臭椿酮抑制急性骨髓性白血病细胞恶性生物学行为的研究

doi:10.19586/j.2095-2341.2021.0189

生物技术进展 ›› 2022, Vol. 12 ›› Issue (5): 769-777.DOI: 10.19586/j.2095-2341.2021.0189

臭椿酮抑制急性骨髓性白血病细胞恶性生物学行为的研究

徐奔(), 覃锐, 向航, 许京淑, 廖子龙, 向金平

恩施土家族苗族自治州中心医院血液科，湖北恩施 445000

收稿日期:2021-11-24 接受日期:2022-03-15 出版日期:2022-09-25 发布日期:2022-09-30
作者简介:徐奔 E-mail: xboo88@tom.com

Study on the Inhibition of Ailanthone on Malignant Biological Behavior of Acute Myeloid Leukemia Cells

Ben XU(), Rui QIN, Hang XIANG, XU Jingshu, Zilong LIAO, Jinping XIANG

Department of Hematology，Central Hospital of Enshi Tujia and Miao Autonomous Prefecture，Hubei Enshi 445000，China

Received:2021-11-24 Accepted:2022-03-15 Online:2022-09-25 Published:2022-09-30

摘要/Abstract

摘要：

为了探讨臭椿酮（ailanthone，AIL）对急性骨髓性白血病（acute myelogenous leukemia，AML）细胞恶性生物学行为的影响，用不同浓度（0.2、0.4、0.8、1.6、3.2 μmol·L^-1）的AIL处理对数生长期的HL-60细胞，将miR-449a mimic质粒、mimic对照质粒、miR-449a inhibitor质粒、inhibitor对照质粒分别转染至未经任何处理的HL-60细胞，并用1.0 μmol·L^-1浓度的AIL处理细胞24 h。采用CCK-8法检测细胞增殖水平，细胞划痕实验检测细胞迁移水平，Transwell小室法检测细胞侵袭水平，Annexin V-FITC/PI双染法检测细胞凋亡水平，qRT-PCR法检测miR-449a mRNA表达水平，Western blot法检测磷脂酰肌醇3-激酶（PI3K）、磷酸化PI3K（p-PI3K）、蛋白激酶B（AKT）、磷酸化AKT（p-AKT）蛋白表达水平。结果显示，AIL干预后HL-60细胞增殖抑制率、凋亡率升高，细胞迁移率及细胞侵袭数降低（P<0.05），miR-449a mRNA表达量升高（P<0.05）。过表达miR-449a可以抑制HL-60细胞增殖、迁移和侵袭，并诱导细胞凋亡（P<0.05），抑制miR-449a的表达可以起到逆转AIL抑制HL-60细胞增殖、迁移和侵袭，诱导细胞凋亡的作用（P<0.05）。AIL能够显著降低HL-60细胞中p-PI3K/PI3K和p-AKT/AKT比值（P<0.05），抑制miR-449a表达可以逆转AIL对HL-60细胞p-PI3K/PI3K和p-AKT/AKT比值的下调作用（P<0.05）。结果表明，AIL可通过上调miR-449a抑制AML细胞的增殖、迁移和侵袭，并诱导细胞凋亡，其作用机制可能与抑制PI3K/AKT信号通路有关。结果表明，AIL有望成为AML治疗的候选药物。

关键词: 臭椿酮, 急性骨髓性白血病, miR-449a, PI3K/AKT信号通路, 增殖, 迁移, 侵袭, 凋亡

Abstract:

To investigate the effect of ailanthone （AIL） on the malignant biological behavior of acute myeloid leukemia （AML） cells， HL-60 cells in logarithmic growth phase were treated with AIL of different concentrations （0.2， 0.4， 0.8， 1.6， 3.2 μmol·L^-1）， miR-449a mimic plasmid， mimic control plasmid， miR-449a inhibitor plasmid and inhibitor control plasmid were transfected into HL-60 cells without any treatment， and the cells were treated with 1.0 μmol·L^-1 AIL for 24 hours. Cell proliferation was detected by CCK-8 assay， cell migration by scratch test， cell invasion by Transwell chamber assay， cell apoptosis by Annexin V-FITC/PI double staining and miR-449a mRNA expression by qRT-PCR. Western blot was used to detect the expression of Phosphatidylinositol 3-kinase （PI3K）， phosphorylated PI3K （P-PI3K）， protein kinase B （AKT） and phosphorylated Akt （p-AKT）. The results showed that after the intervention of AIL， the proliferation inhibition rate and apoptosis rate of HL-60 cells were significantly increased， the cell migration rate and the number of cell invasion were significantly decreased （P<0.05）， and the expression of mir-449a mRNA was significantly increased （P<0.05）. Overexpression of miR-449a could inhibit the proliferation， migration and invasion of HL-60 cells and induce apoptosis （P<0.05）. Inhibition of miR-449a expression could reverse the inhibitory effect of AIL on proliferation， migration and invasion of HL-60 cells and induce apoptosis （P<0.05）. The ratio of p-PI3K/PI3K and p-AKT/AKT in HL-60 cells were significantly decreased by AIL （P<0.05）， inhibition of miR-449a expression could reverse the down-regulation of AIL on p-PI3K/PI3K and p-AKT/AKT ratio in HL-60 cells （P<0.05）. The results indicated that AIL could inhibit the proliferation， migration， invasion and induce apoptosis of AML cells by up-regulating miR-449a， its mechanism may be related to the inhibition of PI3K/Akt signaling pathway. AIL is expected to be a candidate drug for the treatment of AML.

