Decitabine Regulates the Function of Mesenchymal Stem Cells By Restoring MAGEA4-Mediated Apoptosis in Immune Thrombocytopenia

Introduction The impaired immunomodulatory functions of bone marrow mesenchymal stem cells (MSCs) have been demonstrated to play crucial roles in the development of immune thrombocytopenia (ITP). Our previous study showed that enhanced complement activation in bone marrow microenvironment resulted in the dysfunction of ITP-MSCs, which was manifested as reduced capacity to proliferate and increased apoptosis. However, the underlying mechanism of the complement system in the dysfunctional ITP-MSCs remains poorly understood. This study aimed to elucidate the probable regulatory function of complement on ITP-MSCs. Methods ELISA kits were used to quantify the deposition of complement C3a and C5a on the isolated bone morrow MSCs from ITP patients and healthy controls. Illumina HiSeq system was employed for transcriptome profiling to detect the expression profiles of mRNAs and lncRNAs. TMT-based quantitative proteomics was used to characterize the protein profiles in MSCs. The active ITP mice model were constructed to explore the effect of decitabine. Results ITP-MSCs were divided into the group without complement activation (MSCs-C-) if the levels of both C3a and C5a were less than the value of healthy controls. The remaining ITP-MSCs were allocated to the complement activation group (MSCs-C+). RNA sequencing showed that a total of 912 and 1391 lncRNAs were significantly upregulated and downregulated, respectively, in MSCs-C+ compared with MSCs-C-. KEGG analysis demonstrated that the differential lncRNAs were principally enriched in cellular senescence. As shown in the lncRNA-TF-mRNA network, two lncRNAs coexpressed with mRNAs as well as overlapping with the target genes of a specific TF were included. We found that LILRB4, MAGEA4, RGS1, MYL2, MYH6, and SLITRK5 were differentially expressed between MSCs-C+ and MSCs-C-.The decreased expression of MAGEA4 in MSCs-C+ was confirmed by PCR and western blot. We hypothesized that the reduced expression of MAGEA4 may take a part in inducing apoptosis of MSCs-C+. Consistent with our hypothesis, MAGEA4 overexpression decreased apoptosis, as measured by the levels of Bcl-2/Bax ratio and cleaved caspase-3 in MSCs-C+. To identify the pathways driven by MAGEA4 in MSCs, we used proteomics to compare the protein profiles of MSCs and MSCs with MAGEA4 overexpression. KEGG analysis indicated that the differential proteins were mainly enriched in PI3K-Akt signaling pathway. The decreased apoptosis brought by MAGEA4 overexpression was revoked by Akt phosphorylation inhibitor. We further investigated the possible mechanism of decreased MAGEA4 in MSCs-C+. Methylation-specific PCR indicated that the methylation status of MAGEA4 promoter was increased in MSCs-C+. DNA hypomethylating agent decitabine reduced the methylation of the promoter and increased MAGEA4 expression in MSCs-C+. Decitabine restored the impaired proliferation ability of MSCs-C+ and prevented cell cycle arrest at G0/G1 phase. Our previous study demonstrated that ITP-MSCs exhibited defects in immunoregulation. Here, we cocultured CD4+ T cells with MSCs from healthy controls and MSCs-C+ treated with and without decitabine. Compared with MSCs from healthy controls, the percentage of regulatory T cells was reduced when cocultured with MSCs-C+, which was rectified with decitabine. In vivo, decitabine accelerated platelet recovery in ITP mouse model and reduced bleeding symptoms. After treatment with decitabine, the proliferation ability and immunomodulatory function of bone marrow MSCs were restored in ITP mice. Conclusions This study demonstrated that complement activation inhibited the expression of MAGEA4 in MSCs from ITP. Decreased level of MAGEA4 impaired the proliferation ability of MSCs and promoted apoptosis via PI3K-Akt pathway. Decitabine reduced the methylation of the promoter and increased MAGEA4 expression. Decitabine treatment inhibited apoptosis and restored the immunosuppressive capacity of bone marrow MSCs. Our findings may help further understand the specific role of MAGEA gene expression in ITP and provide theoretical basis for the application of decitabine in the treatment of ITP.