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.
题目: circ-SFMBT2调节miR-491-5p/HOXA9轴对急性髓系白血病 细胞增殖、迁移和侵袭的影响.
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-qPCR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、si-circ-SFMBT2、si-circ-SFMBT2+anti-NC和si-circ-SFMBT2+anti-miR-491-5p共5组，用Lipofectamine™ 3000将si-NC、si-circ-SFMBT2、anti-NC、miR-491-5p inhibitor转染至HL-60细胞；采用RT-qPCR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），HOXA9 mRNA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。.
Keywords:
acute myeloid leukemia;