure S7D) and of precursor miRNA (preDNA) clearly showed that only triglycerides (Supplementary information, Figure S7E) mature miRNA was detected in mouse serum and tis- were observed in mice after 3 days of feeding with chow sues while other forms of RNA/DNA were not detected diet, fresh rice, or fresh rice accompanying an injection (probably degraded; Supplementary information, Figure of anti-MIR168a or anti-ncRNA oligonucleotides. As a S2H-S2K). The exclusive presence of mature MIR168a control, mature mammalian miR-150 was added into the in mouse serum and tissues maybe largely dependent on chow diet to feed mice. In a similar fashion, miR-150 its high stability compared to other forms of RNA/DNA levels in mouse liver (Supplementary information, Fig- in mouse GI to serum uptake pathway, although it cannot ure S7F) and serum (Supplementary information, Figure rule out the possibility that there is a specific pathway for S7G) were significantly increased after 3 days of feed- mature miRNA uptake. Our results clearly demonstrate ing, leading to downregulation of liver c-Myb expression that exogenous plant mature miRNAs in food can pass (Supplementary information, Figure S7H).
through the GI tract and be transferred into the blood- stream and tissues. To our knowledge, this is the first re- port indicating that nucleotides with complete functional structure are resistant to digestion in the GI tract and can Mature single-stranded plant miRNAs are stable be delivered to other tissues.
Our previous study demonstrated that mature single- stranded mammalian miRNAs are stably present in Plant miRNAs execute their function in mammalian cells serum or plasma of human and other animals [5] and in a fashion of mammalian miRNA that the circulating miRNAs can serve both as a class Generally, mammalian miRNAs execute its func- of biomarkers of diseases [8, 12] and as active signal tion by base pairing to the complementary sites in the molecules [15]. In the present study, we have detected 3′ UTR of its target genes, thus blocking the translation stable mature single-stranded plant miRNAs in serum or triggering the degradation of the target mRNAs [1, and tissues of mammals using both Solexa sequencing 2]. There are only a few reports showing that mamma- and qRT-PCR (Figure 1A, 1B, 1H and 1I). By perform- lian miRNA can also bind to exon of the target genes ing Solexa sequencing and qRT-PCR after oxidation of [30-32]. Unlike mammalian miRNAs, it is well known small RNAs with periodate, we have found that the plant that plant miRNAs act through RNAi, which requires a miRNAs identified in serum and tissues showed high complete match of their nucleotide sequence with that of levels of resistance to periodate, demonstrating that they their target genes [28]. Plant MIR168 has been reported bear 2′-O-methylated 3′ ends and are therefore genu- to be expressed widely in various species of plant [25, ine plant miRNAs (Figure 1E, 1F, 1J and 1K). Given 26]. In plants, expression of ARGONAUTE1 (AGO1), the fact that plant miRNAs are completely exogenous, the catalytic subunit of the RISC responsible for post- plant miRNAs detected in serum and organs of mam- transcriptional gene silencing, is controlled through a mals should only come from food intake. Food-derived feedback loop involving the MIR168 [37-39]. Here, our mature plant miRNAs such as MIR168a, which can be data showed that MIR168a decreased the LDLRAP1 detected even after cooking the food (Supplementary protein level but did not affect the mRNA level (Figure information, Table S3), are quite stable in animal serum 3C-3F), suggesting that plant MIR168a regulates mam- (Figure 1A-1D), suggesting that they may be also resis- malian LDLRAP1 translation in a fashion of mammalian tant to enzymatic digest in GI. Indeed, we have shown functional miRNA. Results of luciferase reporter assays that acidification mimicking the GI tract environment using multiple plasmids including the plasmid contain- Cell Research Vol 22 No 1 January 2012 Lin Zhang et al. npg ing partial or whole MIR168a complementary site, results of association of MIR168a with AGO2 in MVs, LDLRAP1 BS, human LDLRAP1 exon 4, and human as well as the association of AGO2 with MIR168a and LDLRAP1 CDS (Figure 3G and 3H) and the results that LDLRAP mRNA in HepG2 cells treated with Caco-2 MIR168a was also able to target the artificially expressed cell MVs (Figure 4M-4O) strongly argue that exogenous LDLRAP1 protein in 293T cells (Figure 3I-3K) strongly plant miRNAs cross-kindomly regulate mammalian gene demonstrate that plant MIR168a could bind to its binding through a MV-mediated pathway. Previous study showed site located in exon 4 of mammalian LDLRAP1 gene, that mature miRNA had a low affinity to AGO2 [40], and then inhibit LDLRAP1 protein expression.
