Identification of Dietetically Absorbed Rapeseed (Brassica campestris L.) Bee Pollen MicroRNAs in Serum of Mice

2.1. Rapeseed Bee Pollen

The rapeseed bee pollen was bought from Bee Research Institute of Anhui Agriculture University. The implementation of the standard is GB/T11758-89-bee pollen. Single pollen rates are over 95%, and the production date was November 10, 2015.

2.2. Animal Studies

All animal experiments were performed using male ICR strain mice on a 12 h light/dark cycle in a pathogen-free animal feeding facility at Zhejiang Academy of Traditional Chinese Medicine. The animal study protocols were approved by the Animal Care and Use Committee of Zhejiang Academy of Traditional Chinese Medicine. At 6 weeks of age (weighted 26.37 ± 2.7 g), each mouse was fed rapeseed bee pollen (10 g/kg) by gavage. After a fixed time interval (3 h or 6 h on d1, d4, or d8), serum about 200 μL was collected from each mouse, and then total RNA was extracted using mirVana™ PARIS™ Kit (AM1556, Ambion™).

2.3. Illumina Hiseq2500 Sequencing

The sequencing procedure was conducted according to standard steps provided by Illumina company, Inc. Briefly, a pair of adaptors were ligated to the 3′ and 5′ ends of total RNA. Reverse transcription followed by PCR is used to create cDNA constructs based on the small RNA ligated with 3′ and 5′ adapters. This process selectively enriches those fragments that have adapter molecules on both ends. Then the fragments of around 147–157 bp (22–30 nt length small RNA + adaptors) were purified by PAGE. The purified DNA was directly used for the cluster generation and sequencing using Illumina Hiseq2500 according to the manufacturer's instructions. The image files generated by the sequencer were then processed to produce digital data. The subsequent procedures included removing adapter dimers, junk, low complexity, common RNA families (rRNA, tRNA, snRNA, and snoRNA), and repeats. Subsequently, unique sequences with length in 18–26 nucleotides were mapped onto all plant miRNA precursors in miRBase 20.0 by BLAST search to identify known miRNAs and novel 3p- and 5p-derived miRNAs. Length variation at both 3′ and 5′ ends and one mismatch inside of the sequence were allowed in the alignment. The unique sequences mapping onto specific species mature miRNAs in hairpin arms were identified as known miRNAs. The unique sequences mapping onto the other arm of known specific species precursor hairpin opposite to the annotated mature miRNA-containing arm were considered to be novel 5p- or 3p-derived miRNA candidates.

2.4. Analysis of Level of miRNAs in Rapeseed Bee Pollen by RT-qPCR

Total RNA was extracted from 80 mg rapeseed bee pollen using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Quantitative RT-PCR was performed using Taqman miRNA probes (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. To calculate the absolute expression levels of target miRNAs, a series of synthetic miRNA oligonucleotides at known concentrations were reverse transcribed and amplified. The absolute amount of each miRNA was then calculated with reference to the standard curve. Quantitative PCR was performed using an ABI-StepOnePlus machine (Applied Biosystems).

2.5. Statistical Analysis

Differences are considered statistically significant at P < 0.05, using Student's t-test.

3.1. Raw Data Filtering

We sequenced a small RNA library from blood RNA of mouse fed with rapeseed bee pollen using the Illumina Hiseq2500 system. We acquired a total of 11,089,480 raw sequences. Overview of these reads from raw data to cleaned sequences is shown in Table 1.

We illustrated small RNA reads with Rfam dataset; to remove rRNA, scRNA, snoRNA, snRNA, and tRNA, the pie charts were drawn for total reads and unique reads (Figure 1).

3.2. Plant miRNAs Spectrum in Serum of Mice Fed with Rapeseed Bee Pollen

After removing the junk reads, the clean reads yield 34 plant miRNAs (Table 2). Plant miRNAs are 2′-O-methyl modified on their terminal nucleotide; in contrast, mammalian miRNAs with free 2′ and 3′ hydroxyls render plant miRNAs more difficult to be ligated to the cloning adapter compared with mammalian miRNAs. As a result, in the 11,089,480 raw reads, there were only 132 reads of plant miRNAs. However, some plant miRNAs can be detected even though the mammalian miRNAs caused a strong disturbance; in turn this proves that the content of plant miRNAs in mouse blood was not low. Among the plant miRNAs, miR-166a and miR-159 were with the highest levels in mouse blood; besides, these two miRNAs were both mapped onto rapeseed genome.

3.3. Comparison of Abundance Levels of miR-166a and miR-159 in Rapeseed Bee Pollen

Based on the predominant two miRNAs (miR-166a and miR-159) in the blood, we assumed that miR-166a and miR-159 can be found in rapeseed bee pollen, and the content of miRNAs in the rapeseed pollen also will follow the trend in the serum. To confirm this, the levels of miR-166a and miR-159 in rapeseed bee pollen were assessed by stem-loop quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay. As a result, miR-166a and miR-159 can be detected in RNA of rapeseed bee pollen (Additional Figure 1 in Supplementary Material available online at http://dx.doi.org/10.1155/2016/5413849). Moreover, the CT values of miR-166a and miR-159 were 23.8 ± 0.23 and 31.22 ± 0.33, respectively, suggesting that the abundance level of miR-166a was higher than miR-159. These results suggested that food-derived exogenous miRNA from rapeseed bee pollen could be absorbed by mouse, and the abundance of specific miRNAs is dependent on their origins from the rapeseed bee pollen.

3.4. Comparison of Abundance Levels of miR-166a between Mice Fed with Rapeseed Bee Pollen and Control

Given that miR-166a is the highest abundance rape-encoded miRNA in mice fed with rapeseed bee pollen, and it is rich in rapeseed bee pollen, we speculate that the miR-166a in mouse serum are mainly absorbed from rapeseed bee pollen. To test this speculation, we compared the abundance level of miR-166a in serum of mice fed with rapeseed bee pollen and control. As it is reported that the levels of plant-based miRNAs were elevated in serum of mice for 6 h [4], we compared the levels of miR-166a in serum of mice fed with rapeseed bee pollen after 6 h and control. As shown in Figure 2 and additional Figure 2, the levels of miR-166a were elevated in serum of mice fed with rapeseed bee pollen for 6 h compared with control by RT-qPCR.