Quality assurance of hematopoietic stem cells by macrophages determines stem cell clonality

Wattrus, Samuel J.; Smith, Mackenzie L; Rodrigues, Cecília Pessoa; Hagedorn, Elliott J.; Budnik, Bogdan; Zon, Leonard I.

doi:10.1101/2022.03.07.483327

Cited by 2 publications

(2 citation statements)

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“…Macrophages have heterogeneity and plasticity and can be classi ed in vitro as two types: the classically activated (pro-in ammatory) M1 and the alternatively activated (anti-in ammatory) M2 [18,19] . The polarization and switch between M1 and M2 subtypes of macrophages are important for maintaining normal hematopoietic stem cell proliferation, differentiation, and development [17,[20][21][22] . However, the detection of the two polarized subtypes of macrophages in the bone marrow of PNH patients is currently lacking.…”

Section: Introductionmentioning

confidence: 99%

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Dysregulation of the miRNA-30a-5p/B4GALT5 axis leads to cell proliferation and is associated with M1 Macrophage Polarization in PNH

zeng

Wang

et al. 2023

Preprint

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Background：MicroRNAs (miRNAs) play diverse roles in various biological processes. However, their role in Paroxysmal Nocturnal Hemoglobinuria (PNH) is not well understood. To investigate the involvement of miRNAs in PNH, we conducted a re-analysis of the GEO database and identified miRNA-30a-5p as the most promising candidate for further mechanism research. Methods: We have constructed a PIGA knock-out K562 cell line and PIGAconditional knock-out mice model by CRISPR/cas9 technology. Furthermore, FACS and colony formation assays were used to clarify proliferative and apoptosis abilities of PNH cells. FACS was used to clarify M1 Macrophage Polarization in mouse bone marrow-derived macrophages (BMDMs). Luciferase assay was used to indicate miRNA target gene. RT-PCR and western blot were used to verify the relationship between miR-30a-5p, B4GALT5 and PI3K/AKT signaling. Results: Our study revealed a significant upregulation of miR-30a-5p in Flaer negative cells compared to Flaer positive cells, in a cohort of 10 PNH patients. Cell colony formation and cell cycle analysis demonstrated that the miRNA-30a-5p mimics group exhibited a higher capacity for proliferation, while the miRNA-30a-5p inhibitor group showed opposite effects. Subsequent luciferase assay results confirmed that miRNA-30a-5p targeted the B4GALT5 gene, which was found to be significantly downregulated in Flaer negative cells from 15 PNH patients. In vitro assays showed that the miRNA-30a-5p/B4GALT5 axis significantly promoted cell proliferation by activating PI3K/AKT signaling. Additionally, our study is the first to describe the polarization state of macrophages in the bone marrow microenvironment of PNH patients, where we observed a higher mean fluorescence intensity (MFI) of CD80 and CD86, markers of M1 macrophages, on macrophages from PNH patients compared to normal controls. Furthermore, siB4GALT5 increased the markers of M1 macrophages in BMDMs, and miRNA-30a-5p inhibitor induced M2-like macrophage polarization, which was markedly reversed in the miRNA-30a-5p inhibition+siB4GALT5 group. Conclusion: Our study has identified that the miRNA-30a-5p targets the B4GALT5 gene, and miRNA-30a-5p/B4GALT5 axis facilitates the proliferation of PNH clones via the PI3K/AKT signaling pathway. Additionally, our findings provide initial evidence that the miRNA-30a-5p/B4GALT5 axis may play a potential role in M1 macrophage polarization in the bone marrow of PNH patients.

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Section: Introductionmentioning

confidence: 99%

“…Next, we explored their effects and molecular mechanisms on PNH clone proliferation. Furthermore, the bone marrow microenvironment signi cantly regulates the self-renewal and differentiation of hematopoietic stem cells, in which macrophages are an important component [17] .…”

Section: Introductionmentioning

confidence: 99%