which might provide an explanation why an extreme el- evation of mature MIR168a via the direct transfection of AGO2-associated mature MIR168a in MVs is the func- MIR168a was required to reduce LDLRAP1 expression.
tional form in vivo Endogenous mammalian miRNAs have a dynamic Although a very large amount of single-stranded range of expression, extending from < 1 copy per cell mature plant MIR168a can affect LDLRAP1 levels in to > 10 000 copies per cell, and the regulation of target HepG2 cells (Supplementary information, Figure S3C- genes is highly dependent on miRNA expression levels S3H), it is unlikely that such high concentrations of [41]. It has been shown that miRNAs expressed at low mature plant miRNAs can be achieved in serum, plasma levels (< 100 copies per cell) did not significantly repress and organs of humans or animals via food intake. Our target-containing transcripts [42], since target encounter study here suggested that plant MIR168a derived from occurs by mass action and modestly expressed miR- food intake might execute its function in mouse through NAs have a low probability to meet target-containing a novel pathway. We previously demonstrated that mam- transcripts. Previous measurements of miRNAs in liver malian cells could selectively pack miRNAs into MVs samples revealed that let-7a, miR-16, miR-20, miR-21, in response to different stimuli and then secrete these and miR-22 were present at 700, 3 890, 130, 4 450, and MVs into the circulation of animals or culture medium 310 copies per cell, respectively [43], while miR-181a [15]. Cell-derived MVs could further efficiently deliver was expressed at 810 copies per cell in DP thymocytes miRNAs into the recipient cells where these exogenous [44] and miR-223 and miR-451 were 828 and 1 972 miRNAs regulate the expression of target genes and the copies per cell in CD34(+)/CD133(−) cells [45]. It has biological functions of recipient cells [15]. In this study, been widely demonstrated that miRNAs expressed at we hypothesized that intestinal epithelial cells could take these concentrations are biologically active [46-49]. We up plant miRNAs in food, then pack them into MVs and calculated the amount of MIR168a that was detected in release the miRNA-containing MVs into the circulatory 1 mg of total liver RNA. The amount of MIR168a was system. These MVs secreted from intestinal epithelial 3.2 × 10−6 fmol (1 920 copies) per 100 pg of total RNA, cells would then deliver exogenous plant miRNAs into equivalent to 853 copies per cell. This quantification other organs and regulate the function of recipient cells. revealed that MIR168a is expressed in a similar concen- Supporting this hypothesis, more than half of the plant tration range in liver cells as compared with endogenous MIR168a in the serum was found in MVs (Figure 1G). mammalian miRNAs. Besides the concentration of the The results also showed that MVs contained much less miRNAs, the extent of suppression depends on a number amount of MIR168a but caused stronger effect on LD- of factors, such as the concentration of AGO2-associated LRAP1 level than ‘free' MIR168a did (Figure 4B-4D). miRNAs and the complementarity between the miRNA The efficiency of MV-contained MIR168a was even and its target site [41]. We have identified near-perfect higher than that of transfected pre-MIR168a (Figure complementarity between MIR168a and LDLRAP1 3C and 3D). These results indicated that MIR168a in mRNA. More importantly, we have shown that exog- MVs was more functionally active compared to ‘free' enous plant MIR168a in recipient mammalian liver cells MIR168a. The immunoprecipitation experiments in both is AGO2 associated (Figure 6L and 6M), which repre- MVs and MV-treated cells clearly showed that MIR168a sents an enriched fraction of active and functional miR- was already associated with AGO2 in the MVs derived NAs. Thus, MIR168a-targeting LDLRAP1 fulfills the from Caco-2 cells (Figure 4M), from which we may requirement for a threshold miRNA concentration and draw two conclusions: (1) MIR168a in MVs is more the principle for miRNA-target recognition.
