Potential miRNAs binding to the 3\'-untranslated region (3\'-UTR) of the LILRB3 mRNA were predicted by bioinformatics websites. Then, we screened miRNAs targeting LILRB3 by quantitative real-time PCR, and the dual luciferase reporter assay. The expression of LILRB3 and microRNA (miR)-103a-2-5p in AML were determined and then their interactions were also analyzed. In vitro, the effects of miR-103a-2-5p were determined by CCK8, colony formation assay, and transwell assay, while cell apoptosis and cell cycle were analyzed by flow cytometry. Cationic liposomes (CLPs) were used for the delivery of miR-103a-2-5p in the AML mouse model, which was to validate the potential roles of miR-103a-2-5p in vivo.
LILRB3 was upregulated in AML cells while miR-103a-2-5p was dramatically downregulated. Thus, a negative correlation was found between them. MiR-103a-2-5p directly targeted LILRB3 in AML cells. Overexpressed miR-103a-2-5p significantly suppressed the mRNA and protein levels of LILRB3, thereby inhibiting AML cell growth and reducing CD8 + T cell apoptosis. In addition, overexpressed miR-103a-2-5p reduced both the relative expression of Nrf2/HO-1 pathway-related proteins and the ratio of GSH/ROS, leading to the excessive intracellular ROS that may promote AML cell apoptosis. In the mouse model, the delivery of miR-103a-2-5p through CLPs could inhibit tumor growth.
MiR-103a-2-5p serves as a tumor suppressor that could inhibit AML cell proliferation and promote their apoptosis by downregulating LILRB3 expression, suppressing the Nrf2/HO-1 axis, and reducing the ratio of GSH/ROS. Besides, our findings indicate that miR-103a-2-5p may enhance the CD8 + T cell response by inhibiting LILRB3 expression. Therefore, the delivery of miR-103a-2-5p through CLPs could be useful for the treatment of AML.
方法:通过生物信息学网站预测与LILRB3mRNA的3'-非翻译区(3'-UTR)结合的潜在miRNA。然后,我们通过定量实时PCR筛选了靶向LILRB3的miRNA,和双荧光素酶报告基因测定。测定LILRB3和microRNA(miR)-103a-2-5p在AML中的表达,并分析它们的相互作用。体外,miR-103a-2-5p的影响通过CCK8,集落形成试验,和transwell分析,同时通过流式细胞术分析细胞凋亡和细胞周期。阳离子脂质体(CLPs)用于在AML小鼠模型中递送miR-103a-2-5p,这是为了验证miR-103a-2-5p在体内的潜在作用。
结果:LILRB3在AML细胞中上调,而miR-103a-2-5p显著下调。因此,两者之间呈负相关。MiR-103a-2-5p直接靶向AML细胞中的LILRB3。过表达的miR-103a-2-5p显著抑制LILRB3的mRNA和蛋白水平,从而抑制AML细胞生长并减少CD8+T细胞凋亡。此外,过表达的miR-103a-2-5p降低了Nrf2/HO-1途径相关蛋白的相对表达和GSH/ROS的比例,导致细胞内过量的ROS可能促进AML细胞凋亡。在老鼠模型中,通过CLPs递送miR-103a-2-5p可抑制肿瘤生长.
结论:MiR-103a-2-5p作为一种肿瘤抑制因子,可以通过下调LILRB3的表达来抑制AML细胞增殖并促进其凋亡,抑制Nrf2/HO-1轴,降低GSH/ROS的比值。此外,我们的研究结果表明,miR-103a-2-5p可能通过抑制LILRB3表达增强CD8+T细胞应答.因此,通过CLPs递送miR-103a-2-5p可能有助于AML的治疗.