Integrated mRNA and miRNA Profiling Revealed Deregulation of the Cellular Stress Response in Bone Marrow Mesenchymal Stem Cells Derived from Patients with Immune Thrombocytopenia

Introduction: Immune thrombocytopenia (ITP) is an autoimmune disease characterized by accelerated platelet destruction and decreased platelet production. Our understanding of its pathogenesis remains limited due to the complexity of the immune system and the heterogeneity of the disease (Rivière et al. Br J Haematol. 2015, 170). Recently, we found that the bone marrow mesenchymal stem cells (MSCs) derived from ITP patients (MSC-ITP) exhibited growth defects and functional abnormalities, which may be implicated in the breakdown of self-tolerance in ITP (Zhang et al. Stem Cells Transl Med.2016, 5). The underlying mechanism of these defects remains unclear. In this study, by utilizing microarray technique and bioinformatics analysis, we aimed to profile both the mRNA and microRNA expression and decipher the underlying mechanism involved in the impairment of MSC-ITP.

Methods: MSCs were derived from 4 ITP patients and age-, sex- and BMI-matched healthy individuals served as control. Microarray was performed on total RNA specimens using Affymetrix mRNA and miRNA microarray.

Results: Overall, we identified 740 genes and 32 microRNAs that were differently expressed in ITP patients compared with controls. Function and disease analysis revealed that the differentially expressed genes were most significantly enriched in the processes of cell death and survival, cellular development, cell growth and proliferation and cell cycle, which is in agreement with our previous study demonstrating the excessive apoptosis and senescence of MSCs from ITP patients. Ingenuity canonical pathway annotation enabled us to assign differentially expressed genes to 57 pathways. Most of these top enriched pathways have functions in cell proliferation, cell cycle and cell death. Of note, unfolded protein response (UPR), which is a defense response to protect cells from damage induced by endoplasmic reticulum stress, was identified as the most significantly changed. Furthermore, GSEA identified that UPR was negatively correlated with MSC-ITP. These data suggested that deficient UPR may play key roles in MSC-ITP. Upstream analysis revealed a significantly decreased overall DNA transcription, and many stress response transcriptional factors such as NUPR1 and TGF-b were down-regulated, indicating a potential inability of the cellular stress response. Further, we utilized gene network analysis in an attempt to target the key "cross-road" regulator in MSC-ITP. Network analysis demonstrated that JUN and CDKN1A were two key cross regulators with multiple roles in the biological process of MSC-ITP. JUN and CDKN1A were critical downstream regulators in UPR and the p53 pathway, respectively, implying a potential cross-talk between UPR and the p53 pathway, the latter of which has been verified in MSC-ITP in our previous study. Finally, we utilized microRNA-mRNA interactions analysis to improve our understanding of the regulated genes. Reactome enrichment of the predicted targeted genes of differentially expressed microRNA indicated that the cellular response to stress was deregulated in MSC-ITP. Then, the predicted target genes were cross-referenced against the mRNAs significantly regulated in MSC-ITP and MSC-control, resulting in 163 overlap genes. Again, UPR was identified as the most related process in MSC-ITP by Reactome enrichment, which further confirmed the association between UPR and the defect of MSC-ITP.

Conclusion: Taken together, our results illustrate that the mRNA and microRNA profiles change in MSC-ITP, which revealed a weakened ability to initiate the cellular stress response to protect cells from damage and restore homeostasis. Considering MSCs as an important immunoregulator, these results in MSC-ITP may shed light on the complex ITP bone marrow niche and on the breakdown of self-tolerance.