微小RNA-29b靶向卷曲蛋白6参与急性髓系白血病细胞柔红霉素耐药的分子机制

Molecular mechanism of MicroRNA-29b targeting frizzled-6 involved in daunorubicin resistance of acute myeloid leukemia cells

目的探究微小RNA-29b(miR-29b)靶向卷曲蛋白6(FZD6)参与急性髓系白血病(AML)细胞柔红霉素(DNR)耐药的分子机制。方法CCK-8检测AML细胞系在不同DNR浓度下的生存能力,实时定量聚合酶链反应(qRT-PCR)检测AML细胞系中miR-29b的表达。选取THP-1细胞为研究对象,分为Control组(正常培养)、miR-NC mimics组(转染miR-NC mimics)、miR-29b mimics组(转染miR-29b mimics)、miR-29b mimics+pcDNA组(转染miR-29b mimics和pcDNA)、miR-29b mimics+pcDNA-FZD6组(转染miR-29b mimics和pcDNA-FZD6),转染24 h后使用10 mM DNR处理培养24 h。qRT-PCR和免疫印迹(Western blot)检测各组DNR处理的THP-1细胞中miR-29b和FZD6表达水平。生物信息学和双荧光素酶报告基因预测并检测miR-29b和FZD6靶向关系。CCK-8、Edu法和流式细胞术分别检测DNR处理的各组THP-1细胞增殖和凋亡情况。结果AML细胞系对DNR的耐药性是呈浓度依赖性的,qRT-PCR结果显示,miR-29b在AML细胞系THP-1(1.00±0.05)、KG-1(1.87±0.16)、HL-60(2.96±0.21)及Kasumi-1(3.73±0.29)中的表达逐渐增高(P<0.05)。双荧光素酶报告基因检测、qRT-PCR及Western blot证明miR-29b可直接负靶向FZD6(0.32±0.04,0.37±0.05,0.48±0.06,P<0.05)。miR-29b过表达可使AML细胞对DNR的耐药性减弱,抑制细胞增殖,增强细胞凋亡能力(43.59±5.24,21.97±3.13,P<0.05)。上调FZD6可一定程度上逆转上调miR-29b表达对THP-1细胞增殖和凋亡的影响(65.21±6.39,38.26±4.09,P<0.05)。结论miR-29b通过靶向抑制FZD6降低AML细胞对DNR的耐药性。

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.