functionally active because it has a higher affinity to AGO2 although the underneath mechanism remains Food-derived miRNAs may serve as a novel essential nu- unknown, and (2) plant miRNAs can use mammalian AGO2 to form AGO2-associated RISC and execute It has been widely reported that downregulation of their functions, similar to mammalian miRNAs. The LDLRAP1 increases plasma LDL level [35, 36]. In the www.cell-research.com Cell Research npg Plant MIR168a downregulates mammalian LDLRAP1122 present study, direct reduction of LDLRAP1 in mouse RAP1 in vitro and in vivo, the present study significantly liver by RNAi significantly elevated plasma LDL level extends our understanding of the role of miRNAs. With (Supplementary information, Figure S6A-S6C), confirm- their robust stability and highly conserved sequences, ing that LDLRAP1 is a gene candidate responsible for secretory miRNAs can act not only in a cross-species, plasma LDL removal. Interestingly, an elevated level but also a cross-kingdom fashion. In this sense, miRNAs of MIR168a but a decreased LDLRAP1 in mouse liver may represent a novel class of universal modulators that were detected after just 6 h of rice feeding (Figures 2C play an important role in mediating animal-plant interac- and 5B), indicating that exogenous plant MIR168a from tions at the molecular level. Like vitamins, minerals and food intake can quickly change mouse liver LDLRAP1 other essential nutrients derived from food sources, plant level. Continuous downregulation of mouse liver LD- miRNAs may serve as a novel functional component of LRAP1 level by MIR168a through rice feeding (Figure food and make a critical contribution to maintaining and 6E and 6F) resulted in an elevation of the plasma LDL- shaping animal body structure and function. Extending cholesterol level after 3 days (Figure 6G), implicating a from this concept, the intake of certain plant miRNAs physiological relevance of food-derived plant MIR168a. generation after generation through a particular food Rice feeding-induced reduction of LDLRAP1 protein source may leave an imprint on the genetic map of the and elevation of plasma LDL-cholesterol level could human race. In conclusion, the discovery of plant miR- be largely reversed by anti-MIR168a ASO (Figure 6I- NAs and their roles in the biology of mammalian cells 6K), confirming that the rice feeding-mediated physi- and animal organs represents the first evidence of cross- ological alteration is specifically due to the targeting of kingdom transfer of functionally active miRNAs and mouse liver LDLRAP1 by MIR168a. This conclusion opens a new avenue to explore miRNA-mediated animal- is also supported by the observation that chow diet with plant interactions.
