Objective To explore the molecular mechanism of microRNA-29b (miR-29b) targeting frizzled 6 (FZD6) involved in daunorubicin (DNR) resistance of acute myeloid leukemia (AML) cells. Methods CCK-8 was used to detect the viability of AML cell lines at different DNR concentrations, and quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-29b in AML cell lines. THP-1 cells were selected as the research object, and they were divided into: control group (normally cultured), miR-NC mimics group (transfected with miR-NC mimics), miR-29b mimics group (transfected with miR-29b mimics), miR-29b mimics+pcDNA group (transfected with miR-29b mimics and pcDNA), miR-29b mimics+pcDNA-FZD6 group (transfected with miR-29b mimics and pcDNA-FZD6), and treated with 10 M DNR for 24 h after 24 h of transfection. QRT-PCR and Western blot were used to detect the expression levels of miR-29b and FZD6 in THP-1 cells treated with DNR in each group. Bioinformatics and dual luciferase reporter gene was used to predict and detect the targeting relationship between miR-29b and FZD6. CCK-8, Edu method and flow cytometry were used to detect the proliferation and apoptosis of THP-1 cells in each group treated with DNR. Results The resistance of AML cell lines to DNR was concentration-dependent, qRT-PCR results showed that the expression of miR-29b in AML cell lines THP-1 (1.00±0.05), KG-1 (1.87±0.16), HL-60 (2.96±0.21) and Kasumi-1 (3.73±0.29) gradually increased (P<0.05). Dual luciferase reporter gene detection, qRT-PCR and Western blot proved that miR-29b could directly negatively target FZD6 (0.32±0.04, 0.37±0.05, 0.48±0.06, P<0.05). Over expression of miR-29b could weaken the resistance of AML cells to DNR, inhibit cell proliferation, and enhance cell apoptosis (43.59±5.24, 21.97±3.13, P<0.05). Up-regulation of FZD6 could reverse the effect of up-regulation of miR-29b expression on the proliferation and apoptosis of THP-1 cells to a certain extent (65.21±6.39, 38.26±4.09, P<0.05). Conclusions MiR-29b reduces the resistance of AML cells to DNR by targeting and inhibiting FZD6.
Key words
microRNA-29b /
frizzled-6 /
acute myeloid leukemia /
daunorubicin /
drug resistance
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