齐墩果酸通过miR-18a-5p/STK4轴抑制白血病K562细胞恶性进展

Oleanolic Acid Inhibits Malignant Progression of Leukemia K562 Cells Through The miR-18a-5p/STK4 Axis

慢性粒细胞白血病(chronic myeloid leukemia,CML)是由于骨髓造血干细胞的恶性增生导致的疾病。虽然酪氨酸激酶抑制剂(Tyrosine kinase inhibitors,TKI)对慢性粒细胞性白血病的治疗取得长足进展,但耐药问题仍未解决。因此,寻找新的治疗药物和靶点十分关键。有研究表明,miRNAs与慢性粒细胞白血病在内的多种肿瘤有密切联系,齐墩果酸(oleanolic acid,OA)对白血病细胞有较好的抑制效果。通过对转录物组测序结果发现,齐墩果酸可以显著上调丝氨酸/苏氨酸蛋白质激酶4(serine/threonine-protein kinase 4,STK 4)的水平,而miR1a-5p是STK 4的靶miRNA。因此,本文旨在探究齐墩果酸是否可以通过miR-18a-5p/STK4影响K562细胞恶性进展。K562细胞经齐墩果酸处理后差异表达基因富集在凋亡相关通路上。EdU实验和CCK-8实验结果发现,齐墩果酸降低K562细胞增殖能力(P<0.05)。qRT-PCR,免疫荧光和Western印迹结果显示,齐墩果酸处理后,K562细胞中STK 4蛋白质水平表达显著上调(P<0.05),miR-18a-5p表达下调(P<0.05)。在细胞中转染miR-18a-5p mimics,能显著抑制STK 4的表达(P<0.05);转染miR-18a-5p inhibitor能显著增加STK 4的表达(P<0.05)。活性氧(reactive oxygen species、ROS)检测和线粒体膜电位检测结果显示,齐墩果酸可以促进K562细胞中ROS的增加,降低线粒体膜电位。过表达STK 4后,与Vector组相比,细胞的线粒体膜电位下降;敲降STK 4可以逆转齐墩果酸组导致的线粒体膜电位下降。流式细胞术检测显示,齐墩果酸能明显促进细胞凋亡的发生,而转染miR-18a-5p mimics后凋亡率显著下调(P<0.05);过表达STK 4可以促进细胞从早期凋亡向晚期凋亡转化,而同时转染miR-18a-5p mimics可以抑制这一现象。我们的研究结果证实,齐墩果酸可以通过维持miR-18a-5p的低表达和保持STK 4的高表达状态来促进K562细胞的凋亡。

Chronic myeloid leukemia(CML)is a disease caused by the malignant proliferation of hematopoietic stem cells in the bone marrow.Although tyrosine kinase inhibitors(TKIs)have made significant progress in the treatment of chronic myeloid leukemia,the issue of drug resistance remains unresolved.Therefore,finding new therapeutic drugs and targets is crucial.Studies have shown that miRNAs are closely associated with various tumors,including chronic myeloid leukemia,and oleanolic acid(OA)has a good inhibitory effect on leukemia cells.Through transcriptome sequencing,it was found that oleanolic acid can significantly upregulate the level of serine/threonine protein kinase 4(STK 4),and miR1a-5p is the target miRNA of STK 4.Therefore,this article aims to investigate whether oleanolic acid can affect the malignant progression of K562 cells through miR-18a-5p/STK4.The differentially expressed genes in K562 cells were enriched in apoptosis related pathways after treatment with oleanolic acid.The EdU experiment and CCK-8 experiment results showed that oleanolic acid reduced the proliferation ability of K562 cells(P<0.05).qRT-PCR,immunofluorescence,and Western blot results showed that after treatment with oleanolic acid,the expression of STK 4 protein was significantly upregulated(P<0.05)and miR-18a-5p was downregulated in K562 cells(P<0.05).Transfection of miR-18a-5p mimics into cells significantly inhibited the expression of STK4(P<0.05);Transfection with miR-18a-5p inhibitor significantly increased the expression of STK4(P<0.05).The results of reactive oxygen species(ROS)detection and mitochondrial membrane potential detection showed that oleanolic acid can promote the increase of ROS in K562 cells and reduce mitochondrial membrane potential.After overexpression of STK 4,the mitochondrial membrane potential of the cells decreased compared to the Vector group;Knocking down STK 4 can reverse the decrease in mitochondrial membrane potential caused by the oleanolic acid group.Flow cytometry analysis showed that oleanolic acid can significantly promote the occurrence of cell apoptosis,while the apoptosis rate significantly decreased after transfection with miR-18a-5p mimics(P<0.05);Overexpression of STK 4 can promote the transformation of cells from early apoptosis to late apoptosis,while simultaneous transfection of miR-18a-5p mimics can inhibit this phenomenon.Our research results confirm that oleanolic acid can promote apoptosis of K562 cells by maintaining low expression of miR-18a-5p and high expression of STK 4.