addition of mature MIR168a significantly enhanced the levels of mouse liver MIR168a (Figure 6N) and plasma Materials and Methods
LDL-cholesterol (Figure 6Q) but decreased mouse liver LDLRAP1 protein level (Figure 6O and 6P). Interest- Reagents, cells and antibodies ingly, food intake, possibly via intestinal epithelia of GI The human intestinal carcinoma cell line Caco-2 and the hepa- track, may represent a general pathway for uptake of toma cell line HepG2 were purchased from Institute of Biochemis- food-derived or food-associated miRNAs. As shown in try and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). HepG2 cells Supplementary information, Figure S7F-S7H, we added were maintained at 37 °C in a humidified 5% CO miR-150, an endogenous mammalian miRNA, into chow 2 incubator with Dulbecco's modified eagle medium (Gibco, CA, USA) containing diet and fed mice with miR-150-enriched chow diet and 10% fetal bovine serum (FBS, Gibco), 100 units/ml of penicil- normal chow diet, respectively. We found that miR-150 lin, and 100 µg/ml of streptomycin. Caco-2 cells were grown in could also enter mouse liver and downregulate its target minimum essential medium (Gibco) containing 10% FBS (Gibco), gene, c-Myb. Given that exogenous miRNAs in food or 100 units/ml of penicillin, and 100 µg/ml of streptomycin. Anti- miRNAs that are ‘added' into the food can enter the cir- LDLRAP1 (LS-C20125) antibody was purchased from Lifespan Biosciences (Seattle, WA, USA), anti-Ago2 (ab57113) antibody culation and various organs of animals and play a role in was purchased from Abcam (Cambridge, MA, USA), and anti-c- regulating the physiological or pathophysiological condi- Myb (C19) and anti-α-tubulin (B-7) antibodies were purchased tions, food-derived exogenous miRNAs may be qualified from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Synthetic as a novel nutrient component, like vitamins and miner- RNA molecules, including pre-MIR168a, anti-MIR168a and scrambled negative control oligonucleotides (pre-ncRNA and anti- Previous studies have reported that the transfer of ge- ncRNA), were purchased from Ambion (Austin, TX, USA). Syn- netic material from one species to another may modulate thetic mature MIR168a oligonucleotides and scrambled negative the cellular functions of the recipient species [50, 51]. control oligonucleotides (mature ncRNA) were purchased from Takara (Dalian, China).
Such examples include human miRNAs targeting viral genes [50] and the translocation of host plant mRNAs Serum preparation into dodder (a parasitic plant) [51]. However, to our The recruitment of sera from 11 male (mean age 26.3 ± 1.9) and knowledge, it was still unknown whether plant miRNAs 10 female (mean age 24.2 ± 1.3) healthy Chinese, and 8 pooled could enter mammals and modulate mammalian cell samples (each pooled from 10 healthy Chinese subjects, mean functions. By illustrating that plant miRNAs, such as age 50.9 ± 7.9) was conducted in the Healthy Physical Examina- MIR168a, can be delivered into animal serum and tissues tion Center of the Jinling Hospital. The health condition checkup included detailed history, physical, radiological examinations, through food intake and digestion and that exogenous blood tests, and abdominal sonography. Subjects who showed no MIR168a can target mammalian liver-specific LDL- abnormalities during the medical checkup were enrolled. Written Cell Research Vol 22 No 1 January 2012 Lin Zhang et al. npg informed consent was obtained from all donors prior to the study, using TaqMan miRNA probes (Applied Biosystems, Foster City, and the study was approved by the ethics committee of Nanjing CA, USA) according to the manufacturer's instructions. Briefly, University, China. Venous blood samples ( 5 ml) were collected total RNA was reverse transcribed to cDNA using AMV reverse from each donor and placed in a serum separator tube. Samples transcriptase (Takara) and a stem-loop RT primer (Applied Biosys- were processed within 1 h. Separation of the serum was accom- tems). Real-time PCR was performed using a TaqMan PCR kit and plished by centrifugation at 800× g for 10 min at room tempera- an Applied Biosystems 7300 Sequence Detection System (Applied ture, followed by a 15 min high-speed centrifugation at 10 000× g at room temperature to completely remove the cell debris. The su- All reactions, including no-template controls, were performed pernatant serum was recovered and stored at −80 °C until analysis.
in triplicate. After the reaction, the CT values were determined us- ing fixed threshold settings. To calculate the absolute expression Solexa sequencing levels of the target miRNAs, a series of synthetic miRNA oligo- The sequencing procedure was conducted as previously de- nucleotides at known concentrations was reverse transcribed and scribed [5, 52]. Briefly, total RNA was extracted from 100 ml of amplified. The absolute amount of each miRNA was then calculat- serum using the Trizol Reagent (Invitrogen, Carlsbad, CA, USA) ed in reference to the standard curve. In the experiments presented according to the manufacturer's instructions. After the PAGE puri- here, miRNA expression in cells was normalized to U6 snRNA, as fication of small RNA molecules (under 30 base pairs) and the li- is common in many other reports [53].
