miR-153-3p, a new bio-target, is involved in the pathogenesis of acute graft-versus-host disease via inhibition of indoleamine- 2,3-dioxygenase

Acute graft-versus-host disease (aGVHD) is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Therefore, seeking reliable biomarkers and delineating the potential biological mechanism are important for optimizing treatment strategies and improving their curative effect. In this study, using a microRNA polymerase chain reaction (PCR)-based chip assay, microRNA-153-3p (miR-153-3p) was screened and selected as a potential biomarker of aGVHD. The elevated plasma miR-153-3p levels at +7 d after transplant could be used to predict the upcoming aGVHD. The area under the receiver operating characteristic curve for aGVHD+/aGVHD- patients receiving haploidentical transplant was 0.808 (95% confidence interval, 0.686-0.930) in a training set and 0.809 (95% confidence interval, 0.694-0.923) in a validation set. Interestingly, bioinformatics analysis indicated that indoleamine-2,3-dioxygenase (IDO) is a potential target of miR-153-3p. In vitro study confirmed that IDO could be directly inhibited by miR-153-3p. In a GVHD model, recipient mice injected with a miR-153-3p antagomir exhibited higher IDO expression levels at the early stage after transplantation, as well as delayed aGVHD and longer survival, indicating that the miR-153-3p level at +7 d post-transplant is a good predictor of aGVHD. miR-153-3p participates in aGVHD development by inhibiting IDO expression and might be a novel bio-target for aGVHD intervention.


RNA isolation
Plasma RNA was isolated from a fixed volume of 200 μl of plasma from the patients on +7 d, +14 d, +21 d, +30 d, +45 d, +60 d and +90 d after HSCT. RNA extraction, quality control and normalization control methods were based on a previous study [1,2]. Briefly, plasma RNA was isolated using TRI Reagent (Sigma) following the manufacturer's protocol. To normalize for some of the technical variability in the plasma RNA extraction, 10 μL of a pool of synthetic C. elegans miRNAs, cel-miR-54 and cel-miR-238 (synthetic RNA oligonucleotides synthesized by RIBOBIO, China) were added (0.1 ng of each oligonucleotide) to each sample after the initial plasma denaturation. The RNA was dissolved in 30 μL of RNase-free water.
Total RNA from cells or mouse tissues was extracted using TRI Reagent (Sigma). The RNA concentrations were determined using a NanoDrop spectrophotometer (Thermo Scientific).

Real-time quantitative PCR
Plasma miRNA levels were quantified using TaqMan miRNA qRT-PCR assays as previously described [2]. A fixed volume of 5 μL RNA was reverse transcribed using the Goscript Reverse Transcription System (Promega) and miRNA-specific stem-loop primers (Invitrogen) in a 20-μL RT reaction system. Real-time PCR reactions were performed using a CFX96TM Real-Time PCR detection system (Bio-Rad). Briefly, the reactions consisted of 0.7 μL cDNA and 0.2 μM of Taqman probe. All of the reactions were performed in triplicate. Absolute copies of miR-153-3p were calculated and normalized based on the standard curve. The standard curves were generated by ten-fold serial dilutions of synthetic has-miR-153 (synthesized by RIBOBIO, China) from 10 2 to 10 13 copies. The levels of the synthetic miRNAs were assessed by RT-qPCR assay. The resulting Ct values were plotted versus the log of the amount of synthetic miRNAs. Absolute copies of miR-153-3p from plasma were calculated based on the standard curve and then multiplied by a normalization factor generated from the spiked C. elegans control and a selected endogenous control (has-miR-486) that was selected for its high levels and constant expression in plasma. The normalization factor was calculated for each sample based on the following formula: normalization factor=1/[2^(Median_Control_Ct value)−(Control_Average_Ct value of the given sample).
IDO mRNA expression was detected by SYBRGreen-based qRT-PCR. The relative expression was calculated using the comparative Ct method (2 -ΔΔCt ). Each value was adjusted using GAPDH as the appropriate reference.

Cell culture and transfection
HeLa cells and HEK293T cells (ATCC) were cultured in DMEM (Invitrogen) supplemented with 10% FBS (GIBCO). Plasmids and miRNAs were transfected into HeLa or HEK293T cells using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions.
The cDNA fragments were inserted into a luciferase dualreporter plasmid psiCHECK™-2 Vector (Promega). HEK293T cells were seeded into 35-mm wells (1 x 10 5 / well) and incubated overnight followed by transfection with various plasmids and miRNAs. Twenty-four hours after transfection, the cells were lysed, and the luciferase activity was measured using the Dual Luciferase Reporter Gene Assay Kit (Beyotime, China).

Preparation of cell extracts and western blot analysis
HeLa cells were transfected with 50 nM miR-153-3p mimic or miR mimic control for twelve hours followed by treatment with 1 ng/ml INF-gamma (Peprotech). Twenty-four hours after the treatment, the cells were harvested and lysed in radioimmunoprecipitation assay (RIPA) lysis buffer [150 mM NaCl, 1% (v/v) Nonidet P-40, 0.1% (w/v) SDS, 0.5% deoxycholic acid, 1 x complete protease inhibitor cocktail (Roche)] in 50 mM phosphate buffer, pH 8.0. The proteins were separated by SDS-PAGE and then transferred onto a PVDF membrane (PALL) and probed with IDO antibody (Eptitomics) and GAPDH antibody (Cell Signaling). The proteins were visualized using the Supersignal West Pico Chemiluminescent Substrate kit (Pierce).

Immunohistochemistry
Mouse tissues were formalin-fixed followed by paraffin embedding. The embedded sections were examined by hematoxylin and eosin staining. Granzyme B and IDO immunostains were performed using rabbit polyclonal granzyme B antibody (Genetex) and polyclonal IDO antibody (Novus Biologicals), respectively, and visualized using an AEC kit.

Determination of plasma IDO
Plasma IDO levels were determined using a highly specific and sensitive human indoleamine 2,3-dioxygenase ELISA kit (WuHan Huamei Biotech Co., LTD) according to the manufacturer's instructions.
Briefly, 100 μl of patient plasma was transferred into 96well plates coated with IDO antibody and incubated for 2 h at 37°C. After the wells were washed, 100 μl of HRPlabeled monoclonal antibody was pipetted into the wells, and the plate was incubated for 1 h at 37°C. Next, 90 μl of substrate solution was added to each well, and the plate was incubated in the dark at room temperature for 20 minutes. After the addition of 50 μl of stop solution to each well followed by 10 seconds of agitation, the plate was read at 450 nm in a microplate reader. The sensitivity was 1 U/ml. All of the measurements were performed in duplicate wells.