[1]崔佳奇,魏春梅,邓琳丽,等.miR-143通过抑制AF9调控SKM-1细胞增殖和凋亡[J].第三军医大学学报,2018,40(20):1826-1832.
 CUI Jiaqi,WEI Chunmei,DENG Linli,et al.MiR-143 regulates proliferation and apoptosis of SKM-1 cells in vitro by inhibiting AF9[J].J Third Mil Med Univ,2018,40(20):1826-1832.
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miR-143通过抑制AF9调控SKM-1细胞增殖和凋亡(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
40卷
期数:
2018年第20期
页码:
1826-1832
栏目:
基础医学
出版日期:
2018-10-30

文章信息/Info

Title:
MiR-143 regulates proliferation and apoptosis of SKM-1 cells in vitro by inhibiting AF9
作者:
崔佳奇魏春梅邓琳丽王利
重庆医科大学附属第一医院血液内科
Author(s):
CUI Jiaqi WEI Chunmei DENG Linli WANG Li

Department of Hematology, the First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, China

关键词:
骨髓增生异常综合征miR-143AF9细胞凋亡细胞周期
Keywords:
myelodysplastic syndrome miR-143 AF9 cell apoptosis cell cycle
分类号:
R394-33;R394.12;R551.1
文献标志码:
A
摘要:

目的    探讨miR-143在骨髓增生异常综合征(myelodysplastic syndrome,MDS)中的表达情况及AF9对人骨髓增生异常综合征细胞株SKM-1增殖和凋亡的影响。方法    利用实时荧光定量逆转录PCR(qRT-PCR)检测收集的33位MDS/AML患者以及11位健康人(健康对照)的骨髓标本中miR-143的表达;细胞实验分为过表达组(SKM-1细胞感染含LV-hsa-miR-143的慢病毒载体)、低表达组(SKM-1细胞感染含LV-hsa-miR-143inhibitors的慢病毒载体)和阴性对照组(SKM-1细胞感染含空病毒LV-control的慢病毒载体),分别采用CCK-8法、流式细胞仪检测各组SKM-1细胞增殖、周期和凋亡情况;双荧光素酶报告基因检测miR-143对下游靶基因AF9在转录水平的影响;Western blot测定miR-143对下游靶基因AF9蛋白表达水平的影响。结果与健康对照比较,MDS患者的miR-143相对表达水平明显降低(P<0.05);过表达组的光密度值在第3天为(0.562±0.069),明显低于阴性对照组(P<0.05);低表达组的光密度值在第3天为(1.537±0.094),明显高于阴性对照组(P<0.05);过表达组G0/G1期细胞比例为(57.92±3.58)%,明显高于阴性对照组(P<0.05);低表达组G0/G1期细胞比例为(29.73±2.44)%,明显低于阴性对照组(P<0.05);过表达组细胞凋亡率为(29.80±2.53)%,明显高于阴性对照组(P<0.05);而低表达组细胞凋亡率为(5.17±1.04)%,明显低于阴性对照组(P<0.05);miR143可以显著抑制AF9 3’UTR野生型的报告基因活性;低表达组AF9蛋白表达水平显著高于阴性对照组(P<0.05)。结论    miR-143在MDS患者中的相对表达水平明显减低;过表达miR-143后骨髓增生异常综合征SKM-1细胞增殖活性降低、G0/G1期细胞阻滞增多、凋亡率增加;同时miR143能够负性调节其靶基因AF9。

Abstract:

Objective    To investigate the expression of miR-143 in the bone marrow of patients with myelodysplastic syndrome (MDS) and whether it inhibits SKM-1 cell proliferation and apoptosis by inhibiting AF9. Methods    Real-time quantitative RTPCR (qRT-PCR) was used to detect the expression of miR-143 in bone marrow samples from 33 patients with MDS/acute myeloid leukemia (AML) and 11 healthy individuals. In the in vitro experiment, SKM-1 cell line, a cell model of MDS, was infected with the lentiviral vectors LV-hsa-miR-143, LV-hsa-miR-143-inhibitors, or LV-control, and the changes in the cell proliferation, cell cycle and apoptosis were detected using CCK-8 assay and flow cytometry. Dual-luciferase reporter assay was used to assess the effect of miR-143 expression modulation on the transcriptional level of the downstream target gene AF9; the expression of AF9 protein was detected using Western blotting. Results    Compared with the healthy individuals, MDS patients showed a significantly lowered expression level of miR143 in the bone marrow. In SKM-1 cells, mi-R143 overexpression significantly suppressed cell proliferation, and miR-143 knockdown significantly inhibited cell proliferation as compared with the control cells (P<0.05); mi-R143 overexpression significantly increased the percentage of cells in G0/G1 phase compared with the negative control cells [(57.92±3.58)% vs (38.75±2.78)%, P<0.05], and knockdown of miR-143 expression obviously lowered the percentage of cells in G0/G1 phase [(29.73±2.44)%, P<0.05]; mi-R143 overexpression significantly increased the cells apoptosis rate to (29.80±2.53)% (P<0.05), and miR-143 knockdown decreased the cell apoptosis rate to (5.17±1.04)% (P<0.05), as compared to the rate of (13.92±1.49)% in the negative control cells. Overexpression of miR-143 significantly inhibited the activity of wild-type AF9 3’-UTR reporter gene, and compared with the control cells, the cells with miR-143 knockdown showed significantly increased expression level of AF9 protein (P<0.05). Conclusion    The patients with MDS show obviously lowered expression level of miR-143 in the bone marrow. Overexpression of miR-143 suppresses the proliferation, induces cell cycle arrest in G0/G1 phase and promotes apoptosis of SKM-1 cells by negatively regulating its target gene AF9.

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更新日期/Last Update: 2018-10-31