Downregulation of TANK gene affects erythroid differentiation in PNH patients.

Abstract Background:Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disease of hematopoietic stem cells characterized by complement mediated intravascular hemolysis. Although complement inhibitors significantly improve the quality of life and prolong survival of PNH patients, in the real world, a small number of classic PNH patients who receive complement inhibition therapy cannot restore normal hemoglobin and do not have bone marrow failure despite hemolysis control. The above situation suggests that abnormal development and differentiation of erythroid hematopoiesis in PNH may be involved in the pathogenesis of PNH. Methods:Extract bone marrow mononuclear cells from 5 PNH patients and 5 healthy donors, and perform deep singlecell RNA sequencing on CD59- and CD59+ cells sorted by flow cytometry. Divide the erythroid development stage based on GYPA expression levels to identify differentiation abnormalities and differentially expressed genes. Subsequently, CD34+ cells from PNH and normal bone marrow samples were collected and induced in vitro to the CFU-E stage for qPCR detection of TANK expression levels. Further construct a TANK overexpression cell model based on the established PIGA knockout K562 cell line, and evaluate its regulatory role in erythroid differentiation through flow cytometry, qPCR, and Western blot. Results:The single-cell RNA sequencing results showed that there were abnormal erythroid differentiation trajectories in the bone marrow clones of PNH patients. Based on the expression level of GYPA, we divided red blood cells into seven developmental subgroups, corresponding to the differentiation stages from megakaryoerythroid progenitor cells (MEP) to eosinophils (OrthoE). It was found that in the OrthoE stage, the proportion of cells in the CD59 ⁺ group (P group) and CD59 ⁻ group (N group) of PNH patients was significantly lower than that in the healthy control group (C group). In the PolyE stage, the proportion of cells in the N group was slightly higher than that in the C group, indicating the possibility of cell aggregation or delayed differentiation in this stage. Further analysis of differentially expressed genes revealed that TANK gene was significantly downregulated in CD59 ⁻ clones during the CFU-E stage, while the differences were not significant in other erythroid differentiation stages, suggesting that it may have specific regulatory effects during the CFU-E stage. Subsequently, the primary CD34 ⁺ cell erythroid differentiation system was selected for validation. Extraction of bone marrow mononuclear cells from 7 classic PNH patients and 5 healthy controls, magnetic bead sorting of CD34 ⁺ hematopoietic stem/progenitor cells and induction to CFU-E stage (CD34 ⁻ CD36 ⁺), qPCR results showed that the TANK expression level in PNH group cells was significantly lower than that in the normal control group (P=0.023). by immunomagnetic beads To further validate the function of TANK, we constructed a PNH cell model. The results showed that in PIGA knockout K562 cells, erythroid differentiation was inhibited, and the proportion of CD36 ⁺ and CD235a ⁺ cells was significantly lower than that of wild-type K562 cells. In TANK overexpressing (OE) cells, the proportion of CD36 ⁺ CD235a ⁺ cells significantly increased (P=0.004), partially reversing the differentiation defects caused by knockout. The qPCR and Western blot results further confirmed that TANK expression was significantly increased in the OE group. Conclusion:Single cell sequencing results revealed abnormal erythroid differentiation in PNH patients. In addition, When CD34+cells from PNH patients were induced to the CFU-E stage in vitro, TANK gene expression was significantly downregulated, which may play an important regulatory role in the differentiation of red blood cells in PNH patients.