miR-15b靶向ABCC5对K562/A02细胞增殖、凋亡及阿霉素耐药性的影响

Effects of miR-15b Targeting ABCC5 on Proliferation,Apoptosis and Adriamycin Resistance of K562/A02 Cells

目的探讨微RNA(miRNA/miR)-15b对K562/A02细胞增殖、凋亡及阿霉素耐药性的影响及其机制。方法采用实时荧光定量聚合酶链反应(PCR)检测K562和K562/A02细胞中miR-15b表达情况;将体外培养的K562/A02细胞随机分为对照组(正常培养)、miRNA对照(miR-NC)组(转染模拟物阴性对照)和miR-15b组(转染miR-15b模拟物),实时荧光定量PCR检测K562/A02细胞中miR-15b和ATP结合盒转运蛋白C5(ABCC5)mRNA表达,噻唑蓝法检测K562/A02细胞增殖和阿霉素耐药性,流式细胞仪检测K562/A02细胞凋亡情况,双荧光素酶报告基因实验检测miR-15b与ABCC5的靶向关系,免疫印迹法检测K562/A02细胞中ABCC5蛋白表达情况。结果与K562细胞比较,K562/A02细胞中miR-15b表达水平明显降低[(0.25±0.03)比(0.97±0.05)](P<0.01)。miR-15b组K562/A02细胞miR-15b表达水平及24、48、72 h的细胞增殖抑制率明显高于对照组和miR-NC组(P<0.05),而miR-NC组与对照组K562/A02细胞中miR-15b表达水平以及24、48、72 h的K562/A02细胞增殖抑制率比较差异无统计学意义(P>0.05)。miR-15b组K562/A02细胞凋亡率明显高于对照组和miR-NC组[(23.05±2.85)%比(13.64±2.12)%、(11.58±1.48)%](P<0.05),而miR-NC组与对照组K562/A02细胞凋亡率比较差异无统计学意义(P>0.05)。miR-15b组K562/A02细胞增殖抑制率明显高于对照组及miR-NC组(P<0.05),而miR-NC组与对照组K562/A02细胞增殖抑制率比较差异无统计学意义(P>0.05)。miR-NC组与对照组K562/A02细胞对阿霉素的IC 50值比较差异无统计学意义(P>0.05);miR-15b组K562/A02细胞对阿霉素的IC 50值明显低于对照组和miR-NC组(P<0.05),其相对于对照组和miR-NC组的逆转倍数分别为2.70倍和2.88倍。各组细胞荧光素酶活性比较差异有统计学意义(P<0.01),miR-15b+ABCC5-WT组细胞荧光素酶活性明显低于miR-NC+ABCC5-WT组(P<0.05);miR-NC+ABCC5-WT组与miR-NC+ABCC5-MUT组和miR-15b+ABCC5-MUT组比较差异无统计学意义(P>0.05);且miR-NC+ABCC5-MUT组与miR-15b+ABCC5-MUT组比较差异无统计学意义(P>0.05)。各组细胞中ABCC5蛋白和mRNA表达水平比较差异有统计学意义(P<0.01),miR-NC组与对照组细胞中ABCC5蛋白和mRNA表达水平比较差异无统计学意义(P>0.05);miR-15b组细胞中ABCC5蛋白和mRNA表达水平均明显低于对照组及miR-NC组(P<0.05)。结论miR-15b可能通过抑制ABCC5表达抑制K562/A02细胞增殖,促进其凋亡并降低阿霉素耐药。

Objective To investigate the effects of microRNA(miRNA/miR)-15b on the proliferation,apoptosis and adriamycin resistance of K562/A02 cells and its mechanisms.Methods The expression of miR-15b in K562 and K562/A02 cells was detected by real-time fluorescence quantitative polymerase chain reaction(PCR).K562/A02 cells were randomly divided into a control group(normal cultured),a miR-NC group(transfected with mimic negative control)and a miR-15b group(transfected with miR-15b mimic);the expressions of miR-15b and ATP binding cassette transporter C5(ABCC5)mRNA in K562/A02 cells were detected by real-time fluorescence quantitative PCR;the proliferation and adriamycin resistance of K562/A02 cells were detected by methyl thiazolyl tetrazolium;the apoptosis of K562/A02 cells was detected by flow cytometry;the targeting relationship between miR-15b and ABCC5 was detected by double luciferase reporter gene assay;the expression of ABCC5 protein in K562/A02 cells was detected by Western blot.Results Compared with that in K562 cells,the expression level of miR-15b in K562/A02 cells was significantly lower(0.25±0.03 vs 0.97±0.05)(P<0.01).The expression of miR-15b in K562/A02 cells in the miR-15b group and the inhibition rate of cell proliferation at 24,48 and 72 h were significantly higher than those in the control group and the miR-NC group(P<0.05),but there was no significant difference in the expression of miR-15b and the inhibition rate of cell proliferation at 24,48 and 72 h between the miR-NC group and control group(P>0.05).The apoptotic rate of K562/A02 cells in the miR-15b group was significantly higher than that in the control group and the miR-NC group[(23.05±2.85)%vs(13.64±2.12)%,(11.58±1.48)%](P<0.05),but there was no significant difference in the apoptosis rate between the miR-NC group and the control group(P>0.05).The proliferation inhibition rate of K562/A02 cells in the miR-15b group was significantly higher than that in the control group and the miR-NC group(P<0.05),but there was no significant difference between the miR-NC group and control group(P>0.05).There was no significant difference in IC50 of K562/A02 cells between the miR-NC group and the control group(P>0.05);the IC 50 value of K562/A02 cells to adriamycin in the miR-15b group was significantly lower than that in the control group and the miR-NC group(P<0.05),and the reversal multiple was 2.70 times and 2.88 times compared with the control group and the miR-NC group,respectively.The difference of luciferase activity in each group was statistically significant(P<0.01):the luci-ferase activity in the miR-15b+ABCC5-WT group was significantly lower than that in the miR-NC+ABCC5-WT group(P<0.05);there was no significant difference between the miR-NC+ABCC5-WT group and the miR-NC+ABCC5-MUT group and the miR-15b+ABCC5-MUT group(P>0.05);there was no significant difference between the miR-NC+ABCC5-MUT group and the miR-15b+ABCC5-MUT group(P>0.05).There were significant differences in the expression of ABCC5 protein and mRNA in each group(P<0.01),but there were no significant differences in the expression of ABCC5 protein and mRNA between the miR-NC group and the control group(P>0.05);the expression levels of ABCC5 protein and mRNA in the miR-15b group were significantly lower than those in the control group and the miR-NC group(P<0.05).Conclusion miR-15b may inhibit the proliferation of K562/A02 cells,promote their apoptosis and reduce adriamycin resistance by inhibiting the expression of ABCC5.