Lian Cheng, Liu Yanhui, Lei Pingchong
Department of Hematology, Henan provincial People's Hospital, Zhengzhou, Henan, China.
J Biochem Mol Toxicol. 2025 May;39(5):e70278. doi: 10.1002/jbt.70278.
Acute myeloid leukemia (AML) is a malignant tumor of blood cells, which seriously interferes with the generation of normal cells. Although miR-186-5p is diminished in AML, its exact mechanism is not well understood. miR-186-5p and PD-L1 levels in AML cells (HL-60, KG-1, TF-1a, MOLT-3) and subcutaneous tumor tissue were discovered through qRT-PCR and Western blot. miR-186-5 p and PD-L1 combining sites were foreseen by the database and verified by dual luciferase and immunoprecipitation experiments. AML cells with miR-186-5p overexpression or knockdown and PD-L1 overexpression were cocultured with CD4 and CD8 T cells. The proliferation, migration, invasion and apoptosis of AML cells, CD8 and CD4 T cell growth and apoptosis, and activated markers (Perforin and Granzyme B) and secreted cytokines (IFN-γ, IL-4 and TNF-α) levels were detected by CCK8, Transwell, flow cytometry, CFSE, Western blot and ELISA, respectively. Subcutaneous xenograft magnitude and mass in nude mice were measured. Ki67 level was identified through immunohistochemistry. CD4 and CD8 T cell level and infiltration were detected by immunofluorescence and flow cytometry. miR-186-5p was downregulated, and PD-L1 was boosted in AML cells and subcutaneous tumor tissues (p < 0.05), while miR-186-5p targeted down-regulate PD-L1. miR-186-5p upregulation hindered AML cell multiplication, migration, invasion and facilitate cell death, and enhanced the proliferation activity, activation markers (Perforin and Granzyme B) and secreted cytokines (IFN-γ, IL-4, TNF-α) of CD8 and CD4 T cells, inhibited apoptosis, and inhibited immune escape (p < 0.05). Knockdown of miR-186-5p can promote AML progression, but PD-L1 upregulation weakens the antitumor impact of miR-186-5p overexpression (p < 0.05). Transplanted tumor mice experiments also found that miR-186-5p hindered PD-L1 and tumor growth (p < 0.05). In conclusion, miR-186-5p can target inhibit PD-L1, suppress AML cells multiplication, movement, invasion and immune escape, and then reduce AML, aiming to provide support and basis for the pathological mechanism and prevention and treatment strategy of AML.
急性髓系白血病（AML）是一种血细胞恶性肿瘤，严重干扰正常细胞的生成。尽管miR-186-5p在AML中表达降低，但其确切机制尚不清楚。通过qRT-PCR和蛋白质免疫印迹法检测AML细胞（HL-60、KG-1、TF-1a、MOLT-3）和皮下肿瘤组织中miR-186-5p和PD-L1的水平。通过数据库预测miR-186-5p与PD-L1的结合位点，并通过双荧光素酶和免疫沉淀实验进行验证。将过表达或敲低miR-186-5p以及过表达PD-L1的AML细胞与CD4和CD8 T细胞共培养。分别通过CCK8、Transwell、流式细胞术、CFSE、蛋白质免疫印迹法和ELISA检测AML细胞的增殖、迁移、侵袭和凋亡、CD8和CD4 T细胞的生长和凋亡以及活化标志物（穿孔素和颗粒酶B）和分泌细胞因子（IFN-γ、IL-4和TNF-α）的水平。测量裸鼠皮下异种移植瘤的大小和质量。通过免疫组织化学鉴定Ki67水平。通过免疫荧光和流式细胞术检测CD4和CD8 T细胞水平及浸润情况。AML细胞和皮下肿瘤组织中miR-186-5p下调，而PD-L1上调（p<0.05），同时miR-186-5p靶向下调PD-L1。miR-186-5p上调可抑制AML细胞的增殖、迁移、侵袭并促进细胞死亡，增强CD8和CD4 T细胞的增殖活性、活化标志物（穿孔素和颗粒酶B）和分泌细胞因子（IFN-γ、IL-4、TNF-α），抑制凋亡并抑制免疫逃逸（p<0.05）。敲低miR-186-5p可促进AML进展，但PD-L1上调会削弱miR-186-5p过表达的抗肿瘤作用（p<0.05）。移植瘤小鼠实验还发现miR-186-5p可抑制PD-L1和肿瘤生长（p<0.05）。总之，miR-186-5p可靶向抑制PD-L1，抑制AML细胞的增殖、运动、侵袭和免疫逃逸，进而减轻AML，旨在为AML的病理机制及防治策略提供支持和依据。
