Circulating Mir-153 Participates in Occurrence of Acute Graft-Versus-Host Disease By Down-Regulating Indoleamine-2,3-Dioxygenase

Background Indoleamine-2,3-dioxygenase (IDO) is able to catalyze the first and rate-limiting step in the catabolism of tryptophan, which is the essential amino acid for T cell proliferation. Many previous studies have proved that IDO is able to play important roles in immunosuppression-associated with tumor immunity, autoimmunity and chronic infection. Since donor T cells will be activated, proliferate, differentiate and finally lead to tissue injury, growing research paid more attention to the immunoregulatory role of IDO in the process of aGVHD. Our previous work also demonstrated that plasma IDO level was correlated with the severity of aGVHD. Increased IDO expression would serve as a protective molecular during aGVHD. However, the exact regulatory mechanism of IDO is still unclear. MicroRNAs (miRNAs) are a class of small non-coding RNA that negatively regulate gene expression by translational repression or induction of mRNA degradation. Recently, circulating miRNAs have been reported to be the promising biomarkers for various kinds of diseases. We speculated that miRNA that might regulate those GVHD-related molecules/cytokines expression including IDO, and the circulating miRNA that directed at IDO would serve as new potential biomarker for aGVHD. This study is to find the potential regulatory miRNA of IDO and investigate the correlation between plasma miRNA expression and aGVHD.

Methods  The potential regulatory miRNA for IDO was identified through bioinformatics analysis using TargetScan and Miranda. The regulatory function of target miRNA on IDO protein and gene was confirmed by Western Blot analysis and luciferase assay. Human plasma samples from 70 patients who underwent allo-HSCT from Sep 2012 to Jun 2013 were prospectively collected at +7 day(d), +14d, +21d, +30d, +45d, +60d, +90d and the occurrence of aGVHD from Peking University Institute of Hematology. All patients provided informed consent. This study was approved by the institutional review board at the Peking University Institute of Hematology (Protocol number 2013-42). Real-time quantitative PCR (RQ-PCR) analysis was performed to examine the miRNA expression of plasma at different time points posttransplant. Receiver operating characteristic (ROC) analysis was employed to determine the optimal cut-off value for predict the occurrence of aGVHD.

Results  Through bioinformatics analysis, we found the predicted target sites in its 3’UTR binding miRNA-153. The intracellular IDO protein would be reduced when miRNA-153 was transfected into interferon-gamma-treated Hela cells. Surprisingly, the mRNA expression level of IDO was also affected in miRNA-153 transfected cells obviously (P<0.001). In addition, luciferase assay in 293T cells showed that miRNA-153 was able to bind directly to the promoter region of IDO gene and affect its transcription. Luciferase activity dropped around 44% when wild-type construct was co-transfected into 293T cells with miRNA-153 (P=0.009). However, when the potential binding sites were mutated, this phenomenon could not be observed. This indicates that the IDO 3’UTR is normally targeted by endogenous miR-153. Among the 70 patients who underwent allo-HSCT, 35 patients developed into aGVHD and 35 patients did not. A significant difference in miRNA-153 expression levels between the two groups was confirmed at +7d (P<0.001). Furthermore, we used ROC analysis to evaluate the diagnostic accuracy. Plasma levels of miRNA-153 at day 7 could predict an impending aGVHD with a sensitivity of 38.71% and a specificity of 100%, and the area under the curve (AUC) was 0.898 (95%CI: 0.821-0.974, P<0.0001).

Conclusions  Our study suggested that miRNA-153 might participate in occurrence of aGVHD by down regulating IDO and might be a putative target for novel aGVHD therapy. The plasma level of miRNA-153 at +7d after allo-HSCT would be a good biomarker for predicting the occurrence of aGVHD.