Reagents, cells and antibodies
The human intestinal carcinoma cell line Caco-2 and the hepatoma cell line HepG2 were purchased from Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). HepG2 cells were maintained at 37 °C in a humidified 5% CO2 incubator with Dulbecco's modified eagle medium (Gibco, CA, USA) containing 10% fetal bovine serum (FBS, Gibco), 100 units/ml of penicillin, and 100 μg/ml of streptomycin. Caco-2 cells were grown in minimum essential medium (Gibco) containing 10% FBS (Gibco), 100 units/ml of penicillin, and 100 μg/ml of streptomycin. Anti-LDLRAP1 (LS-C20125) antibody was purchased from Lifespan Biosciences (Seattle, WA, USA), anti-Ago2 (ab57113) antibody was purchased from Abcam (Cambridge, MA, USA), and anti-c-Myb (C19) and anti-α-tubulin (B-7) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Synthetic RNA molecules, including pre-MIR168a, anti-MIR168a and scrambled negative control oligonucleotides (pre-ncRNA and anti-ncRNA), were purchased from Ambion (Austin, TX, USA). Synthetic mature MIR168a oligonucleotides and scrambled negative control oligonucleotides (mature ncRNA) were purchased from Takara (Dalian, China).
The recruitment of sera from 11 male (mean age 26.3 ± 1.9) and 10 female (mean age 24.2 ± 1.3) healthy Chinese, and 8 pooled samples (each pooled from 10 healthy Chinese subjects, mean age 50.9 ± 7.9) was conducted in the Healthy Physical Examination Center of the Jinling Hospital. The health condition checkup included detailed history, physical, radiological examinations, blood tests, and abdominal sonography. Subjects who showed no abnormalities during the medical checkup were enrolled. Written informed consent was obtained from all donors prior to the study, and the study was approved by the ethics committee of Nanjing University, China. Venous blood samples (~5 ml) were collected from each donor and placed in a serum separator tube. Samples were processed within 1 h. Separation of the serum was accomplished by centrifugation at 800× g for 10 min at room temperature, followed by a 15 min high-speed centrifugation at 10 000× g at room temperature to completely remove the cell debris. The supernatant serum was recovered and stored at −80 °C until analysis.
The sequencing procedure was conducted as previously described 5, 52
. Briefly, total RNA was extracted from 100 ml of serum using the Trizol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. After the PAGE purification of small RNA molecules (under 30 base pairs) and the ligation of a pair of Solexa adaptors to their 5′ and 3′ ends, the small RNA molecules were amplified using the adaptor primers for 17 cycles and the fragments of around 90 bp (small RNA + adaptors) were isolated from PAGE gels. The purified DNA was directly used for the cluster generation and sequencing analysis using an Illumina Genome Analyzer according to the manufacturer's instructions. The image files generated by the sequencer were then processed to produce digital data. The subsequent procedures included summarizing data production, evaluating sequencing quality and depth, calculating length distribution of small RNAs, and filtrating contaminated reads. After masking the adaptor sequences, the clean reads were aligned against the miRBase database 16.0 based on the Smith-Waterman algorithm. Only candidate with identical sequence and length compared to reference miRNA was counted as miRNA matching. For normalization, the sequencing frequency of each plant miRNA was normalized to the total amount of mammalian miRNAs.
Transfection of cells with ncRNA, pre-MIR168a, or mature MIR168a
HepG2 or Caco-2 cells were seeded on 12-well plates or 10-mm dishes overnight and transfected the following day using Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions. For the overexpression of MIR168a, 20 pmol per 1 × 105 cells of pre-MIR168a or mature MIR168a was used. Scrambled negative control pre-miRNA (pre-ncRNA) and mature ncRNA were used as controls for pre-MIR168a and mature MIR168a, respectively. Cells were harvested 24 or 48 h after transfection for semi-quantitative RT-PCR, real-time PCR analysis, and western blotting.
MVs were isolated from the cell culture medium by differential centrifugation according to previous publications 16, 17
. Briefly, after removing cells and other debris by centrifugation at 300× g
, 1 200× g
, and 10 000× g
, the supernatant was centrifuged at 110 000× g
for 2 h (all steps were performed at 4 °C). MVs were collected from the pellet and resuspended in FBS-free medium.
RNA isolation and qRT-PCR of mature miRNAs
Total RNA was extracted from the serum, cells, or tissues using TRIzol Reagent or Trizol LS Reagent (Invitrogen) according to the manufacturer's instructions. Quantitative RT-PCR was performed using TaqMan miRNA probes (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. Briefly, total RNA was reverse transcribed to cDNA using AMV reverse transcriptase (Takara) and a stem-loop RT primer (Applied Biosystems). Real-time PCR was performed using a TaqMan PCR kit and an Applied Biosystems 7300 Sequence Detection System (Applied Biosystems).