gation of a pair of Solexa adaptors to their 5′ and 3′ ends, the small RNA molecules were amplified using the adaptor primers for 17 Deep sequencing and qRT-PCR after oxidation of small cycles and the fragments of around 90 bp (small RNA + adaptors) RNAs with periodate were isolated from PAGE gels. The purified DNA was directly The methylation status of small RNAs was evaluated by treat- used for the cluster generation and sequencing analysis using an Il- ment of the small RNAs with sodium periodate followed by Solexa lumina Genome Analyzer according to the manufacturer's instruc- sequencing or qRT-PCR with the miScript PCR system. Periodate tions. The image files generated by the sequencer were then pro- oxidation was performed as previously described with slight modi- cessed to produce digital data. The subsequent procedures included fications [22, 54]. Briefly, total RNA was extracted from 200 ml summarizing data production, evaluating sequencing quality and of human serum or 100 mg mouse liver using the Trizol Reagent depth, calculating length distribution of small RNAs, and filtrat- (Invitrogen) according to the manufacturer's instructions. Small ing contaminated reads. After masking the adaptor sequences, the RNA fractions (fewer than 30 base pairs) were enriched by PAGE clean reads were aligned against the miRBase database 16.0 based purification. A 100 µl mixture consisting of 20 µg of small RNA on the Smith-Waterman algorithm. Only candidate with identical fraction and 10 mM NaIO4 was incubated at 0 °C for 40 min in sequence and length compared to reference miRNA was counted dark. The oxidized RNA was precipitated twice by ethanol, rinsed as miRNA matching. For normalization, the sequencing frequency once with 80% ethanol, aired dried, dissolved in ddH2O, and then of each plant miRNA was normalized to the total amount of mam- subjected to Solexa sequencing or qRT-PCR assay. The qRT-PCR malian miRNAs.
assay was conducted using the miScript PCR system (QIAGEN, Valencia, CA, USA) according to the manufacturer's instructions. Transfection of cells with ncRNA, pre-MIR168a, or mature Briefly, miRNAs are polyadenylated by poly(A) polymerase and subsequently converted into cDNA by reverse transcriptase with HepG2 or Caco-2 cells were seeded on 12-well plates or 10- oligo-dT. The cDNA is then used for real-time PCR quantification mm dishes overnight and transfected the following day using of mature miRNAs.
Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions. For the overexpression of MIR168a, 20 pmol per 1 × Northern blotting analysis 105 cells of pre-MIR168a or mature MIR168a was used. Scrambled Oligonucleotide probes complementary to mature miRNAs negative control pre-miRNA (pre-ncRNA) and mature ncRNA were end-labeled with γ-32P-ATP using T4 Polynucleotide Kinase were used as controls for pre-MIR168a and mature MIR168a, (Takara). Labeled probes were purified using a Sephadex G25 spin respectively. Cells were harvested 24 or 48 h after transfection for column (Roche). Total RNA was extracted from 100 ml human semi-quantitative RT-PCR, real-time PCR analysis, and western serum or 40 ml calf serum using TRIzol Reagent (Invitrogen) ac- cording to the manufacturer's instructions. Synthetic oligonucle- otides (1 pmol) were loaded as positive control. Total RNA was MV isolation fractionated by PAGE using a 15% denaturing polyacrylamide gel. MVs were isolated from the cell culture medium by differential The RNA was then transferred onto a nylon membrane (Hybond centrifugation according to previous publications [16, 17]. Briefly, N+, Amersham Biosciences) by electroblotting at 200 mA in 0.5× after removing cells and other debris by centrifugation at 300× g, TBE (Tris-Borate-EDTA) buffer for 2 h. The membrane was dried 1 200× g, and 10 000× g, the supernatant was centrifuged at 110 and cross-linked. A prehybridization step was performed by in- 000× g for 2 h (all steps were performed at 4 °C). MVs were col- cubating the membrane with 10 ml of ULTRAhyb-Oligo solution lected from the pellet and resuspended in FBS-free medium.
