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北京大学学报（医学版） ›› 2025, Vol. 57 ›› Issue (5): 884-894. doi: 10.19723/j.issn.1671-167X.2025.05.012

• 论著 • 上一篇    下一篇

HDAC2介导H3K27ac修饰促进肝癌细胞的增殖和迁移

唐少海1, 杨宝明1, 李建坤1, 赵丽丽2, 王仪凡3, 王顺祥1,*()   

  1. 1. 河北医科大学第四医院肝胆外科, 石家庄 050000
    2. 河北医科大学药学院, 石家庄 050000
    3. 河北医科大学第四医院结直肠外科, 石家庄 050000
  • 收稿日期:2024-06-28 出版日期:2025-10-18 发布日期:2025-06-18
  • 通讯作者: 王顺祥
  • 基金资助:
    河北省自然科学基金(H2020206455); 河北省2023年度医学科学研究课题计划(20230875); 河北省2018年度医学科学研究重点课题计划(20180537); 以及2015年和2016年河北省政府资助临床医学优秀人才培养和基础课题研究项目计划(361006)

HDAC2-mediated H3K27 acetylation promotes the proliferation and migration of hepatocellular carcinoma cells

Shaohai TANG1, Baoming YANG1, Jiankun LI1, Lili ZHAO2, Yifan WANG3, Shunxiang WANG1,*()   

  1. 1. Department of Hepatobiliary Surgery, the Fourth Hospital of Hebei Medical University, Shijiazhuang 050000, China
    2. College of Pharmacy, Hebei Medical University, Shijiazhuang 050000, China;
    3. Department of Colorectal Surgery, the Fourth Hospital of Hebei Medical University, Shijiazhuang 050000, China
  • Received:2024-06-28 Online:2025-10-18 Published:2025-06-18
  • Contact: Shunxiang WANG
  • Supported by:
    the Natural Science Foundation Project of Hebei Province(H2020206455); the 2023 Annual Medical Science Research Project Plan of Hebei Province(20230875); the Key Medical Science Research Project Plan of Hebei Province in 2018(20180537); the 2015 and 2016 Hebei Provincial Government' s Plan for Funding the Cultivation of Outstanding Clinical Medicine Talents and Basic Research Projects(361006)

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摘要： 目的: 探究组蛋白去乙酰化酶2(histone deacetylase 2, HDAC2)介导组蛋白H3第27位赖氨酸乙酰化(histone H3 lysine 27 acetylation, H3K27ac)修饰促进肝癌细胞增殖、迁移的作用机制。方法: 收集2021年1月至2023年1月经手术切除的40例肝癌、癌旁组织样本, 免疫组化和Western blotting检测肝癌、癌旁组织、细胞系HDAC2、H3K27ac表达, 分析HDAC2与H3K27ac表达水平的相关性, 以及HDAC2表达与肝癌患者临床病理特征的关系。Hep3B、HepG2细胞分为sh-NC组(转染sh-NC)、sh-HDAC2组(转染sh-HDAC2)、空质粒组(转染dCas9-空质粒)、HDAC2组(转染dCas9-HDAC2)、iHDAC2组(转染dCas9-失活HDAC2)和iHDAC2+740Y-P组(转染dCas9-失活HDAC2, 培养基加入30 μmol/mL 740Y-P)。通过MTS、克隆形成、划痕、Transwell实验测定各组Hep3B、HepG2细胞增殖、迁移、侵袭能力, 通过Western blotting、实时荧光定量PCR(real-time fluorescence quantitative PCR, qRT-PCR)、HDAC2活性检测、染色质免疫共沉淀-高通量测序(chromatin immunoprecipitation high-throughput sequencing, ChIP-seq)检测验证HDAC2介导H3K27ac修饰, 体内异种移植实验测定各组细胞成瘤能力, 检测磷脂酰肌醇-3激酶/第10号染色体磷酸酶和张力蛋白同源缺失基因/蛋白激酶B/哺乳动物雷帕霉素靶蛋白(phosphoinositide 3-kinases/phosphatase and tensin homolog deleted on chromosome ten/protein kinase B/mammalian target of rapamycin, PI3K/PTEN/AKT/mTOR)信号通路相关蛋白表达。结果: 肝癌组织、细胞系HDAC2呈高表达(P＜0.05), H3K27ac呈低表达(P＜0.05), 二者表达水平呈负相关(r=-0.477, P=0.002)。HDAC2表达水平与肿瘤大小、感染乙肝病毒、TNM分期、门静脉癌栓相关(P＜0.05)。与Hep3B、HepG2细胞sh-NC组相比, sh-HDAC2组增殖、克隆形成、迁移、侵袭能力降低(P＜0.05)。与空质粒组相比, HDAC2组HDAC2表达水平、活性、细胞增殖、克隆形成、迁移、侵袭能力, 体内成瘤体积、质量, p-PI3K、p-AKT、p-mTOR表达水平均升高(P＜0.05), H3K27ac富集程度、H3K27ac、PTEN表达水平降低(P＜0.05), iHDAC2组HDAC2表达水平、活性、增殖、克隆形成、迁移、侵袭能力, 体内成瘤体积、质量, p-PI3K、p-AKT、p-mTOR表达水平降低(P＜0.05), H3K27ac、PTEN表达水平升高(P＜0.05)。引入PI3K激活剂740Y-P验证PI3K/PTEN/AKT/mTOR信号通路参与HDAC2介导H3K27ac修饰的肝癌细胞恶性行为调控, 与iHDAC2组相比, iHDAC2+740Y-P组增殖、克隆形成、迁移、侵袭能力, 体内成瘤体积、质量, p-PI3K、p-AKT、p-mTOR表达水平升高(P＜0.05), PTEN表达水平降低(P＜0.05)。结论: HDAC2通过介导H3K27ac修饰启动PI3K/PTEN/AKT/mTOR信号通路, 促进肝癌发生发展。

