Circ_0035381 contributes to the progression of acute myeloid leukemia via regulating miR-186-5p/CDCA3 pathway

Circular RNAs (circRNAs) are involved in acute myeloid leukemia (AML) and may be useful for AML therapy. Herein, the project aimed to explore the functions and mechanisms of circ_0035381 in AML. Circ_0035381, microRNA-186-5p (miR-186-5p), and cell division cycle associated 3 (CDCA3) expression were determined using quantitative real-time polymerase chain reaction (qRT-PCR) assay. Western blot assay was used to measure protein levels. 5’-ethynyl-2’-deoxyuridine (EdU) and flow cytometry were adopted to measure cell proliferation and apoptosis. Glucose consumption and lactate uptake were examined with commercial kits. The relationships between miR-186-5p and circ_0035381 or CDCA3 were validated using dual-luciferase reporter and RNA pull-down assays. Circ_0035381 was increased in the AML subject and AML cell line. Circ_0035381 deficiency hindered the proliferation and glycolysis level and promoted apoptosis in the AML cell line. Circ_0035381 sponged miR-186-5p and miR-186-5p inhibition reversed the effect of circ_0035381 knockdown on AML cell progression. CDCA3 was the target gene of miR-186-5p. CDCA3 overexpression reversed circ_0035381 knockdown-mediated AML cell proliferation and glycolysis inhibition and apoptosis promotion. Circ_0035381 promoted AML progression by elevating CDCA3 through sponging miR-186-5p, providing some clues for the diagnosis and treatment of AML.

环状RNA（Circular RNAs, circRNAs）参与急性髓系白血病（acute myeloid leukemia, AML）的发生发展，或可用于AML的治疗。本研究旨在探究circ_0035381在AML中的功能与作用机制。采用实时荧光定量聚合酶链反应（quantitative real-time polymerase chain reaction, qRT-PCR）检测circ_0035381、微小RNA-186-5p（microRNA-186-5p, miR-186-5p）及细胞分裂周期关联蛋白3（cell division cycle associated 3, CDCA3）的表达水平；采用蛋白质印迹法（Western blot assay）检测蛋白表达量。通过5'-乙炔基-2'-脱氧尿苷（5’-ethynyl-2’-deoxyuridine, EdU）染色与流式细胞术分别检测细胞增殖与凋亡情况；采用商品化试剂盒检测葡萄糖消耗量与乳酸摄取量。采用双荧光素酶报告基因实验与RNA下拉实验验证miR-186-5p分别与circ_0035381、CDCA3的靶向结合关系。实验结果显示，circ_0035381在AML患者样本与AML细胞系中表达上调；敲低circ_0035381可抑制AML细胞的增殖与糖酵解水平，并促进细胞凋亡。circ_0035381可通过海绵吸附作用靶向结合miR-186-5p，而抑制miR-186-5p可逆转敲低circ_0035381对AML细胞进程的影响。CDCA3是miR-186-5p的靶基因；过表达CDCA3可逆转敲低circ_0035381介导的AML细胞增殖与糖酵解抑制效应，以及细胞凋亡促进效应。综上，circ_0035381可通过海绵吸附miR-186-5p上调CDCA3的表达，进而促进AML的疾病进程，为AML的临床诊断与治疗提供了潜在研究线索。