摘要
目的:探究长非编码RNA(long non-coding RNA,LncRNA)GA结合蛋白转录因子β亚基 1的反义RNA(antisense RNA of GA binding protein transcription factor β subunit 1,GABPB1-AS1)靶向微小 RNA-497-5p/热休克蛋白 8(microRNA-497-5p/heat shock protein 8,miR-497-5p/HSPA8)轴调控急性髓系白血病(acute myeloid leukemia,AML)细胞增殖、侵袭迁移及凋亡的作用和机制.方法:HL-60细胞分为正常对照组、si-NC组、si-GABPB1-AS1组、si-GABPB1-AS1+NC inhibitor组、si-GABPB1-AS1+miR-497-5p inhibitor组.qRT-PCR检测LncRNA GABPB1-AS1、miR-497-5p和HSPA8表达;双荧光素酶报告基因实验验证LncRNA GABPB1-AS1、miR-497-5p和HSPA8的靶向关系;MTT检测细胞活力;Edu检测细胞增殖;Transwell小室实验检测侵袭和迁移;流式细胞仪检测细胞凋亡;Western blot检测增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)、HSPA8、肿瘤转移相关蛋白 2(metastasis-associated proteins,MTA2)、酵母Atg6同系物(homolog of yeast Atg6,Beclin-1)和Caspase-3蛋白水平;建立小鼠移植瘤模型验证LncRNA GABPB1-AS1对AML移植瘤生长的影响.结果:与人骨髓单核细胞相比,不同AML细胞系 THP-1、NB4、U-937、HL-60中 LncRNA GABPB1-AS1高表达[(1.29±0.10)、(1.58±0.12)、(2.02±0.17)、(3.17±0.24)vs.(1.02±0.07)]、miR-497-5p低表达[(0.94±0.07)、(0.75±0.03)、(0.57±0.03)、(0.25±0.01)vs.(1.05±0.09)].由于HL-60细胞中LncRNA GABPB1-AS1表达最高,故选择该细胞系进行功能验证实验.敲低LncRNA GABPB1-AS1能降低HL-60细胞活力、Edu阳性率、细胞侵袭数、迁移数、HSPA8 mRNA及HSPA8、PCNA和MTA2蛋白表达,增加凋亡率、miR-497-5p及Caspase-3和Beclin-1蛋白表达(P<0.05).miR-497-5p与LncRNA GABPB1-AS1、HSPA8之间分别存在靶向关系;抑制miR-497-5p表达能逆转LncRNA GABPB1-AS1敲低对HL-60细胞恶性生物学行为的抑制作用;抑制LncRNA GABPB1-AS1表达可抑制移植瘤生长.结论:LncRNA GABPB1-AS1敲低对AML细胞进展抑制作用,可能与上调miR-497-5p表达,下调HSPA8表达有关.
Abstract
Objective:To explored the role and mechanism of antisense RNA of long non-coding RNA(LncRNA)GA binding protein transcrip-tion factor β subunit 1(GABPB1-AS1)in regulating the proliferation,invasion,migration,and apoptosis of acute myeloid leukemia(AML)cells by targeting the microRNA-497-5p/heat shock protein 8(miR-497-5p/HSPA8)axis.Methods:HL-60 cells were divided into the normal con-trol,si-NC,si-GABPB1-AS1,si-GABPB1-AS1+NC,and si-GABPB1-AS1+miR-497-5p inhibitor groups.Quantitative real time polymerase chain re-action(RT-qPCR)was used to detect the expression levels of LncRNA GABPB1-AS1,miR-497-5p,and HSPA8.Double luciferase reporter gene assay was used to verify the targeting relationship between LncRNA GABPB1-AS1,miR-497-5p,and HSPA8;MTT was used to detect cell viab-ility;while 5-ethynyl-2'-deoxyuridine(EdU)was used to detect proliferation.Transwell chamber experiments were used to detect invasion and migration while flow cytometry was used to detect apoptosis.Western blot was used to detect the levels of proliferating cell nuclear an-tigen(PCNA),HSPA8,metastasis-associated proteins(MTA2),homolog of yeast Atg6(Beclin-1),and Caspase-3 proteins.A mouse trans-planted tumor model was established to verify the effect of LncRNA GABPB1-AS1 on the growth of AML transplanted tumors.Results:Com-pared to human bone marrow monocytes,LncRNA GABPB1-AS1 was highly expressed[(1.29±0.10),(1.58±0.12),(2.02±0.17),(3.17±0.24)vs.(1.02±0.07)]while miR-497-5p was lowly expressed[(0.94±0.07),(0.75±0.03),(0.57±0.03),(0.25±0.01)vs.(1.05±0.09)]in different AML cells(THP-1,NB4,U-937,and HL-60,respectively).The HL-60 cell line was selected for functional verification experiments since LncRNA GABPB1-AS1 expression was highest in the HL-60 cells.Knockdown of LncRNA GABPB1-AS1 reduced HL-60 cell viability,the EdU positive rate,cell in-vasion and migration,the expression of HSPA8 mRNA,and HSPA8,PCNA,and MTA2 protein contents.It increased the apoptosis rate,and the expression of miR-497-5p,Caspase-3 and Beclin-1 protein(P<0.05).miR-497-5p had a targeting relationship with LncRNA GABPB1-AS1 and HSPA8;inhibiting the expression of miR-497-5p reversed the inhibitory effect of LncRNA GABPB1-AS1 knockdown on the malignant bio-logical behavior of HL-60 cells.Meanwhile,inhibiting the expression of LncRNA GABPB1-AS1 constrained the growth of transplanted tumors.Conclusions:Knockdown of LncRNA GABPB1-AS1 inhibits the progression of AML cells,which may be related to the upregulation of miR-497-5p expression and downregulation of HSPA8 expression.
基金项目
山东省医药卫生科技发展计划项目(202006010355)
NETL
NSTL
万方数据