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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front. Med.    2017, Vol. 11 Issue (1) : 87-96     DOI: 10.1007/s11684-016-0469-4
RESEARCH ARTICLE |
Regulatory mechanism and functional analysis of S100A9 in acute promyelocytic leukemia cells
Yonglan Zhu1,Fang Zhang1,Shanzhen Zhang2,Wanglong Deng1,Huiyong Fan1,Haiwei Wang1,2,Ji Zhang1,2()
1. State Key Laboratory of Medical Genomics, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
2. Medical Institute of Health Sciences, Chinese Academy of Sciences, Shanghai 200025, China
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Abstract  

S100A9, a calcium-binding protein, participates in the inflammatory process and development of various tumors, thus attracting much attention in the field of cancer biology. This study aimed to investigate the regulatory mechanism of S100A9 and its function involvement in APL. We used real-time quantitative PCR to determine whether PML/RARα affects the expression of S100A9 in NB4 and PR9 cells upon ATRA treatment. ChIP-based PCR and dual-luciferase reporter assay system were used to detect how PML/RARα and PU.1 regulate S100A9 promoter activity. CCK-8 assay and flow cytometry were employed to observe the viability and apoptosis of NB4 cells when S100A9 was overexpressed. Results showed that S100A9 was an ATRA-responsive gene, and PML/RARα was necessary for the ATRA-induced expression of S100A9 in APL cells. In addition, PU.1 could bind to the promoter of S100A9, especially when treated with ATRA in NB4 cells, and promote its activity. More importantly, overexpression of S100A9 induced the apoptosis of NB4 cells and inhibited cell growth. Collectively, our data indicated that PML/RARα and PU.1 were necessary for the ATRA-induced expression of S100A9 in APL cells. Furthermore, S100A9 promoted apoptosis in APL cells and affected cell growth.

Keywords S100A9      PU.1      PML/RARα      ATRA      APL     
Corresponding Authors: Ji Zhang   
Just Accepted Date: 14 December 2016   Online First Date: 05 January 2017    Issue Date: 20 March 2017
URL:  
http://academic.hep.com.cn/fmd/EN/10.1007/s11684-016-0469-4     OR     http://academic.hep.com.cn/fmd/EN/Y2017/V11/I1/87
Fig.1  ATRA-induced expression of S100A9 is dependent on PML-RARa in APL. (A) S100A9 expression in APL patients was significantly lower than that in other AML patients. The expression values are the absolute intensities after log transformation. Data were extracted from GSE10358. (B) The expression level of S100A9 was significantly lower in NB4 cells than in U937 and HL-60 cells. (C) RNA and protein expression of S100A9 gradually increased in NB4 cells when treated with ATRA at a series of time points. (D) The relative expression of S100A9 gradually increased after PML/RARa induction of ZnSO4-treated PR9 cells in the presence of ATRA at the indicated time points. However, the expression did not change in PR9 cells without ZnSO4 treatment even in the presence of ATRA. The data represent the mean of three replicates±S.D. **P<0.01, ***P<0.001.
Fig.2  PML/RARa binds to the promoter of S100A9 in NB4 cells. (A) Schematic representation of the promoter regions of S100A9. PU.1 binding sites (rhombus) and RARE half-sites (rectangle) were defined using TRANSFAC with core and matrix similarity. The S100A9-1 region spanned PU.1 and RARE sites. The S100A9-2 region spanned PU.1 sites. (B) Schematic shows the binding of PML/RARa to the promoter regions of S100A9. ChIP assays of the NB4 cells were conducted using anti-PML and anti-RARa antibodies. The peaks represented the PML/RARa-enriched ChIP regions. S100A9 was located on the Watson (+) strand of chromosome 1. (C) ChIP was conducted on NB4 cells using anti-RARa, anti-PML, or nonspecific (normal immunoglobulin G (IgG)) antibodies. ChIP-qPCR was conducted using S100A9-1 and negative (BLNK) or positive (PLCB2) primers. Results were presented as fold enrichment of chromatin, normalized to nonspecific IgG. (D) Promoter activities of S100A9 were repressed by PML/RARa in a dose-dependent manner. Luciferase reporter assays were conducted on 293T cells. The data represent the mean of three replicates±S.D.
Fig.3  PU.1 regulates the basal expression of S100A9 by targeting the promoter regions of S100A9. (A) A positive correlation was found between S100A9 and PU.1 mRNA levels in 415 AML (M1–M6) patients from GSE14468. (B) Knockdown of PU.1 reduced ATRA-mediated activation of S100A9 at the mRNA expression level. The mRNA levels of PU.1 and S100A9 were detected in NB4 cells by RT-PCR at 48 h post-transfection with siRNA (3 µg) and in the absence or presence of ATRA for 24 h. (C) PU.1 bound to the promoter of S100A9 in APL cells. The pattern on the left is a schematic that shows PU.1 binding to the promoter regions of S100A9. ChIP assays of the NB4 cells were conducted using anti-PU.1 antibody. The peaks represent the PU.1-enriched ChIP regions. The image on the right shows the ChIP results of the assays for the NB4 cells that were conducted using anti-PU.1 or IgG antibodies. ChIP-qPCR was undertaken with S100A9-1, S100A9-2, BLNK, and PLCB2 primers. (D) PU.1 increased the promoter activities of S100A9 in a dose-dependent manner. Luciferase reporter assays were conducted on 293T cells. The data represent the mean of three replicates±S.D. *P<0.05.
Fig.4  Overexpression of S100A9 induces cell apoptosis and growth inhibition in NB4 cells. (A) Relative expression of S100A9 in NB4 cells that were harvested 72 h after MigR1 (Ctrl) or MigR1-S100A9 infection. (B) S100A9 overexpression inhibited the proliferation of NB4 cells. NB4 cells were infected with MigR1 (Ctrl) or MigR1-S100A9 retroviral component, and cell proliferation was detected by CCK-8 assay. Results showed that the proliferation rate of NB4 cells infected with MigR1-S100A9 was reduced significantly at days 4 and 5 when compared with the MigR1 (Ctrl) group and blank group. *P<0.05. (C) Overexpression of S100A9 induced apoptosis of NB4 cells. GFP-positive cells were analyzed by flow cytometry. The left panel depicts the apoptosis of MigR1-infected NB4 cells, and the right panel shows the result for MigR1-S100A9-infected NB4 cells. The values represent the three replicates±S.D. (D) Western blot analysis of cleaved caspase-3 and Bcl-2 expression in total cell lysates.
Fig.5  Possible model for the transcription regulation of S100A9 is proposed in APL.
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