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[Knocking Out DNMT1 Enhances the Inhibitory Effect of NK Cells on Acute Myeloid Leukemia]
Kun Wu1,2,3, Jia-Li Huang4,5, Shen-Ju Cheng1,2,3
1Yunnan Key Laboratory of Laboratory Medicine,Kunming 650032, Yunnan Province, China.
Objective:
To explore the effect and mechanism of DNA methyltransferase 1 (DNMT1) knockout on the inhibition of acute myeloid leukemia (AML) by natural killer (NK) cells.
Methods:
The peripheral blood NK cells of AML patients and controls were collected, and the mRNA and protein level of DNMT1 were measured by PCR and Western blot, respectively. The DNMT1 knockout mice were constructed to obtain NKDNMT1-/- cells. The NK cells were stimulated with interleukin (IL)-12, IL-15, and IL-18 to construct memory NK cells, and then the interferon-γ (IFN-γ) levels were measured by ELISA. After co-culturing with memory NK cells and HL60 cells, the killing effect of NKDNMT1-/- cells on HL60 cells was detected by LDH assay. Then, the HL60 cell apoptosis and NK cell NKG2D level were measured by flow cytometry. The perforin and granzyme B protein levels of NK cells were measured by Western blot. The AML model mice were constructed by injecting HL60 cells into the tail vein, meanwhile, memory NK cells were also injected, and then the mouse weights, CD33 positive rates, and survival time were detected.
Results:
The mRNA and protein levels of DNMT1 in NK cells of AML patients were significantly higher than those in the control group (both P < 0.01), while the IFN-γ level induced by interleukin was significantly lower than that in the control group (P < 0.05). Compared with NKDNMT1+/+ cells, the ability of NKDNMT1-/- cells to secrete IFN-γ after interleukin stimulation was significantly increased (P < 0.05). The killing and apoptosis-inducing effects of NKDNMT1-/- cells on HL60 cells were significantly stronger than those of NKDNMT1+/+ cells (both P < 0.05). The NKG2D level and expression of perforin and granzyme B of NKDNMT1-/- cells were significantly increased compared with NKDNMT1+/+ cells (all P < 0.05). Compared with AML mice injected with NKDNMT1+/+ cells, AML mice injected with NKDNMT1-/- cells showed significantly increased body weight, decreased CD33 positive rate, and prolonged survival time (all P < 0.05).
Conclusion:
Knocking out DNMT1 can enhance the inhibitory effect of NK cells on AML, which may be related to enhancing NK cell memory function.
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