关键词: 组蛋白去乙酰化酶2, 组蛋白H3第27位赖氨酸乙酰化, 肝癌, 增殖, 迁移

Abstract: Objective: To explore the specific mechanism of histone deacetylase 2 (HDAC2) mediated histone H3 lysine 27 acetylation (H3K27ac) modification in promoting the proliferation and migration of hepatocellular carcinoma cells. Methods: Samples of 40 cases of hepatocellular carcinoma and paracancerous tissues resected from January 2021 to January 2023 were collected. The expressions of HDAC2 and H3K27ac in hepatocellular carcinoma, paracancerous tissues and cell lines were detected by immunohistochemistry and Western blotting. The correlation between the expression levels of HDAC2 and H3K27ac and the relationship between HDAC2 expression and clinicopathological characteristics of patients with hepatocellular carcinoma were analyzed. The proliferation, migration and invasion of Hep3B and HepG2 cells were determined by MTS, clone formation, scratch and Transwell experiments. The acetylation of H3K27 mediated by HDAC2 was verified by Western blotting, real-time fluorescence quantitative PCR (qRT-PCR) and chromatin immunoprecipitation high-throughput sequencing (ChIP-seq). In vivo xenotransplantation experiment, the tumorigenicity of cells in each group was measured, and the expression of proteins related to phosphoinositide 3-kinases/phosphatase and tensin homolog deleted on chromosome ten/protein kinase B/mammalian target of rapamycin (PI3K/PTEN/AKT/mTOR) signal pathway was detected. Results: High expression of HDAC2 and low expression of H3K27ac were found in hepatocellular carcinoma tissues and cell lines (P < 0.05), and there was a negative correlation between them (r=-0.477, P=0.002). The expression of HDAC2 was related to tumor size, hepatitis B virus infection, TNM stage and portal vein tumor thrombus (P < 0.05). Compared with the sh-NC group of Hep3B and HepG2 cells, the proliferation, clone formation, migration and invasion ability of sh-HDAC2 group were decreased (P < 0.05). Compared with the Empty group, the HDAC2 group exhibited increased expression levels and activity of HDAC2, as well as enhanced cell proliferation, clone formation, migration, invasion ability, tumor volume and mass in vivo, and elevated expression levels of p-PI3K, p-AKT, and p-mTOR (P < 0.05). Conversely, the enrichment and expression levels of H3K27ac, along with the expression level of PTEN, were decreased (P < 0.05). In the iHDAC2 group, the expression levels and activity of HDAC2, as well as the proliferation, clone formation, migration, invasion ability, tumor volume and mass in vivo, and expression levels of p-PI3K, p-AKT, and p-mTOR were reduced (P < 0.05). Additionally, the expression levels of H3K27ac and PTEN were increased (P < 0.05). To validate the involvement of the PI3K/PTEN/AKT/mTOR signaling pathway in HDAC2-mediated regulation of malignant behaviors in liver cancer cells through H3K27ac, the PI3K activator 740Y-P was introduced. Compared with the iHDAC2 group, the iHDAC2+740Y-P group exhibited increased proliferation, clone formation, migration, invasion ability, tumor volume and mass in vivo, and elevated expression levels of p-PI3K, p-AKT, and p-mTOR (P < 0.05). Conversely, the expression level of PTEN was decreased (P < 0.05). Conclusion: HDAC2 initiates PI3K/PTEN/AKT/mTOR signal pathway by mediating H3K27 acetylation, which promotes the occurrence and development of hepatocellular carcinoma.