All reactions, including no-template controls, were performed in triplicate. After the reaction, the CT
values were determined using fixed threshold settings. To calculate the absolute expression levels of the target miRNAs, a series of synthetic miRNA oligonucleotides at known concentrations was reverse transcribed and amplified. The absolute amount of each miRNA was then calculated in reference to the standard curve. In the experiments presented here, miRNA expression in cells was normalized to U6 snRNA, as is common in many other reports 53
Deep sequencing and qRT-PCR after oxidation of small RNAs with periodate
The methylation status of small RNAs was evaluated by treatment of the small RNAs with sodium periodate followed by Solexa sequencing or qRT-PCR with the miScript PCR system. Periodate oxidation was performed as previously described with slight modifications 22, 54
. Briefly, total RNA was extracted from 200 ml of human serum or 100 mg mouse liver using the Trizol Reagent (Invitrogen) according to the manufacturer's instructions. Small RNA fractions (fewer than 30 base pairs) were enriched by PAGE purification. A 100 μl mixture consisting of 20 μg of small RNA fraction and 10 mM NaIO4
was incubated at 0 °C for 40 min in dark. The oxidized RNA was precipitated twice by ethanol, rinsed once with 80% ethanol, aired dried, dissolved in ddH2
O, and then subjected to Solexa sequencing or qRT-PCR assay. The qRT-PCR assay was conducted using the miScript PCR system (QIAGEN, Valencia, CA, USA) according to the manufacturer's instructions. Briefly, miRNAs are polyadenylated by poly(A) polymerase and subsequently converted into cDNA by reverse transcriptase with oligo-dT. The cDNA is then used for real-time PCR quantification of mature miRNAs.
Northern blotting analysis
Oligonucleotide probes complementary to mature miRNAs were end-labeled with γ-32P-ATP using T4 Polynucleotide Kinase (Takara). Labeled probes were purified using a Sephadex G25 spin column (Roche). Total RNA was extracted from 100 ml human serum or 40 ml calf serum using TRIzol Reagent (Invitrogen) according to the manufacturer's instructions. Synthetic oligonucleotides (1 pmol) were loaded as positive control. Total RNA was fractionated by PAGE using a 15% denaturing polyacrylamide gel. The RNA was then transferred onto a nylon membrane (Hybond N+, Amersham Biosciences) by electroblotting at 200 mA in 0.5× TBE (Tris-Borate-EDTA) buffer for 2 h. The membrane was dried and cross-linked. A prehybridization step was performed by incubating the membrane with 10 ml of ULTRAhyb-Oligo solution (Ambion) pre-heated to 65 °C. Prehybridization was performed for 1 h at 37 °C in a standard rotating hybridization oven. The radio-labeled probe was added directly to the ULTRAhyb-Oligo solution and the membrane was incubated overnight at 37 °C with rotation in a hybridization oven. After hybridization, the membrane was washed 3 × 10 min at room temperature in 2× SSC (Saline-sodium citrate), 0.5% sodium dodecyl sulfate (SDS) and then 1 × 15 min at 42 °C in 2 × SSC, 0.5% SDS. The membrane was wrapped in plastic wrap and exposed to an X-ray film at −80 °C.
Western blot analysis
Samples of tissues and cultured cells were lysed in a buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.1% SDS), sonicated (6 × 1.5 s, 30% power), and centrifuged at 12 000× g 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 separated on 10% SDS-polyacrylamide gels (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked for 1 h at room temperature with 5% non-fat milk in Tris-buffered saline (TBS) plus Tween 20 (TBST), followed by an overnight incubation at 4 °C with antibodies (diluted in blocking buffer) against LDLRAP1 and α-tubulin. Following 3 × 10 min washes with TBST, the blots were incubated at room temperature for 1 h with the appropriate secondary antibody conjugated to horseradish peroxidase (HRP) and detected with an enhanced chemiluminescence reagent (Cell Signaling Technology Inc., USA). The autoradiographic intensity of each band was scanned and quantified using BandScan software (Glyko Inc., Novato, CA, USA). Values were normalized to α-tubulin and the ratio to the control values was calculated.
Plasmid construction and luciferase assay
A mammalian expression vector encoding the human LDLRAP1 ORF (pReceiver-M02-LDLRAP1) and its C-terminally GFP-tagged form (pReceiver-M03-LDLRAP1) were purchased from GeneCopoeia (Germantown, MD, USA). To introduce mutations into the MIR168a target site in the LDLRAP1 coding region, primers were designed for site-directed mutagenesis that resulted in the destruction of the MIR168a target site without altering the amino-acid sequence of LDLRAP1. The sites (LCTKR) were mutated as follows: prior to mutagenesis, CTC TGC ACC AAG CGG; following mutagenesis, CTg TGt ACg AAa CGc. To generate luciferase reporters, the ampliﬁed fragments were cloned into the 3′ UTR region of the pMIR-report plasmid (Ambion). Efficient insertion was confirmed by sequencing. For luciferase reporter assays, 0.2 μg of firefly luciferase reporter plasmid, 0.1 μg of β-galactosidase expression vector (Ambion), and equal amounts (20 pmol) of pre-MIR168a, mature MIR168a or scrambled negative control RNA were transfected into cells in 24-well plates. The β-galactosidase vector was used as a transfection control. At 24 h post-transfection, cells were analyzed using a luciferase assay kit (Promega).
