NRG4基因对急性髓系白血病细胞增殖、凋亡及周期的影响

doi:10.19586/j.2095-2341.2022.0181

生物技术进展 ›› 2023, Vol. 13 ›› Issue (2): 305-310.DOI: 10.19586/j.2095-2341.2022.0181

• 研究论文 • 上一篇

NRG4基因对急性髓系白血病细胞增殖、凋亡及周期的影响

黄琬玲¹^,²(), 朱文琦¹^,², 郭妮妮¹^,², 王楠¹^,², 任倩¹^,², 马小彤¹^,²()

^1.中国医学科学院血液病医院（中国医学科学院血液学研究所），北京协和医学院，实验血液学国家重点实验室，国家血液系统疾病临床医学研究中心，细胞生态海河实验室，天津 300020
^2.天津医学健康研究院，天津 301600

收稿日期:2022-11-04 接受日期:2022-12-16 出版日期:2023-03-25 发布日期:2023-04-07
通讯作者: 马小彤
作者简介:黄琬玲 E-mail:huangwanling@ihcams.ac.cn；
基金资助:
国家自然科学基金项目(82270122)

Effects of NRG4 on Proliferation, Apoptosis and Cell Cycle of Acute Myeloid Leukemia Cells

Wanling HUANG¹^,²(), Wenqi ZHU¹^,², Nini GUO¹^,², Nan WANG¹^,², Qian REN¹^,², Xiaotong MA¹^,²()

^1.State Key Laboratory of Experimental Hematology，National Clinical Research Center for Blood Diseases，Haihe Laboratory of Cell Ecosystem，Institute of Hematology & Blood Diseases Hospital，Chinese Academy of Medical Sciences & Peking Union Medical College，Tianjin 300020，China
^2.Tianjin Institutes of Health Science，Tianjin 3016000，China

Received:2022-11-04 Accepted:2022-12-16 Online:2023-03-25 Published:2023-04-07
Contact: Xiaotong MA

摘要/Abstract

摘要：

为了分析、检测造血系统恶性肿瘤患者和细胞系中NRG4基因的表达情况，探讨其对急性髓系白血病（acute myeloid leukemia, AML）细胞系增殖、凋亡、周期和集落形成的影响。运用基因表达综合数据库（gene expression omnibus，GEO）和OncoLnc数据库分析NRG4在造血系统恶性肿瘤患者中的表达情况及其对患者预后的影响，实时荧光定量PCR检测NRG4在造血系统恶性肿瘤各细胞系中的表达情况。构建包含NRG4 shRNA及scramble对照慢病毒，感染AML细胞系Molm-13、KG-1a和Kasumi，采用生长曲线、AnnexinV/7-AAD和PI染色、集落形成等方法评价NRG4对AML细胞增殖、凋亡、周期和集落形成的影响。结果表明，NRG4在AML患者骨髓单核细胞（bone marrow mononuclear cells，BMMCs）中的表达水平显著高于健康对照，高表达NRG4的白血病患者的生存率显著低于低表达患者（P<0.05）。在Molm-13、KG-1a和Kasumi细胞中敲降NRG4能够显著抑制细胞的增殖、集落形成，促进细胞凋亡，并使细胞阻滞于S期（P<0.05）。总之，NRG4基因在AML患者和细胞系中高表达，并与患者预后不良相关，对AML细胞系具有显著的促癌作用。研究结果有助于阐明AML的发生机制，为新治疗靶点的开发奠定了基础。

关键词: NRG4基因, 急性髓系白血病, 细胞增殖, 细胞凋亡

Abstract:

The aim of the study is to detect the expression of NRG4 in patients with hematopoietic malignancies and malignant hematopoietic cell lines and to investigate its effect on proliferation， apoptosis and clone formation of acute myeloid leukemia （AML） cells．The expression of NRG4 in patients with hematopoietic malignancies and its correlation with the prognosis of patients were analyzed with gene expression omnibus （GEO） and OncoLnc database. Expression of NRG4 mRNA of malignant hematopoietic cell lines were detected by real-time fluorescent quantitative PCR. Lentiviral vector for knockdown of NRG4 were constructed and transduced into AML cell lines Molm-13， KG-1a and Kasumi．The roles of NRG4 in cell proliferation， apoptosis， cell cycle， colony formation were evaluated by cell counting， flow cytometric analysis of Annexin V/7-AAD staining， PI staining， colony-forming assay， respectively. Expression of NRG4 was significantly higher in bone marrow mononuclear cells （BMMCs） from patients with AML compared to those from healthy donors. The survival of AML patients with higher NRG4 expression were significantly shorter than those of AML patients with low NRG4 level（P<0.05）. Knockdown of NRG4 inhibited cell growth and colony formation， promoted apoptosis， and induced cell cycle arrest（P<0.05）. In summary， NRG4 is highly expressed in BMMCs from patients with AML and AML cell lines， and its expression was correlated with poor prognosis in AML patients. NRG4 exerted tumor-promoting roles in AML cells. These findings shed insight into the underlying mechanism of development of AML and provide novel target for AML treatment.

