内科理论与实践

内科理论与实践 ›› 2025, Vol. 20 ›› Issue (02): 146-151.doi: 10.16138/j.1673-6087.2025.02.08

• 论著 • 上一篇    下一篇

PML::RARα融合蛋白对白介素6受体的转录调控研究

赵玲玲1, 崔灿琦2, 糜坚青1,3()   

  1. 1.上海交通大学医学院附属瑞金医院血液科 上海血液学研究所，上海 200025
    2.杜克大学医学院分子生理学研究所，美国北卡罗来纳州 达勒姆 27703
    3.上海市血液病基因编辑与细胞免疫治疗重点实验室，上海 200025
  • 收稿日期:2025-03-25 出版日期:2025-04-28 发布日期:2025-07-08
  • 通讯作者: 糜坚青 E-mail:jianqingmi@shsmu.edu.cn
  • 基金资助:
    国家重点研发计划(2023YFC2508900);上海交通大学医学院“上海市高水平地方高校”协同创新团队项目

Transcriptional regulation of interleukin-6 receptor by PML::RARα fusion protein

ZHAO Lingling1, CUI Canqi2, MI Jianqing1,3()   

  1. 1. Department of Hematology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Institute of Hematology, Shanghai 200025, China
    2. Duke Molecular Physiology Institute, Duke University School of Medicine, Durham, NC 27703, USA
    3. Shanghai Key Laboratory of Gene Editing and Cell-based Immunotherapy for Hematological Diseases, Shanghai 200025, China
  • Received:2025-03-25 Online:2025-04-28 Published:2025-07-08
  • Contact: MI Jianqing E-mail:jianqingmi@shsmu.edu.cn

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摘要：

目的：探讨PML::RARα融合蛋白对白介素6受体（interleukin-6 receptor ，IL-6R）的调控机制,及IL-6R对急性早幼粒细胞白血病（acute promyelocytic leukemia，APL）细胞增殖和分化的影响。方法：采用GSE12662和GSE10358数据集分析APL细胞中IL-6R的表达水平；采用逆转录实时定量PCR（reverse transcription quantitative real-time PCR，RT-qPCR）检测全反式维A酸（all-trans retinoic acid，ATRA）处理前后的NB4细胞及Zn²⁺诱导前后PR9细胞中IL-6R的mRNA表达；通过染色质免疫共沉淀（chromatin immunoprecipitation，ChIP）-seq数据分析、ChIP-qPCR实验及双荧光素酶报告基因活性检测，研究PML::RARα对IL-6R的调控机制；构建IL-6R过表达质粒并通过逆转录病毒转染至NB4细胞，利用细胞计数试剂盒-8（cell counting kit-8，CCK-8）实验检测细胞增殖，流式细胞术检测NB4细胞的分化情况。结果：GSE12662数据集分析结果显示，APL患者早幼粒细胞中IL-6R的表达水平（12.20±0.41）显著低于正常早幼粒细胞（13.14±0.47,t=4.289,P<0.001）和中性粒细胞（14.82±0.40,t=12.35,P<0.001）；GSE10358数据集分析结果显示,APL患者白血病细胞中IL-6R表达水平（5.93±0.84）显著低于非APL急性髓系白血病（acute myeloid leukemia，AML）患者（6.50±0.87,t=3.91,P<0.001）。APL患者细胞中IL-6R表达受PML::RARα融合蛋白的抑制，在APL中呈低表达模式，其机制为PML::RARα直接结合在IL-6R启动子区域，从而抑制其转录。在APL细胞株NB4中过表达IL-6R后，细胞增殖显著被抑制，转染4 d后，CCK-8检测到的吸光度值分别为0.86 ± 0.01和0.40 ± 0.01（t=32.66，P<0.001）；同时细胞分化显著增强，CD11b阳性细胞比例由（3.10±1.22）%升高至（14.4±1.11）%（t=11.84，P<0.001）。结论：IL-6R是PML::RARα的靶基因，PML::RARα通过直接结合IL-6R启动子抑制其转录；明确IL-6R通过抑制APL细胞增殖并诱导其部分分化发挥其生物学功能。

关键词: 白介素6受体, 急性早幼粒细胞白血病, PML::RARα, 细胞增殖, 细胞分化

Abstract:

Objective To investigate the regulatory mechanism of PML::RARα fusion protein on interleukin-6 receptor (IL-6R) and the effects of IL-6R on the proliferation and differentiation of acute promyelocytic leukemia (APL) cells.Methods The expression levels of IL-6R in APL cells were analyzed using the GSE12662 and GSE10358 datasets. Reverse transcription quantitative real-time quantitative PCR（RT-qPCR） was performed to detect IL-6R mRNA expression in NB4 cells before and after all-trans retinoic acid (ATRA) treatment, as well as in PR9 cells before and after Zn²⁺ induction. Chromatin immunoprecipitation (ChIP)-seq data analysis, ChIP-qPCR experiments, and luciferase reporter gene activity assays were performed to explore the regulatory mechanism of PML::RARα on IL-6R. An IL-6R expression plasmid was constructed for NB4 cells via retrovirus. Cell proliferation was assessed using the cell counting kit-8 (CCK-8) assay, and CD11b expression was detected by flow cytometry.Results Analysis of the GSE12662 dataset revealed that the expression level of IL-6R in APL cells (12.20 ± 0.41) was significantly lower than that in normal promyelocytes (13.14 ± 0.47, t = 4.289, P < 0.001) and polymorphonuclear cells (14.82 ± 0.40, t = 12.35, P < 0.001). Moreover, analysis of the GSE10358 dataset showed that IL-6R expression in APL patients (5.93 ± 0.84) was significantly lower than that in non-APL AML patients (6.50 ± 0.87, t = 3.91, P < 0.001). PML::RARα directly bound to the promoter region of IL-6R to inhibit its transcriptional activity resulting in the low expression. Overexpression of IL-6R in the APL-derived NB4 cells significantly inhibited cell proliferation. Four days after transfection, the optical density values measured by the CCK-8 assay were 0.86 ± 0.01 and 0.40 ± 0.01, respectively (t = 32.66, P < 0.001). Simultaneously, cell differentiation was significantly enhanced. The ratio of the CD11b positive cells increased from 3.10 % ± 1.22 % to 14.4 % ± 1.11 % (t = 11.84, P < 0.001).Conclusions IL-6R is a target gene of PML::RARα, demonstrating that PML::RARα can suppress IL-6R transcription by binding to its promoter region. It is illustrated that IL-6R inhibited the cell proliferation and induced partial differentiation in APL cells.

Key words: Interleukin-6 receptor, Acute promyelocytic leukemia, PML::RARα, Cell proliferation, Cell differentiation

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