Immunoprecipitation assays were performed using a Chromatin Immunoprecipitation (ChIP) Assay Kit (Millipore) according to the manufacturer's instructions. Briefly, cells were washed three times with cold PBS (4 °C), scraped from each dish and then collected by centrifugation at 1 000 rpm for 5 min at 4 °C. Cells were then resuspended in an appropriate volume of complete RIP lysis buffer. Mouse monoclonal anti-AGO2 antibody (5 μg) was used to immunoprecipitate RNA-binding proteins. After purification, immunoprecipitated RNA was analyzed by real-time RT-PCR for MIRNA168a using TaqMan miRNA probes (Applied Biosystems) according to the manufacturer's instructions or by semi-quantitative RT-PCR using primers specific for human LDLRAP1. Primer sequences for human LDLRAP1 were as follows: 5′-AGAGCCAGCACAACCAGA-3′ (forward primer) and 5′-CTTGGACACCTGCCAAAA-3′ (reverse primer). Primer sequences for mouse LDLRAP1 were as follows: 5′-AAGTATCTTGGTATGACGCTGGTG-3′ (forward primer) and 5′-TCCTGGTTGGCTTTCTCCCT-3′ (reverse primer).
All experimental animals were maintained on a C57BL/6J background on a 12-h light/dark cycle in a pathogen-free animal facility at Nanjing University. The Institutional Review Board of Nanjing University approved all housing and surgical procedures. At 10 weeks of age, each mouse was fed fresh rice total RNA (80 μg), synthetic MIR168a (300 pmol), or synthetic methylated MIR168a (300 pmol) by gavage after fasting overnight. After a fixed time interval (i.e., 0.5 h, 3 h, or 6 h), serum and tissues were collected and total RNA was extracted. In a separate experiment, after fasting overnight, two groups of 10-week-old male mice were placed on a diet of either mouse chow (the fundamental ingredients of the chow diet are listed in Supplementary information, Table S5
) or fresh rice. Fresh food was administered every 3 days from a supply stored at −20 °C, and food consumption and body 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 which serum and tissues were collected. Furthermore, the mice fed on rice received tail vein injections of ncRNA or anti-MIR168a (10 nmol each). After a fixed time interval (i.e., 0.5, 3, or 6 h, or 1, 3, or 7 days), sera and tissues were collected. Several mice were euthanized directly after overnight fasting, and their sera and tissues were collected as a control. In a separate experiment, synthetic MIR168a or ncRNA (100 pmol) were mixed evenly with 5 g of mouse chow. To avoid miRNA degradation and contamination, this food was prepared before feeding. qRT-PCR was used to determine the level of MIR168a in the chow diet with the addition of MIR168a or ncRNA. After fasting overnight, two groups of 10-week-old male mice were placed on a diet of mouse chow plus either MIR168a or ncRNA. Fresh food was administered every day, and food consumption and body weight were measured. Generally, mouse consumes 5-7 g of food per day. The mice were maintained on the diets for 3 days, after which serum and tissues were collected.
Analysis of serum lipids and lipoproteins
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 Laboratories, Ltd. (Crumlin, UK) and reagent kits for LDL cholesterol and lipoprotein A were obtained from Daiichi Pure Chemicals (Japan).
Full-length cDNAs of the human genes were obtained from the NCBI Genebank database. A program was developed and implemented to identify MIR168a-matched sites in the entire CDS/UTR of the transcripts. This program used several common criteria to determine whether a transcript was a target for MIR168a. The first criterion for target recognition named “seed rules” was base pairing between the “seed” (the core sequence that encompassed the first 2 to 8 bases of the mature miRNA) and the target 55
. Second, the free energy of the hybrid was expected to be within the range of the authentic miRNA-target pairs, typically lower than −17 kcal/mol. Third, an optional rule for target prediction required interspecies conservation of the putative binding sites. Using these rules, ~50 human genes were identified as putative targets of MIR168a.
All photo images of western blotting and semi-quantitative RT-PCR are representative of at least three independent experiments. Real-time PCR were performed in triplicate and each experiment was repeated several times. Data shown are presented as the mean ± SEM of at least three independent experiments; differences are considered statistically significant at P < 0.05, using a Student's t-test.