[Effect of Knocking Out HOXA5 Gene by CRISPR-Cas9-Mediated Gene Editing Technique on Proliferation of Acute Myeloid Leukemia Cells].

OBJECTIVE: To construct a acute myeloid leukemia (AML) cell line in which HOXA5 gene is stably knocked out by CRISPR-Cas9-mediated gene editing technique, so as to clarify the effect of HOXA5 gene knockout on the proliferation of AML cells, and preliminarily explore the role of HOXA5 gene in the pathogenesis of AML. METHODS: The expression of HOXA5 in bone marrow mononuclear cells (BMMC) of non-tumor hematological patients and newly diagnosed AML patients was detected by quantitative real-time PCR (qRT-PCR) and Western blot, respectively. The AML cell line KO-HOXA5-THP-1 was constructed in which HOXA5 gene was knocked out by CRISPR-Cas9-Mediated gene editing technique, and the knockout of HOXA5 gene was verified by qRT-PCR and Western blot, and the cell proliferation was detected by CCK-8 assay. RESULTS: Compared with non-tumor hematological patients, the levels of HOXA5 gene and protein in BMMC of newly diagnosed AML patients were significantly increased (P <0.05). The stable HOXA5 knockout cell line can be obtained by CRISPR-Cas9-Mediated gene editing technique, and the proliferation ability of THP-1 cells with HOXA5 gene knockout was significantly decreased (P <0.05). CONCLUSION: HOXA5 is highly expressed in AML cells, and knocking out HOXA5 can significantly affect the proliferation ability of AML cells, which provides a new potential therapeutic target for the precise treatment of AML.

Salinomycin sodium exerts anti diffuse large B-cell lymphoma activity through inhibition of LRP6-mediated Wnt/ß-catenin and mTORC1 signaling.

Low-density lipoprotein receptor-related protein-6 (LRP6) is overexpressed in various cancers. The small molecule salinomycin sodium inhibits LRP6. We observed a higher proportion of subjects with non-germinal center B (non-GCB) subtypes having high LRP6 expression than those with GCB subtypes by immunohistochemistry. The PCR and Western blot assays demonstrated increased LRP6 expression in non-GCB subtype cells. In addition, CCK-8 assays and transwell cell migration assays revealed that salinomycin sodium exhibited dose- and time-dependent inhibition of proliferation and migration in non-GCB subtype cells. Furthermore, Western blot assays showed that salinomycin sodium decreased the expression of Bcl2, while increasing the expression of Bax. Additionally, salinomycin sodium suppressed LRP6 expression, blocked LRP6 phosphorylation, and inhibited the Wnt/ß-catenin and mTORC1 signaling pathways. Our results suggest that LRP6 is highly expressed in non-GCB subtype. Furthermore, salinomycin sodium inhibited LRP6 expression and the Wnt/ß-catenin and mTORC1 signaling in non-GCB subtype cells, and displayed potent anticancer activity.

CXCL2 benefits acute myeloid leukemia cells in hypoxia.

INTRODUCTION: Drug resistance and relapse of acute myeloid leukemia (AML) is still an important problem in the treatment of leukemia. Leukemia outbreak causes severe hypoxia in bone marrow (BM), remolding BM microenvironment (niche), and transforming hematopoietic stem cell (HSC) niche into leukemia stem cell (LSC) niche. AML cells and the microenvironment usually conduct "cross-talk" through cytokines to anchor resistant AML cells into LSC niche, thus supporting their survival. Therefore, this study was aimed to investigate the role of CXCL2 in the hypoxic AML niche. METHODS: AML hypoxic niche was simulated by hypoxic culture of THP-1 and HL-60 cells in vitro, thus to study the effects of CXCL2 on the proliferation and migration of AML cells. The expression of hypoxia-inducible factor-1α (HIF-1α) and the activation of survival-related kinases such as PIM2 and mTOR under CoCl2 -simulated hypoxic conditions were detected. The correlation between CXCL2 and the prognosis of AML with big data was verified. RESULTS: (a) CXCL2 promoted the proliferation and migration of AML cells. (b) CXCL2 up-regulated the expression of PIM2 by enhancing the transcriptional activity of HIF-1α. (c) CXCL2 activated mTOR in AML cells. (d) CXCL2 was associated with poor prognosis in AML. CONCLUSION: CXCL2 promotes survival, migration, and drug resistance pathway of AML cells in hypoxia and is associated with poor prognosis in AML. Therefore, CXCL2 can be considered as an important factor in promoting the development of AML cells in hypoxia.

Hypoxia-CXCL6 axis affects arteriolar niche remodeling in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a malignant clonal disease derived from hematopoietic stem/progenitor cell. Leukemia blasts cause extensive hypoxia of bone marrow (BM), which lead to disorder and remodeling of BM niche, thereby becoming "leukemic niche" to support the development and drug-resistance of AML as well as the maintenance of normal hematopoietic stem cells. In this study, the biological characteristics (such as self-renewal, apoptosis, migration, autocrine) and function (vascularization) of mesenchymal stem cells (MSCs) and human umbilical artery endothelial cells (HUAECs) that make up BM arteriolar niche in simulated hypoxia AML context were investigated. It was found that moderate hypoxia enhanced the viability of the arteriolar niche cells, but severe hypoxia of AML BM resulted in the damage of arteriolar niche cells and the disorder of vascular cytokines C-X-C motif chemokine ligand 6 (CXCL6). The dynamic changes of CXCL6 in the system as well as its anti-apoptotic and promoting angiogenic effects suggested that CXCL6 played an important role in the remodeling of BM arteriolar niche in AML. Taking advantage of CXCL6 can save the damaged MSCs and HUAECs, which is the hope of rescuing arteriolar niche. It is suggested that CXCL6 may be an assistant strategy for microenvironment targeted therapy of AML.