(Ambion) pre-heated to 65 °C. Prehybridization was performed for 1 h at 37 °C in a standard rotating hybridization oven. The radio- RNA isolation and qRT-PCR of mature miRNAs labeled probe was added directly to the ULTRAhyb-Oligo solution Total RNA was extracted from the serum, cells, or tissues using and the membrane was incubated overnight at 37 °C with rotation TRIzol Reagent or Trizol LS Reagent (Invitrogen) according to the in a hybridization oven. After hybridization, the membrane was manufacturer's instructions. Quantitative RT-PCR was performed washed 3 × 10 min at room temperature in 2× SSC (Saline-sodium www.cell-research.com Cell Research npg Plant MIR168a downregulates mammalian LDLRAP1124 citrate), 0.5% sodium dodecyl sulfate (SDS) and then 1 × 15 min according to the manufacturer's instructions or by semi-quantita- at 42 °C in 2 × SSC, 0.5% SDS. The membrane was wrapped in tive RT-PCR using primers specific for human LDLRAP1. Primer plastic wrap and exposed to an X-ray film at −80 °C.
sequences for human LDLRAP1 were as follows: 5′-AGAGC- CAGCACAACCAGA-3′ (forward primer) and 5′-CTTGGACAC- Western blot analysis CTGCCAAAA-3′ (reverse primer). Primer sequences for mouse Samples of tissues and cultured cells were lysed in a buffer LDLRAP1 were as follows: 5′-AAGTATCTTGGTATGACGCT- (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.1% SDS), GGTG-3′ (forward primer) and 5′-TCCTGGTTGGCTTTCTC- sonicated (6 × 1.5 s, 30% power), and centrifuged at 12 000× g CCT-3′ (reverse primer).
for 10 min at 4 °C. The supernatant fraction was removed, and the protein concentration was determined by BCA assay (Pierce, Rockford, USA). Aliquots of proteins (60 to 100 µg) were separat- All experimental animals were maintained on a C57BL/6J ed on 10% SDS-polyacrylamide gels (SDS-PAGE) and transferred background on a 12-h light/dark cycle in a pathogen-free animal to polyvinylidene difluoride (PVDF) membranes. The membranes facility at Nanjing University. The Institutional Review Board of were blocked for 1 h at room temperature with 5% non-fat milk in Nanjing University approved all housing and surgical procedures. Tris-buffered saline (TBS) plus Tween 20 (TBST), followed by an At 10 weeks of age, each mouse was fed fresh rice total RNA overnight incubation at 4 °C with antibodies (diluted in blocking (80 µg), synthetic MIR168a (300 pmol), or synthetic methylated buffer) against LDLRAP1 and α-tubulin. Following 3 × 10 min MIR168a (300 pmol) by gavage after fasting overnight. After a washes with TBST, the blots were incubated at room tempera- fixed time interval (i.e., 0.5 h, 3 h, or 6 h), serum and tissues were ture for 1 h with the appropriate secondary antibody conjugated collected and total RNA was extracted. In a separate experiment, to horseradish peroxidase (HRP) and detected with an enhanced after fasting overnight, two groups of 10-week-old male mice were chemiluminescence reagent (Cell Signaling Technology Inc., placed on a diet of either mouse chow (the fundamental ingre- USA). The autoradiographic intensity of each band was scanned dients of the chow diet are listed in Supplementary information, and quantified using BandScan software (Glyko Inc., Novato, CA, Table S5) or fresh rice. Fresh food was administered every 3 days USA). Values were normalized to α-tubulin and the ratio to the from a supply stored at −20 °C, and food consumption and body control values was calculated.
