In this study, we used the 44 k intron-exon oligoarray and stringent statistical criteria to determine the protein-coding and intronic non-coding transcript expression profiles in CD34+ and stromal cells of MDS-RARS patients and healthy individuals. We herein validated the expression of a set of selected transcripts by real time RT-PCR in five MDS-RARS patients, however future confirmation in a larger group of MDS-RARS cases is warranted. Pathway analyses of differential protein-coding transcripts pointed to new genetic networks that are altered in both CD34+ and stromal cells of MDS-RARS patients (Additional file 2).
MDS are characterized by hematopoietic insufficiency associated with cytopenia, leading to severe morbidity in addition to increased risk of leukemia transformation . The exact stage of CD34+ progenitor cells involved in the process of MDS and transformation to AML are still in debate. Bone marrow microenvironment contributes to regulate self-renewal, commitment, differentiation, proliferation and the dynamics of apoptosis of hematopoietic progenitors , and CD34+ progenitor cells are known to be severely impacted in MDS by the composition of micro environmental stimuli . Detection of differentially expressed transcripts in MDS-RARS stromal cells suggests that these transcripts could contribute to maintain CD34+ cells.
Pathways analysis of the set of protein-coding transcripts altered in CD34+ cells of MDS-RARS patients revealed an important gene network related to hematological disease, including BLNK, CD19, CD72, CD81, EBF1, F5, FLT3, LY96, MPDZ and THBS1. Down-regulation in MDS-RARS of genes related to the B cell receptor signaling pathway were found in our results (BLNK, CD19 and CD72), and the low expression of several genes involved in B lymphocyte development, corroborate the hypothesis that early MDS can be defined by a B-cell progenitor defect .
The abnormal expression of genes encoding mitochondrial proteins involved in iron metabolism has been characterized in MDS-RARS [7, 50]. The present study found 3 different ncRNA transcripts with altered expression in the CD34+ cells of MDS-RARS patients, from gene loci of mitochondrial proteins (AASS, GCDH and PPIF) and 6 modulated mitochondrial protein-coding transcripts. In addition, corroborating our findings, the down regulation of iron transporter ABCB7 has been described in CD34+ cells of patients MDS-RARS, indicating that low ABCB7 levels would contribute to abnormal mitochondrial iron homeostasis . Furthermore, genes related to heme biosynthesis pathway and transferrin trafficking (PXDN and MYO5C, respectively), were down regulated in MDS-RARS CD34+ cells. These differentially expressed genes could be related to the iron accumulation observed in mitochondria of RARS patients.
One of the mechanisms that contribute to hypercellular marrow and peripheral blood cytopenia of patients with early stage MDS is the significant increase in apoptosis of hematopoietic cells . The higher expression of Fas-FasL system found in MDS plays a role in inducing MDS bone marrow apoptosis and works in both an autocrine (hematopoietic cell-hematopoietic cell interaction) or paracrine (hematopoietic cell-stromal cell interaction) pattern . The protein encoded by SEMA3A (Class 3 semaphorins), a secreted member of the semaphorin family involved in axonal guidance, organogenesis, angiogenesis, and highly expressed in several tumor cells [53, 54], has recently been demonstrated to be an important determinant of leukemic cells sensitivity to Fas-mediated apoptosis signal . Furthermore, Sema3A has already been described to act through different signaling pathways to control neural progenitor cell repulsion activating Erk1/2 or apoptosis process involving p38MAPK . Surprisingly, SEMA3A is present in both affected networks of MDS-RARS stromal cells (see Additional file 2), suggesting participation of this gene in diverse abnormalities implicated in the modification of hematopoietic cells development and apoptosis in MDS [12, 13].
The non-coding expression profiles of CD34+ and stromal cells of MDS-RARS were clearly distinct from those obtained from CD34+ and stromal cells of healthy controls, representing 30% and 25% of the total amount of differentially expressed genes in CD34+ and stromal cells of MDS-RARS patients, respectively. Currently, evidence of the biological roles played by ncRNA have increased, especially those transcribed from partially conserved introns of protein-coding genes . Recently, eosinophil granule ontogeny (EGO) has been shown to involve an ncRNA expressed during IL-5 stimulation, whose function is to regulate MBP granule protein and EDN mRNA levels .
Interestingly, our results showed 13 differentially expressed ncRNA transcripts in CD34+ cells of MDS-RARS patients for which there was a simultaneous change in expression of the protein-coding gene in the corresponding locus: for 7 of them both the ncRNA and the protein-coding gene were simultaneously down-regulated in MDS-RARS, 5 were up-regulated, and in one gene locus the TIN ncRNA was up-regulated whereas the protein-coding gene was down-regulated. Expression of both, protein-coding and non-coding pairs in the same locus, suggest that these intronic ncRNAs may act upon cis-regulatory factors, modulating the stability and/or processing of the corresponding protein-coding transcript, or even directly affecting the levels and/or the splicing of protein-coding isoforms [27, 59]. We found 2 altered genes of the nuclear receptor subfamily 4, group A (NR4A2 e NR4A3), known to be involved in T-cell apoptosis, brain development, and vascular disease , and both showed a simultaneous up-regulation of the protein-coding and the ncRNA from the same locus in MDS-RARS, suggesting that these ncRNAs could be involved in the control of protein coding expression of this gene family in MDS-RARS patients.