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Background: Altered T‐cell repertoire with an aberrant T‐cell activation and imbalance of the Th17/Treg cells has been reported in acquired aplastic anemia (aAA). miRNAs are well known to orchestrate T‐cell activation and differentiation, however, their role in aAA is poorly characterized. The study aimed at identifying the profile of miRNAs likely to be involved in T‐cell activation and the Th17/Treg‐cell imbalance in aAA, to explore newer therapeutic targets. Methods: Five milliliters peripheral blood samples from 30 patients of aAA and 15 healthy controls were subjected to flow cytometry for evaluating Th17‐ and Treg‐cell subsets. The differential expression of 7 selected miRNAs viz; hsa‐miR‐126‐3p, miR‐146b‐5p, miR‐155‐5p, miR‐16, miR‐17, miR‐326, and miR‐181c was evaluated in the PB‐MNCs. Expression analysis of the miRNAs was performed using qRT‐PCR and fold change was calculated by 2−ΔΔCt method. The alterations in the target genes of deregulated miRNAs were assessed by qRT‐PCR. The targets studied included various transcription factors, cytokines, and downstream proteins. Results: The absolute CD3+ lymphocytes were significantly elevated in the PB of aAA patients when compared with healthy controls (p < 0.0035), however, the CD4:CD8 ratio was unperturbed. Th17: Treg‐cell ratio was altered in aAA patients (9.1 vs. 3.7%, p value <0.05), which correlated positively with disease severity and the PNH positive aAA. Across all severities of aAA, altered expression of the 07 miRNAs was noted in comparison to controls; upregulation of miR‐155 (FC—2.174, p‐value‐0.0001), miR‐146 (FC—2.006, p‐value‐0.0001), and miR‐17 (FC—3.1, p‐value‐0.0001), and downregulation of miR‐126 (FC—0.329, p‐value‐0.0001), miR‐181c (FC—0.317, p‐value‐0.0001), miR‐16 (FC—0.348, p‐value‐0.0001), and miR‐326 (FC—0.334, p‐value‐0.0001). Target study for these miRNAs revealed an increased expression of transcription factors responsible for Th1 and Th17 differentiation (T‐bet, RORϒt, IL‐17, IL‐6, and IFN‐ϒ), T‐cell activation (NFκB, MYC, and PIK3R2), downregulation of FOX‐P3, and other regulatory downstream molecules like SHIP‐1, ETS‐1, IRAK‐1, TRAF‐6, and PTEN. Conclusion: The study for the first time highlights the plausible role of different miRNAs in deregulating the Th17/Treg‐cell imbalance in aAA, and comprehensively suggest the role of altered NF‐kB and mTOR pathways in aAA. The axis may be actively explored for development of newer therapeutic targets in aAA. [ABSTRACT FROM AUTHOR]

Introduction: MicroRNAs (miRNAs) play a critical role in orchestrating T cell differentiation and activation and may thus play a vital role in acquired aplastic anemia (aAA). The study aimed to evaluate the differential expression of selected miRNAs and their relevant target genes in bone marrow samples of aAA patients. Methods: Differential expression of 8 miRNAs viz; hsa‐miR‐126‐3p, miR‐145‐5p, miR‐155‐5p, miR‐150‐5p, miR‐146b‐5p, miR‐34a, miR‐29a, and miR‐29b was evaluated in the bone marrow mononuclear cells of aAA patients. TaqMan microRNA assay was performed for preparing the cDNA of specific miRNA, followed by expression analysis using qRT‐PCR. Data were normalized using two endogenous controls, RNU6B and RNU48. Delta‐delta CT method was used to calculate the fold change (FC) of miRNA expression in individual samples, and a FC of >1.5 was taken as significant. Target genes of these miRNAs were evaluated by qRT‐PCR. Results: Thirty five samples of aAA patients and 20 controls were evaluated. Irrespective of the disease severity, five miRNAs were found to be deregulated; miR‐126 (FC‐0.348; P‐value‐.0001) and miR‐145 (FC‐0.31; P‐value‐.0001) were downregulated, while miR‐155 (FC‐3.50; P‐value‐.0067), miR‐146 (FC‐3.13; P‐value‐.0105), and miR‐150 (FC‐5.78; P‐value‐.0001) were upregulated. Target gene study revealed an upregulation of PIK3R2, MYC, SOCS1, and TRAF‐6, and downregulation of MYB. Conclusion: This is the first study from the Indian subcontinent demonstrating the presence of altered miRNA expression in the bone marrow samples of aAA patients, suggesting their role in the pathogenesis of the disease. A comprehensive study focusing on the effect of these miRNA‐mRNA interactions is likely to open new avenues of management. [ABSTRACT FROM AUTHOR]

We have investigated the expression of RNA transcripts of hematopoiesis regulatory molecules, viz., macrophage inflammatory protein (MIP)-1α, tumor necrosis factor (TNF)-α, granulocyte colony-stimulating factor (G-CSF), stromal cell-derived factor (SDF)-1α, stem cell factor (SCF), and transforming growth factor (TGF)-β in lipopolysaccharide-induced bone marrow mesenchymal stem cells (BM-MSCs) and levels of their soluble forms in the culture supernatants of BM-MSCs and BM plasma of patients with acquired aplastic anemia (AA) (n=29) and controls (n=29). The BM-MSCs of AA patients as compared to controls had markedly lower expression of MIP-1α transcripts (p<0.001), higher expression of TNF-α (p<0.001), G-CSF (p<0.001), and SDF-1α (p<0.01) transcripts, and no difference in the expression of SCF and TGF-β transcripts. The culture supernatants of BM-MSCs and BM plasma of AA patients in comparison to controls also had lower levels of MIP-1α (p<0.01 and p<0.001, respectively) and higher levels of TNF-α (p<0.05 for both) and G-CSF (p<0.05 and p<0.001, respectively) but with no difference in the levels of SDF-1α and SCF. The levels of TGF-β were although similar in culture supernatants of BM-MSCs of both the groups, but they were significantly lower in BM plasma of the patients than controls (p<0.001). Our data shows that BM-MSCs of AA patients have altered expression of hematopoiesis regulatory molecules suggesting that they may have a role in the pathogenesis of the disease. [ABSTRACT FROM AUTHOR]