miR-21反义寡核苷酸增强地西他滨体外抗白血病效应

目的:研究miR-21反义寡核苷酸(anti-miR-21 oligonucleotide,AMO)对地西他滨(decitabine,DCA)抗白血病效应的影响及可能机制。方法:将AMO和无义寡核苷酸(scramble oligonucleotide,SCR)通过脂质体转染导入HL-60细胞,实时荧光定量PCR(real-time PCR)验证转染效率,再分别与DCA 0.5、2.0和4.0μmol/L作用48 h。Real-time PCR分别检测人周期节律蛋白3(h Per3)mRNA表达,Annexin V/PI法检测凋亡,流式细胞术检测CD117和CD11b平均荧光强度(MFI)。结果:AMO转染组miR-21表达(0.35±0.07)低于空白组(0.71±0.07)和SCR转染组(0.66±0.05),差异有统计学意义(P0.05)。AMO转染组的HL-60细胞DCA的IC50低于空白组和SCR转染组(P0.01)。同一浓度下,AMO组的早期凋亡率、CD11b的MFI和h Per3 mRNA均高于同一浓度药物作用的空白组和SCR组,CD117 MFI均低于同一浓度药物作用的空白组和SCR组,差异均具有统计学意义(P0.01)。结论:AMO能显著促进DCA体外抗白血病效应,其机制可能与其协助激活h Per3的表达有关。

Enhanced anti-leukemic activity of decitabine to leukemia HL-60 cells by anti-miR-21 oligonucleotide

AIM: To investigate the role of anti-miR-21 oligonucleotide (AMO) in the anti-leukemic activity of decitabine (DCA) in vitro.METHODS: AMO and scramble oligonucleotide (SCR) were constructed and transfected into HL-60 cells. The miR-21 expression was analyzed by real-time PCR to identify the transfection efficiency. The cells were treated with DCA at gradient concentrations (0.5, 2.0 and 4.0 μmol/L) for 48 h. The mRNA expression of human period circadian protein 3 (hPer3) was detected by real-time PCR. The early apoptotic rates were determined by flow cytometry with Annexin V/PI staining. Mean fluorescence intensities (MFI) of CD117 and CD11b were also measured by flow cytometry. RESULTS: The miR-21 relative expression level in AMO group was significantly lower than that in blank group and SCR group (P<0.01). IC₅₀ of DCA in AMO group was significantly lower than that in blank group and SCR group (P<0.01).With the same concentration of DCA, the early apoptotic rate, the mRNA expression of hPer3 and the MFI of CD11b in AMO group were significantly higher than those in blank group and SCR group (P<0.01). The MFI of CD117 in AMO group were significantly lower than those in blank group and SCR group (P<0.01). CONCLUSION: Activation of hPer3 expression plays an important role in enhanced anti-leukemic activity of decitabine by AMO in vitro.