SUPPLEMENTAL DIGITAL CONTENT (SDC) Materials and Methods
Animals
Inbred male Lewis and BN rats weighing 200-250g were obtained from Peking Vital River Laboratory Animal, Ltd. (Beijing, China) and maintained in laminar flow cages in a specific pathogen-free animal facility at the Zhongshan Hospital of Fudan University (Shanghai, China) with a standard diet and water.
Lewis to BN (AR) and BN to BN (control) OLT were carried out. OLT was performed as previously described [1] without reconstruction of the hepatic artery. All procedures in this study were performed according to the guidelines of the Council of Animal Care at Fudan University. In the FK506 (tacrolimus)-treated group (AR+FK506 group, n=10), 1 mg/kg/day FK506 was applied by intramuscular injection from the first day after OLT to the day of sacrifice. In the delay FK506-treated group (AR+delay FK506 group, n=10), FK506 was applied from the third day after OLT to the day of sacrifice. After sacrifice, formalin-fixed and paraffin-embedded liver tissue was collected and histology examination was performed (Fig S4).
Samples Collection and RNA Extraction
Peripheral blood was collected fromthe tail vein on the 3rd, 7th, and 10th days after OLT in Ethylenediaminetetraacetic acid (EDTA)-treated tubes.
Within 30 minutes, the tubes were centrifuged at 820g for 10 min. Then, 1-ml aliquots of plasma were transferred to 1.5-ml tubes and centrifuged at 16,000g for 10 min to pellet any remaining cellular debris. Subsequently, the supernatant was transferred to RNase-free tubes and stored at –80 °C. Tissue samples from the brain, thymus, lung, heart, liver graft, spleen, and kidney of each animal were obtained and conserved in liquid nitrogen until use. PBMCs were purified with Ficoll separating solution (Biochrom AG, Berlin, Germany).
Total RNA was extracted from plasma samples with the mirVana PARIS miRNA Isolation Kit (Ambion, Austin, TX) and from tissue samples with the mirVana miRNA Isolation Kit (Ambion). RNA concentrations were determined with a NanoDrop 1000 Spectrophotometer (NanoDrop Technologies, Waltham, MA).
MicroRNA Microarray
RNA labeling
The miRNAs were labeled with Cy3 using the miRNA Complete Labeling and Hyb Kit (Agilent Technologies, Santa Clara, CA) following the manufacturer’s instructions.
Array hybridization
Each slide was hybridized with 100 ng Cy3-labeled RNA with the miRNA Complete Labeling and Hyb Kit (Agilent Technologies, Santa Clara, CA) in a hybridization oven (Agilent Technologies) at 55°C and 20 rpm for 20 hours according to the manufacturer’s instructions. After hybridization, slides were
washed in staining dishes (Thermo Shandon, Waltham, MA) with the Gene Expression Wash Buffer Kit (Agilent Technologies).
Data Acquisition
Microarray slides were scanned by an XDR Scan (PMT100, PMT5). The labeling and hybridization were performed according to the protocols of the Agilent microRNA microarray system. The microarray image data were converted into spot intensity values with Feature Extraction Software Rev.
9.5.3 (Agilent Technologies). The signal minus background values were exported directly into GeneSpring GX10 software (Agilent Technologies), and the raw data were normalized by the Quantile algorithm, Gene Spring Software 11.0 (Agilent Technologies).
RT-qPCR
Specific stem-loop PCR primers was used to evaluate the expression levels of the candidate microRNAs with Taqman microRNA assays (Applied Biosystems, Foster City, CA) according to the manufacturer’s protocol. Levels of microRNAs were measured by the fluorescentsignal produced from the Taqman probes on ABI 7900HT real-time PCR system (Applied Biosystems).
All samples were assayed in triplicate. The results were analyzed by SDS2.3.
U6 snRNA and miR-16 were used as internal controls in the RT-qPCR reactions for tissue and plasma samples, respectively [2].
We used U6 snRNA as internal control for tissue microRNA assay. The
choice of stable normalization control for plasma microRNA detection currently varies between laboratories. In our previous study we used miR-1228 as stable internal control for human plasma microRNA detection [3]. Nevertheless, there is no rodent homologue of miR-1228. Other studies revealed that miR-16 was a candidate of internal control for circulating microRNA assay [4]. In this case, we evaluated the stability of miR-16 in the plasma of NR and AR rats.
After treated with denaturing solution, 5 µl of syn-cel-miR-54 miScript miRNA Mimic (5 nM,Qiagen, GmbH) was added to 500µL rat plasma. Total RNA was extracted from plasma samples with the mirVana PARIS miRNA Isolation Kit (Ambion, Austin, TX).The expression level of miR-16 was compared between NR rats and AR rats after normalized by syn-cel-miR-54. We found the plasma expression level of miR-16 was stable across NR rats and AR rats (fold change=1.089 and P=0.786). Thus, we used miR-16 as an endogenous control for the present study.