Key words: NRG4 gene, acute myeloid leukemia, cell proliferation, apoptosis

中图分类号:

R733.71

黄琬玲, 朱文琦, 郭妮妮, 王楠, 任倩, 马小彤. NRG4基因对急性髓系白血病细胞增殖、凋亡及周期的影响[J]. 生物技术进展, 2023, 13(2): 305-310.

Wanling HUANG, Wenqi ZHU, Nini GUO, Nan WANG, Qian REN, Xiaotong MA. Effects of NRG4 on Proliferation, Apoptosis and Cell Cycle of Acute Myeloid Leukemia Cells[J]. Current Biotechnology, 2023, 13(2): 305-310.

图/表 8

表1 本研究的引物序列

Table 1 Primer sequences used in this study

基因		引物序列（5'→3'）
shNRG4	F	5'-CCGGACCTGGTAGAGACGAGCAGTACTCGAGTACTGCTCGTCTCTACCAGGTTTTTTG-3'
shNRG4	R	5'-AATTCAAAAAACCTGGTAGAGACGAGCAGTACTCGAGTACTGCTCGTCTCTACCAGGT-3'
NRG4^*	F	5'-ATGCCAACAGATCACGAAGAG-3'
NRG4^*	R	5'-AATGGGCTGGGAATAGTAGGT-3'
ACTIN^*	F	5'-AGTATCCATGAAAATAAGTGGTTACAGG-3'
ACTIN^*	R	5'-TTTGCAGTACATAATTTACACAGAAGC-3'

图 1 NRG4在AML患者细胞系中的相对表达量A：NRG4在AML患者及健康对照中的相对表达量；B：NRG4在造血系统恶性肿瘤细胞系中的相对表达量。AML—急性髓系白血病；ALL—急性淋巴细胞白血病；T-ALL—T急性淋巴细胞白血病；CML—慢性髓系白血病；MDS—骨髓增生异常综合征；MLL—混合谱系白血病；Pro-B ALL—早期前体B细胞急性淋巴细胞白血病；Pre-B ALL—前体B细胞急性淋巴细胞白血病；c-ALL—普通急性淋巴细胞白血病。*、***分别表示与健康对照(A)或NB4(B)相比，差异在P<0.05， P<0.000 1水平上有统计学意义。

Fig. 1 Relative mRNA expression of NRG4 in AML patients and hematopoietic malignant cell lines

图2 NRG4表达水平与AML患者生存率的相关关系

Fig. 2 Correlational relationship of the percentage of survival of AML patients stratified by NRG4 expression

图3 RT-qPCR检测Molm-13、KG-1a和Kasumi细胞敲降组NRG4 mRNA水平注：*、***分别表示与shCtrl组相比，差异在P<0.05、P<0.000 1水平上有统计学意义。

Fig. 3 RT-PCR evaluation of NRG4 expression in Molm-13,KG-1a and Kasumi cells with NRG4 knockdown

图4 敲降NRG4对Molm-13、KG-1a和Kasumi细胞增殖的影响注：***表示与shCtrl组相比，差异在P<0.000 1水平上有统计学意义。

Fig. 4 The effects of knockdown of NRG4 on proliferation of Molm-13, KG-1a and Kasumi cells

图5 敲降NRG4对Molm-13、KG-1a和Kasumi细胞集落形成能力的影响注：**、***表示与shCtrl组相比，差异在P<0.01、P<0.000 1水平上有统计学意义。

Fig. 5 The effects of knockdown of NRG4 on colony formation of Molm-13, KG-1a and Kasumi cells

图6 敲降NRG4对Molm-13、KG-1a和Kasumi细胞凋亡的影响注：**、***表示与shCtrl组相比，差异在P<0.01、P<0.0001水平上有统计学意义。

Fig. 6 The effects of knockdown of NRG4 on the apoptosis of Molm-13, KG-1a and Kasumi cells

图7 敲降NRG4对Molm-13、KG-1a和Kasumi细胞周期的影响注：*表示与shCtrl组相比，差异在P<0.05水平上有统计学意义。

Fig. 7 Effect of NRG4 knockdown on cell cycle of Molm-13, KG-1a and Kasumi cells

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NRG4基因对急性髓系白血病细胞增殖、凋亡及周期的影响

Effects of NRG4 on Proliferation, Apoptosis and Cell Cycle of Acute Myeloid Leukemia Cells

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