miR-5581-5p/TRIM22在急性早幼粒细胞白血病细胞分化中的作用

Effect of miR-5581-5p/TRIM22 on acute promyelocytic leukemia cell differentiation

目的探究在急性早幼粒白血病(APL)细胞向粒系细胞终末分化过程中,miR-5581-5p的功能及其与三基序蛋白22(TRIM22)的相互作用。方法利用全反式维甲酸(ATRA)诱导APL细胞(NB4)向粒系细胞分化,以二甲基亚砜作为对照,实时荧光定量PCR(qRT-PCR)及Western blotting检测分化前后TRIM22的表达变化,qRT-PCR检测分化前后miR-5581-5p的表达变化。通过细胞转染miR-5581-5p mimic和inhibitor调控miRNA表达,并设阴性对照,qRT-PCR验证调控效果,荧光素酶结合实验检测二者是否存在靶向结合,Western blotting检测miRNA差异表达后对TRIM22的表达影响。流式细胞术检测调控miR-5581-5p对ATRA诱导的NB4细胞分化的影响。结果ATRA诱导NB4细胞向粒系细胞分化后,TRIM22在基因水平表达量较对照组明显升高(24.56±2.80 vs.1.02±0.13;t=8.392,P=0.001),在蛋白水平表达量也较对照组明显升高(0.80±0.01 vs.0.17±0.01;t=44.900,P<0.001)。而NB4细胞诱导分化后miR-5581-5p在基因水平表达量明显低于对照组(0.14±0.02 vs.1.01±0.08;t=10.840,P<0.001)。双荧光素酶报告基因结果显示,共转染miR-5581-5p mimic和TRIM22 WT组的荧光素酶活性明显低于共转染miR-5581-5p mimic和TRIM22 MUT组(0.73±0.02 vs.0.98±0.03;t=7.534,P=0.002)。Western blotting检测发现,转染miR-5581-5p inhibitor后,TRIM22表达较阴性对照明显升高(0.44±0.01 vs.0.21±0.01;t=18.290,P<0.001);转染miR-5581-5p mimic后,TRIM22表达较阴性对照明显下降(0.62±0.01 vs.0.80±0.02;t=6.402,P=0.003)。ATRA作用后miR-5581-5p mimic组CD11b表达量明显低于对照组(45.80±1.80 vs.56.61±1.88;t=4.159,P=0.014),而miR-5581-5p inhibitor组CD11b表达量明显高于对照组(66.48±2.54 vs.52.60±1.70;t=4.539,P=0.011)。结论miR-5581-5p靶向结合TRIM223'UTR区,通过负性调控机制影响TRIM22的表达。下调miR-5581-5p可增加TRIM22的表达,从而促进ATRA诱导的NB4细胞粒系分化。

Objective To investigate the function of miR-5581-5p and its interaction with tripartite motif 22(TRIM22)during the terminal differentiation of human acute promyelocytic leukemia(APL)cells into granulocytes.Methods APL cells(NB4)were differentiated into granulocytes by all-trans retinoic acid(ATRA),using dimethylsulfoxide(DMSO)as the control.The expression of TRIM22 was detected by real-time fluorescent quantitative PCR(qRT-PCR)and Western blotting,and the expression of miR-5581-5p was detected by qRT-PCR during cell differentiation.miRNA expression was regulated by cell transfection with miR-5581-5p mimic and inhibitor,and negative control was set,and qRT-PCR was used to verify the regulatory effect.Luciferase binding assay was performed to detect the presence of targeted binding.Western blotting was used to detect the expression of TRIM22 after miRNA differential expression.Flow cytometry was used to detect the effects of the regulation of miR-5581-5p on the differentiation of NB4 cells induced by ATRA.Results After ATRA induced NB4 cells to differentiate into granulocytes,the gene expression level of TRIM22 was significantly higher than that of the control group(24.56±2.80 vs.1.02±0.13;t=8.392,P=0.001).The level of protein expression was also significantly higher than that of the control group(0.80±0.01 vs.0.17±0.01;t=44.900,P<0.001).The expression level of miR-5581-5p in NB4 cells differentiation group was significantly lower than that in the control group(0.14±0.02 vs.1.01±0.08;t=10.840,P<0.001).The results of the dual luciferase reporter gene showed that the luciferase activity of the co-transfected miR-5581-5p mimic and TRIM22 WT group was significantly lower than that of the co-transfected miR-5581-5p mimic and TRIM22 MUT group(0.73±0.02 vs.0.98±0.03;t=7.534,P=0.002).Western blotting showed that after transfection with miR-5581-5p inhibitor,the expression of TRIM22 was significantly higher than that of the negative control(0.44±0.01 vs.0.21±0.01;t=18.290,P<0.001).While after transfection with miR-5581-5p mimic,the expression of TRIM22 decreased significantly compared with the negative control(0.62±0.01 vs.0.80±0.02;t=6.402,P=0.003).CD11b expression of miR-5581-5p mimic group after ATRA treatment was significantly lower than that of the control group(45.80±1.80 vs.56.61±1.88;t=4.159,P=0.014).The expression of CD11b in miR-5581-5p inhibitor group was significantly higher than that in the control group(66.48±2.54 vs.52.60±1.70;t=4.539,P=0.011).Conclusion miRNA-5581-5p can bind to TRIM223'UTR and negatively regulate TRIM22 expression.The decrease of miR-5581-5p can increase the expression of TRIM22,then promote the differentiation of ATRA-induced NB4 cells into granulocytes.