Statistical Analysis
GeneSpring GX10 software (Agilent Technologies Inc., Santa Clara, CA) was used for microarray analysis.The expression levels were compared between AR group and NR group using Unpaired T test. Hierarchical clustering analysis was performed by significantly expressed microRNAs. The microRNA expression levels determined by RT-qPCR were compared between groups using One-Way ANOVA, and P<0.05 (two-tailed) was considered
statisticallysignificant.
Plasma Microvesicles(MVs) Isolation and microRNA Extraction
MVs from the plasma of AR rats and NR rats were isolated using the protocol previously described [5,6]. Briefly, 500µl plasma was passed through a 0.22-µm filter (Milipore, Billerica, MA) to remove anylarge cell particles or cell debris.And the suspension was centrifuged at 110,000g for 2 h at 4°C to pellet the MVs. Carefully remove the supernatant, saving the MVs pellet. Wash the MVs pellet, gently resuspend in PBS, and Centrifuge at 110,000 g for 70 min at 4°C.Resuspend the washed MVs pellet in a 100 µl of PBS. Transmission electron microscopy (TEM) was used to observe the morphology of MVs (Fig.
S3). Samples were treated with Rnase A (Ambion, Austin, TX, USA) before RNA extraction in order toensure that RNA was derived from MVs. After treated with denaturing solution, 3µl of syn-cel-miR-54 miScript miRNA Mimic (3 nM, Qiagen, GmbH) was added. Then, total RNA was extracted using mirVana PARIS miRNA Isolation Kit (Ambion, Austin, TX). MiR-122, miR-192 and miR-146a level was detected using Taqman microRNA assays (Applied Biosystems, Foster City, CA) and syn-cel-miR-54 was used as control to normalize the expression of these three microRNAs.
MiR-146a in Situ Hybridization (ISH)
We used ISH to localize candidate microRNA expression in FFPE graft sections. Liver tissue samples were obtained at the time of sacrifice, and each sample was fixed in 10% buffered neutralformalin and embedded in paraffin.
Sections4μm thick were stained with hematoxylin and eosin (H&E), and sections with 6 μm thick were prepared for ISH. The deparaffinization and rehydration of sections were performed with xylene and ethanol. Then, a 300-µL protease K (10ng/µL; Qiagen GmbH) preparation was applied to each section for 6 min at 37°C. Slides were placed in coplin jars that contained PBS in a slide rack, washed twice with PBS, dehydrated in new ethanol solutions, and then air-dried for 15 min. Slides were placed on a flat surface, and 25 µL of hybridization mix (with 50 nM 5’-DIG LNA™ microRNA probe, Exiqon, Vedbaek, Denmark) was applied to them. A sterile cover slip was then applied onto each section, and fixogum was used to seal all four edges. The slides were placed in the Hybridizer and hybridized for 1 hour at 55°C. The fixogum was removed, and the cover slip was carefully removed. The slides were then placed in a coplin jar that contained 5xSSC at RT. The slides were then washed in coplin jars containing 1x SSC and 0.2xSSC at 4°C and then transferred to PBS-containing coplin jars. The slides were placed in a humidifying chamber and incubated with blocking solution for 15 minutes at 25°C. Next, anti-Digoxigenin reagent (sheep anti-Digoxigenin-AP, Roche Applied Science) diluted 1:800 in a blocking solution that contained 2% sheep serum was applied to the slides, and the slides were incubated for 60 minutes.
After the incubation with reagent, AP substrate (NBT-BCIP, Sigma) was added, and the slides were further incubated for 2 hours at 30°C. Nuclear fast red was used for nuclear counter staining. Photographs were taken under amicroscope (Leica Corporation) for both H&E sections and ISH sections.
Multiple Model Validation
To validate our results, we adopted another rat OLT model system. A rat OLT model with AR, using Lewis rats that received OLT with liver graft from ACI rats (ACI to Lewis), was performed. OLT with Lewis rats as the donors and recipients were performed (Lewis to Lewis), and these rats served as the control group. OLT with BN rats as the donors and Lewis rats as recipients were also performed (BN to Lewis), and this group was used as spontaneous liver tolerance model [7,8]. Peripheral blood was collected from the tail vein on the 3rd, 7th, and 10th days after OLT in EDTA-treated tubes. Plasma microRNAs were extracted and quantified using Taqman microRNA assays (Applied Biosystems, Foster City, CA).
miR-122 and miR-192 continued to show an upward trend from the 3rd to 10th day after OLT in the ACI to Lewis group. In BN to Lewis group, miR-122 and miR-192 peaked on the 7rd day after OLT and slightly decreased on the 10th day after OLT (Fig. S2 A and B). miR-146a showed significantly higher expression on the 3rd day after OLT both in ACI to Lewis group and BN to Lewis group when compared with Lewis to Lewis group. The expression level
of miR-146a slightly decreased from the 7th day to 10th day in ACI to Lewis group. While, in BN to Lewis group, miR-146a reduced significantly to a level that showed no significantly difference from Lewis to Lewis group on the 10th day after OLT (Fig. S2 C).