Key words: Histone deacetylase 2, Histone H3 lysine 27 acetylation, Hepatocellular carcinoma, Proliferation, Migration

中图分类号: 

  • R735.7

表1

引物序列"

Gene Primer sequences (5′-3′)
HDAC2 Forward：ATGGCGTACAGTCAAGGAGGC
Reverse：AAATCAGAACAGCTCAGCAAC
GAPDH Forward：ATGGGGAAGGTGAAGGTCGG
Reverse：TTACTCCTTGGAGGCCATGT

图1

HDAC2、H3K27ac在肝癌中的表达情况"

表2

HDAC2表达水平与肝癌患者临床病理特征间的关系"

Items Total HDAC2 expression χ2 P
High (n=20) Low (n=20)
Age/years
  ≥59 25 (62.50) 14 (35.00) 11 (27.50) 0.960 0.327
  ＜59 15 (37.50) 6 (15.00) 9 (22.50)
Gender
  Male 21 (52.50) 13 (32.50) 8 (20.00) 2.506 0.113
  Female 19 (47.50) 7 (17.50) 12 (30.00)
Tumor size/cm
  ≥3 28 (70.00) 17 (42.50) 11 (27.50) 4.286 0.038
  ＜3 12 (30.00) 3 (7.50) 9 (22.50)
HBV infection
  Positive 30 (75.00) 19 (47.50) 11 (27.50) 8.533 0.003
  Negative 10 (25.00) 1 (2.50) 9 (22.50)
Alpha-fetoprotein/(μg/L)
  ≥400 27 (67.50) 12 (30.00) 15 (37.50) 1.026 0.311
  ＜400 13 (32.50) 8 (20.00) 5 (12.50)
Histologic grade
  Well and moderate 25 (62.50) 10 (25.00) 15 (37.50) 2.667 0.102
  Low 15 (37.50) 10 (25.00) 5 (12.50)
TNM stage
  Ⅰ to Ⅱ 19 (47.50) 6 (15.00) 13 (32.50) 4.912 0.027
  Ⅲ to Ⅳ 21 (52.50) 14 (35.00) 7 (17.50)
Portal vein tumor thrombus
  Yes 11 (27.50) 9 (22.50) 2 (5.00) 6.144 0.013
  No 29 (72.50) 11 (27.50) 18 (45.00)
Cirrhosis
  Yes 15 (37.50) 6 (15.00) 9 (22.50) 0.960 0.327
  No 25 (62.50) 14 (35.00) 11 (27.50)

图2

抑制HDAC2对肝癌细胞增殖、迁移和侵袭的影响"

图3

HDAC2介导H3K27ac修饰"

图4

体外细胞实验验证HDAC2介导H3K27ac修饰促进肝癌细胞增殖、迁移和侵袭"

图5

体内细胞实验验证HDAC2介导H3K27ac修饰启动PI3K/PTEN/AKT/mTOR信号通路增强肝癌细胞成瘤能力"

图6

HDAC2介导H3K27ac修饰促进肝癌进展的作用机制"

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ISSN 1671-167X
CN 11-4691/R
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