摘要
目的:探讨circ-SFMBT2对急性髓系白血病(AML)细胞增殖、迁移和侵袭的影响及分子机制。方法:收集2015年4月至2017年4月在河南省儿童医院确诊的35例儿童AML患者和35名健康对照者的骨髓样本以及人骨髓基质细胞系(HS-5)和AML细胞系(HL-60、THP-1、U-937和Kasumi-1),RT-q PCR和Western blot检测骨髓样本和细胞中circ-SFMBT2、miR-491-5p、同源盒基因A9(HOXA9)的表达,采用Pearson法分析AML患儿骨髓样本中circ-SFMBT2、miR-491-5p和HOXA9 mRNA表达水平的相关性。体外培养HL-60细胞并分为对照、si-NC、sicirc-SFMBT2、si-circ-SFMBT2+anti-NC和si-circ-SFMBT2+anti-miR-491-5p共5组,用Lipofectamine^(TM)3000将si-NC、si-circ-SFMBT2、anti-NC、miR-491-5p inhibitor转染至HL-60细胞;采用RT-q PCR和Western blot检测各组细胞中circ-SFMBT2、miR-491-5p、HOXA9的表达水平;CCK-8法分别在转染24、48和72 h检测各组HL-60细胞增殖活性;流式细胞术检测细胞凋亡率;Transwell小室实验检测细胞的迁移、侵袭能力;通过双荧光素酶报告基因、RNA pull-down和RNA结合蛋白免疫沉淀实验验证AML细胞中circ-SFMBT2、miR-491-5p和HOXA9的调控作用。结果:AML患儿骨髓样本和细胞系(HL-60、THP-1、U-937和Kasumi-1)中circ-SFMBT2、HOXA9 mRNA表达水平升高(P<0.001),而miR-491-5p表达水平明显降低(P<0.001);Pearson相关分析显示,AML患儿骨髓样本中circ-SFMBT2与miR-491-5p的表达水平呈负相关(r=-0.905),miR-491-5p与HOXA9 mRNA的表达水平呈负相关(r=-0.930),HOXA9mRNA与circ-SFMBT2的表达水平呈正相关(r=0.911),且circ-SFMBT2高表达的AML患儿的总生存率较低表达患儿显著降低(P<0.05)。circ-SFMBT2的沉默可显著上调miR-491-5p表达,降低HOXA9表达,抑制AML细胞增殖、迁移和侵袭,促进细胞凋亡(P<0.05);下调miR-491-5p表达可显著减弱circ-SFMBT2沉默对细胞增殖、迁移和侵袭的抑制作用(P<0.05)。双荧光素酶报告基因、RNA pull-down和RNA结合蛋白免疫沉淀实验证实circ-SFMBT2可以靶向miR-491-5p并在AML中负调控miR-491-5p的表达,HOXA9是miR-491-5p的靶标。结论:circ-SFMBT2的沉默可能通过调节miR-491-5p/HOXA9轴来抑制AML细胞的增殖、迁移与侵袭。
Objective:To explore the effects and molecular mechanism of circ-SFMBT2 on the proliferation,migration and invasion of acute myeloid leukemia(AML)cells.Methods:Bone marrow samples from 35 pediatric AML patients and 35 healthy controls in Henan Provincial Children's Hospital from April 2015 to April 2017 and human bone marrow stromal cell lines(HS-5)and AML cell lines(HL-60,THP-1,U-937 and Kasumi-1)were collected.The expressions of circ-SFMBT2,miR-491-5p and homeobox A9(HOXA9)in bone marrow samples and cells were detected by RT-qPCR and Western blot.The Pearson method was used to analyze the correlation of circ-SFMBT2,miR-491-5p and HOXA9 mRNA expression levels in bone marrow samples of AML patients.HL-60 cells were cultured in vitro and divided into 5 groups:Control,si-NC,si-circ-SFMBT2,si-circ-SFMBT2+anti-NC and si-circ-SFMBT2+anti-miR-491-5p,HL-60 cells were transfected with si-NC,si-circ-SFMBT2,anti-NC,and miR-491-5p inhibitor with Lipofectamine™3000.RT-qPCR and Western blot were performed to detect the expression levels of circ-SFMBT2,miR-491-5p and HOXA9 in cells of each group.The proliferation activity of HL-60 cells in each group was detected by CCK-8 assay at 24,48 and 72 h after transfection,respectively.The apoptosis rate was detected by flow cytometry.The migration and invasion abilities of cells were detected by Transwell assay.The regulatory roles of circ-SFMBT2,miR-491-5p and HOXA9 in AML cells were verified by dual-luciferase reporter gene assay,RNA pull-down and RNA-binding protein immunoprecipitation(RIP)experiments.Results:The expression levels of circ-SFMBT2 and HOXA9 mRNA were increased in bone marrow samples and cell lines(HL-60,THP-1,U-937 and Kasumi-1)of children with AML(P<0.001),while the expression level of miR-491-5p was significantly decreased(P<0.001).Pearson correlation analysis showed that the expression levels of circ-SFMBT2 and miR-491-5p in bone marrow samples of AML children were negatively correlated(r=-0.905),miR-491-5p was also negatively correlated with HOXA9 mRNA(r=-0.930),while the expression levels of HOXA9 mRNA and circ-SFMBT2 was positively correlated(r=0.911).The overall survival rate of AML children with high expression of circ-SFMBT2 was significantly decreased than those with low expression of circ-SFMBT2(P<0.05).Silencing of circ-SFMBT2 could greatly up-regulate the expression of miR-491-5p,decrease the expression of HOXA9,inhibit the proliferation,migration and invasion of AML cells,and promote cell apoptosis(P<0.05).Down-regulation of miR-491-5p expression greatly attenuated the inhibitory effects of circ-SFMBT2 silencing on cell proliferation,migration and invasion(P<0.05).Dual-luciferase reporter gene assay,RNA pull-down and RIP experiments confirmed that circ-SFMBT2 could target miR-491-5p and negatively regulate the expression of miR-491-5p in AML,and HOXA9 was the target of miR-491-5p.Conclusion:Silencing of circ-SFMBT2 may inhibit the proliferation,migration and invasion of AML cells by regulating the miR-491-5p/HOXA9 axis.
作者
李妍
史利欢
谢昕
范朋凯
LI Yan;SHI Li-Huan;XIE Xin;FAN Peng-Kai(Henan Provincial Children's Hospital,Zhengzhou Children's Hospital,Henan Provincial Key Laboratory of Pediatric Hematology,Zhengzhou Henan Province,China)
出处
《中国实验血液学杂志》
CAS
CSCD
北大核心
2023年第6期1599-1607,共9页
Journal of Experimental Hematology
基金
2021年河南省医学科技攻关计划联合共建项目(LHGJ20210659)。
