miR-520c-3p/CXCL8信号轴在慢性髓系白血病细胞红系分化中的调控作用

Regulatory role of the miR-520c-3p/CXCL8 axis in erythroid differentiation of chronic myeloid leukemia cells

目的:探讨miR-520c-3p/CXCL8信号轴在慢性髓系白血病(CML)细胞红系分化中的作用及其机制。方法:用氯化血红素诱导人CML细胞系K562细胞红系分化,逆转录实时荧光定量PCR(RT-qPCR)法检测CXCL8 mRNA的表达水平。用不同浓度CXCL8(0、20、40、60、80和100 ng/mL)处理K562细胞72 h,RT-qPCR实验检测γ-globin mRNA的表达情况,据此分析CXCL8对K562细胞红系分化的影响。将miR-520c-3p模拟物和inhibitor转染到K562细胞以增强或抑制miR-520c-3p的功能,RT-qPCR法检测γ-globin mRNA的表达水平,分析miR-520c-3p对K562细胞红系分化的影响。分别采用Western blot和RT-qPCR实验检测CXCL8 mRNA和蛋白的表达水平,观察miR-520c-3p对CXCL8表达的影响。细胞分为实验组(共转染miR-520c-3p模拟物与CXCL8 3′-UTR)和对照组(共转染模拟物对照与CXCL8 3′-UTR),用双荧光素酶报告基因实验检测miR-520c-3p是否靶向CXCL8的3′-UTR。结果:与对照组相比,氯化血红素诱导分化处理后,K562细胞中CXCL8 mRNA的表达水平升高(P<0.01)。与未处理组相比,不同剂量CXCL8处理组K562细胞中γ-globin mRNA的表达水平均明显升高(P<0.01)。与阴性对照组细胞相比,miR-520c-3p模拟物转染组K562细胞中γ-globin mRNA的表达水平、CXCL8 mRNA和蛋白表达水平均下降(均为P<0.01);miR-520c-3p抑制物转染组细胞中γ-globin mRNA表达水平、CXCL8 mRNA和蛋白表达水平均上升(均为P<0.01)。双荧光素酶报告基因实验检测发现,与对照组相比,实验组的相对荧光素酶活性明显下降(P<0.01)。结论:本研究揭示了miR-520c-3p/CXCL8信号轴在CML细胞红系分化中的关键作用。miR-520c-3p通过靶向调控CXCL8的表达,在K562细胞的红系分化过程中发挥抑制效应。

OBJECTIVE:To explore the role and mechanism of the miR-520c-3p/CXCL8 axis in erythroid differentiation of chronic myeloid leukemia(CML).METHODS:K562 cells were induced to differentiate with hemin and treated with CXCL8(0,20,40,60,80 and 100 ng/mL)for 72 h.Expression ofγ-globin mRNA was examined by RT-qPCR to analyze the effect of CXCL8 on erythroid differentiation.miR-520c-3p mimics and inhibitor were transfected into K562 cells to enhance or inhibit miR-520c-3p function,respectively.Expression ofγ-globin mRNA was measured by RT-qPCR to assess the impact of miR-520c-3p on erythroid differentiation of K562 cells.Western blot and RT-qPCR experiments were performed to detect CXCL8 expression and observe the influence of miR-520c-3p on CXCL8 expression.The cells were divided into the experiment group(co-transfected with miR-520c-3p mimics and CXCL83′-UTR)and the control group(co-transfected with mimics-control and CXCL83′-UTR),a dual luciferase reporter gene assay was conducted to examine the targeting relationship between miR-520c-3p and the 3′-UTR of CXCL8.RESULTS:Compared to the control group,expression of CXCL8 mRNA was significantly increased after hemin-induced differentiation(P<0.01).Compared to the untreated group,theγ-globin mRNA expression of CXCL8 treatment groups were significantly elevated(P<0.01).Compared with the negative control group,the expression levels ofγ-globin mRNA,CXCL8 mRNA,and CXCL8 protein in K562 cells transfected with miR-520c-3p mimics were all decreased(P<0.01).In cells transfected with the miR-520c-3p inhibitor,expression levels ofγ-globin mRNA,CXCL8 mRNA,and CXCL8 protein were all increased(P<0.01).The dual luciferase reporter gene assay revealed a significant decrease in relative fluorescence activities in the experimental group compared to the control group(P<0.01).CONCLUSION:This study reveals the key role of the miR-520c-3p/CXCL8 signaling axis in the erythroid differentiation of CML cells.miR-520c-3p exerted an inhibitory effect on the erythroid differentiation of K562 cells by targeting the expression of CXCL8.