weight were measured. The mice were maintained on the diets for a fixed time interval (i.e., 0.5, 3, or 6 h, or 1, 3, or 7 days), after Plasmid construction and luciferase assay which serum and tissues were collected. Furthermore, the mice fed A mammalian expression vector encoding the human LDL- on rice received tail vein injections of ncRNA or anti-MIR168a RAP1 ORF (pReceiver-M02-LDLRAP1) and its C-terminally (10 nmol each). After a fixed time interval (i.e., 0.5, 3, or 6 h, GFP-tagged form (pReceiver-M03-LDLRAP1) were purchased or 1, 3, or 7 days), sera and tissues were collected. Several mice from GeneCopoeia (Germantown, MD, USA). To introduce muta- were euthanized directly after overnight fasting, and their sera tions into the MIR168a target site in the LDLRAP1 coding region, and tissues were collected as a control. In a separate experiment, primers were designed for site-directed mutagenesis that resulted synthetic MIR168a or ncRNA (100 pmol) were mixed evenly with in the destruction of the MIR168a target site without altering the 5 g of mouse chow. To avoid miRNA degradation and contamina- amino-acid sequence of LDLRAP1. The sites (LCTKR) were tion, this food was prepared before feeding. qRT-PCR was used mutated as follows: prior to mutagenesis, CTC TGC ACC AAG to determine the level of MIR168a in the chow diet with the addi- CGG; following mutagenesis, CTg TGt ACg AAa CGc. To gener- tion of MIR168a or ncRNA. After fasting overnight, two groups ate luciferase reporters, the amplified fragments were cloned into of 10-week-old male mice were placed on a diet of mouse chow the 3′ UTR region of the pMIR-report plasmid (Ambion). Efficient plus either MIR168a or ncRNA. Fresh food was administered ev- insertion was confirmed by sequencing. For luciferase reporter ery day, and food consumption and body weight were measured. assays, 0.2 µg of firefly luciferase reporter plasmid, 0.1 µg of Generally, mouse consumes 5-7 g of food per day. The mice were β-galactosidase expression vector (Ambion), and equal amounts maintained on the diets for 3 days, after which serum and tissues (20 pmol) of pre-MIR168a, mature MIR168a or scrambled nega- were collected.
tive control RNA were transfected into cells in 24-well plates. The β-galactosidase vector was used as a transfection control. At 24 h Analysis of serum lipids and lipoproteins post-transfection, cells were analyzed using a luciferase assay kit Serum lipids and lipoproteins were assayed using commercially available kits and a clinical chemistry analyzer (HITACHI 7600, Hitachi Koki Co. Ltd., Hitachinaka City, Japan). Reagent kits for total cholesterol and triglycerides were obtained from Randox Immunoprecipitation assays were performed using a Chromatin Laboratories, Ltd. (Crumlin, UK) and reagent kits for LDL choles- Immunoprecipitation (ChIP) Assay Kit (Millipore) according to terol and lipoprotein A were obtained from Daiichi Pure Chemicals the manufacturer's instructions. Briefly, cells were washed three times with cold PBS (4 °C), scraped from each dish and then col- lected by centrifugation at 1 000 rpm for 5 min at 4 °C. Cells were then resuspended in an appropriate volume of complete RIP lysis Full-length cDNAs of the human genes were obtained from the buffer. Mouse monoclonal anti-AGO2 antibody (5 µg) was used NCBI Genebank database. A program was developed and imple- to immunoprecipitate RNA-binding proteins. After purification, mented to identify MIR168a-matched sites in the entire CDS/UTR immunoprecipitated RNA was analyzed by real-time RT-PCR for of the transcripts. This program used several common criteria to MIRNA168a using TaqMan miRNA probes (Applied Biosystems) determine whether a transcript was a target for MIR168a. The first Cell Research Vol 22 No 1 January 2012 Lin Zhang et al. npg criterion for target recognition named "seed rules" was base pair- ing between the "seed" (the core sequence that encompassed the 10 Resnick KE, Alder H, Hagan JP, Richardson DL, Croce CM, first 2 to 8 bases of the mature miRNA) and the target [55]. Sec- Cohn DE. The detection of differentially expressed microR- ond, the free energy of the hybrid was expected to be within the NAs from the serum of ovarian cancer patients using a novel range of the authentic miRNA-target pairs, typically lower than real-time PCR platform. Gynecol Oncol 2009; 112:55-59.
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