Key words: ailanthone, acute myeloid leukemia, miR-449a, PI3K/AKT signaling pathway, proliferation, migration, invasion, apoptosis

中图分类号:

R733.7

徐奔, 覃锐, 向航, 许京淑, 廖子龙, 向金平. 臭椿酮抑制急性骨髓性白血病细胞恶性生物学行为的研究[J]. 生物技术进展, 2022, 12(5): 769-777.

Ben XU, Rui QIN, Hang XIANG, XU Jingshu, Zilong LIAO, Jinping XIANG. Study on the Inhibition of Ailanthone on Malignant Biological Behavior of Acute Myeloid Leukemia Cells[J]. Current Biotechnology, 2022, 12(5): 769-777.

图/表 6

图1 AIL对HL-60细胞增殖的影响视图注：*表示与Control组相比差异在P<0.05水平具有统计学意义。

Fig. 1 Effect of AIL on proliferation of HL-60 cells

图2 AIL对HL-60细胞迁移、侵袭能力和凋亡的影响A：不同浓度的AIL对HL-60细胞迁移能力的影响； B：不同浓度的AIL对HL-60细胞侵袭能力的影响； C：不同浓度的AIL对HL-60细胞凋亡的影响。*表示与Control组相比差异在P<0.05水平具有统计学意义。

Fig. 2 Effects of AIL on migration, invasion and apoptosis of HL-60 cells

图3 AIL对HL-60细胞miR-449a mRNA表达水平的影响注：*表示与Control组相比差异在P<0.05水平具有统计学意义。

Fig. 3 Effect of AIL on miR-449a mRNA expression in HL-60 cells

图4 过表达miR-449a对HL-60细胞增殖、凋亡、迁移和侵袭的影响A：过表达miR-449a各处理miR-449a的表达量；B：过表达miR-449a对HL-60细胞增殖抑制率的影响；C：过表达miR-449a对HL-60细胞迁移能力的影响；D：过表达miR-449a对HL-60细胞侵袭能力的影响；E：过表达miR-449a对HL-60细胞凋亡的影响。#表示与mimic NC组相比差异在P<0.05水平具有统计学意义。

Fig. 4 Effects of overexpression of miR-449a on proliferation, apoptosis, migration and invasion of HL-60 cells

图5 抑制miR-449a表达逆转AIL对HL-60细胞增殖、凋亡、迁移和侵袭的影响A：抑制miR-449a表达状态下各组处理miR-449a的表达量；B：抑制miR-449a表达状态下各组处理的细胞增殖抑制率；C：抑制miR-449a表达对HL-60细胞迁移能力的影响；D：抑制miR-449a表达对HL-60细胞侵袭能力的影响；E：抑制miR-449a表达对HL-60细胞凋亡的影响。*表示与Control组相比差异在P<0.05水平具有统计学意义；&表示与AIL+inhibitor NC组相比差异在P<0.05水平具有统计学意义。

Fig. 5 Effects of inhibition expression of miR-449a reverses the effects of AIL on proliferation, apoptosis, migration and invasion of HL-60 cells

图6 抑制miR-449a表达逆转AIL对HL-60细胞PI3K/AKT信号通路相关蛋白的表达A：抑制miR-449a表达状态下Western blot法检测结果；B：抑制miR-449a表达状态下PI3K/AKT信号通路相关蛋白的表达水平。*表示与Control组相比差异在P<0.05水平具有统计学意义；&表示与AIL+inhibitor NC组相比差异在P<0.05水平具有统计学意义。

Fig. 6 Effects of inhibition expression of miR-449a reverses the regulatory effect of AIL on the expression of PI3K/Akt signaling pathway related proteins in HL-60 cells

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臭椿酮抑制急性骨髓性白血病细胞恶性生物学行为的研究

Study on the Inhibition of Ailanthone on Malignant Biological Behavior of Acute Myeloid Leukemia Cells

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