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Table S1. Representative target genes of miR-122, miR-192, and miR146a and their pathways.
Validated Target* Pathway
miR-122 AKT3 VEGF signaling, osteoclast differentiation, MAPK signaling, tight junctions, insulin signaling, carbohydrate digestion and absorption MAPK11 VEGF signaling, osteoclast differentiation, MAPK signaling NFATC1 VEGF signaling, osteoclast differentiation
ALDOA Metabolic pathways
CS Metabolic pathways
GALNT10 Metabolic pathways
CLDN18 Tight junction, cell adhesion molecules DUSP2 MAPK signaling
GYS1 Insulin signaling
NCAM1 Cell adhesion molecules
ATP1A2 Carbohydrate digestion and absorption
miR-192 DHFR Metabolic pathways, folate biosynthesis, one carbon pool by folate miR-146a CXCR4 Cytokine-cytokine receptor interactions, chemokine signaling,
leukocyte transendothelial migration LTB Cytokine-cytokine receptor interactions FADD Toll-like receptor signaling
STAT1 Toll-like receptor signaling, chemokine signaling, JAK-STAT signaling
LTB Rheumatoid arthritis
*from miRecords database
Table S2. Change of plasma microRNA expression in CCl4-induced liver injury rats.
miR-122 miR-192 miR-146a ALT
FC 22.126 8.833 1.181 18.655
P value 0.002 <0.001 0.594 0.003
CCl4-induced liver injury rats vs. control. FC, fold change
Table S3. Comparisons of microRNA expression between AR (Lewis to BN) and NR (BN to BN) rats.
miR-122 miR-192 miR-146a
Tissues FC P FC P FC P
Brain 1.086 0.909 0.918 0.770 0.970 0.913
Heart 0.937 0.901 0.489 0.116 0.581 0.222
Lung 1.287 0.726 1.331 0.326 0.920 0.784
Spleen 1.434 0.504 1.481 0.464 1.480 0.711
Kidney 0.960 0.170 1.858 0.112 1.445 0.296
Thymus 0.747 0.691 1.090 0.889 1.580 0.347
PMBCs 1.574 0.565 0.824 0.774 1.113 0.799
Liver graft 0.324 <0.001 0.247 0.002 3.209 0.014 FC, fold change; PBMCs, peripheral blood mononuclear cells
Figure S1: The timeline model of rejection. Horizontal axis is time line and
Y-axis represents treatment efficiency. The development of AR is acontinuum, with the initial events being molecular perturbations (A) following histomorphological changes (B) and clinical manifestations (C) occurring relatively late in the disease timeline. The treatment efficiency may decrease if initiated later in the course of the disease.
Figure S2: Dynamic change of plasma microRNAs in another rat model.
miR-122 (A), miR-192 (B), and miR-146a (C) showed significant increases on the 3rd, 7th, and 10th days after OLT in the ACI to Lewis group when compared with Lewis to Lewis group. miR-122 and miR-192 increased stepwise from the 3rd to 10th day in the ACI to Lewis group, but got plateau at the 7th day in the BN to Lewis group. miR-146a reached its peak level on the 3rd day after OLT. The expression level of miR-146a slightly decreased from the 7th day to 10th day in ACI to Lewis group. While, in BN to Lewis group, miR-146a reduced significantly to a level that showed no significantly difference with Lewis to Lewis group on the 10th day after OLT.
Figure S3: Histomorphological change of carbon tetrachloride(CCL4)-induced liver injury rats. Representative H&E-stained, histological sections of liver from rats receiving olive oil alone (C and D) or CCL4 in olive oil (A and B).(magnification:A,C×100; B, D×400)
Figure S4: Histomorphological examination of AR (Lewis to BN) rat and
control. After sacrifice on the 10th day post OLT, formalin-fixed and paraffin-embedded liver tissue was collected and histology examination was performed. Representative H&E-stained, histological sections of liver from normal BN rat (A), BN to BN group (B), AR+FK506 group (C) and AR group (D). (magnification:×100)
