Huang Fengxia, Tang Wei, Lei Yan
Department of Medical Technology Clinical and Hematological Laboratory Office, Xi'an Medical University, Xi'an, Shaanxi, China.
Department of Radiology, Affiliated Hospital of North Sichuan Medical College, Nanchong, Sichuan, China.
Arch Med Sci. 2020 Feb 4;17(4):1044-1055. doi: 10.5114/aoms.2020.92860. eCollection 2021.
This study aimed to investigate the role of microRNA (miRNA) that affects acute myelocytic leukemia (AML) and its potential molecular mechanism by constructing a miRNA-mRNA interaction network using bioinformatics methods.
MicroRNA expression data of AML were retrieved from Gene Expression Omnibus (GEO) and analyzed by microarray analysis. Expression levels of miR-107 and RAD51 mRNA were detected by quantitative real time polymerase chain reaction (qRT-PCR). Protein expression of RAD51, pro-apoptotic protein Bax, apoptosis related protein CytC and anti-apoptotic protein Bcl-2 were determined by Western blot. The rate of cell apoptosis was detected by Annexin-V/PI. The predicted targeting relationship between miR-107 and the 3'UTR of RAD51 was first predicted by the online application TargetScan and then verified by dual-luciferase assay.
Acute myelocytic leukemia-associated genes ( = 197) and miRNAs ( = 1701) were retrieved from the database, the interaction network of miRNA-mRNA was constructed and the core position was occupied by RAD51. miR-107 exhibited a regulatory effect on RAD51 in which the mRNA and protein expression of RAD51 were both significantly inhibited by miR-107 mimics . Additionally, down-regulated expression of miR107 as well as up-regulated expression of RAD51 were detected not only in the plasma of AML patients compared to healthy volunteers, but also in AML cell lines compared to the normal bone marrow stromal cell line. Further study found that increased expression of miR-107 and the consequent down-regulation of RAD51 could aggravate the apoptosis of AML cells .
Our present results showed that the crucial role of RAD51 and miR-107 in the apoptosis of AML cells, i.e., miR-107 promotes the apoptosis of AML cells through down-regulating the expression of RAD51.
本研究旨在通过生物信息学方法构建miRNA- mRNA相互作用网络，探讨影响急性髓细胞白血病（AML）的微小RNA（miRNA）的作用及其潜在分子机制。
从基因表达综合数据库（GEO）中检索AML的miRNA表达数据，并通过微阵列分析进行分析。采用定量实时聚合酶链反应（qRT-PCR）检测miR-107和RAD51 mRNA的表达水平。通过蛋白质免疫印迹法测定RAD51、促凋亡蛋白Bax、凋亡相关蛋白CytC和抗凋亡蛋白Bcl-2的蛋白表达。采用Annexin-V/PI检测细胞凋亡率。首先通过在线应用程序TargetScan预测miR-107与RAD51的3'UTR之间的预测靶向关系，然后通过双荧光素酶测定进行验证。
从数据库中检索到急性髓细胞白血病相关基因（n = 197）和miRNA（n = 1701），构建了miRNA- mRNA相互作用网络，RAD51占据核心位置。miR-107对RAD51具有调节作用，其中miR-107模拟物显著抑制RAD51的mRNA和蛋白表达。此外，与健康志愿者相比，不仅在AML患者血浆中检测到miR107表达下调以及RAD51表达上调，而且与正常骨髓基质细胞系相比，在AML细胞系中也检测到上述情况。进一步研究发现，miR-107表达增加以及随之而来的RAD51下调可加重AML细胞的凋亡。
我们目前的结果表明RAD51和miR-107在AML细胞凋亡中起关键作用，即miR-107通过下调RAD51的表达促进AML细胞凋亡。
