EZH2-H3K27me3 介导的 miR-139-5p 沉默通过激活 TOP2A 抑制肝癌细胞衰老。

EZH2-H3K27me3-mediated silencing of mir-139-5p inhibits cellular senescence in hepatocellular carcinoma by activating TOP2A.

机构信息

Department of digestive surgery, Xijing Hospital, Fourth Military Medical University, Xi'an, 710032, China.

Department of General Surgery, Central Theater Command General Hospital of the Chinese People's Liberation Army, Wuhan, 430064, China.

出版信息

J Exp Clin Cancer Res. 2023 Nov 27;42(1):320. doi: 10.1186/s13046-023-02855-2.

DOI:10.1186/s13046-023-02855-2

PMID:38008711

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10680220/

Abstract

BACKGROUND

Epigenetic alterations play an important role in hepatocellular carcinoma (HCC) development. Enhancer of zeste homolog 2 (EZH2) is a well-known epigenetic modifier that functions as an oncogene in tumors by promoting the H3K27me3-mediated transcriptional repression of tumor suppressor genes. "Senescent cells" has been proposed as a possible core component of the hallmarks of cancer conceptualization. Induction of cell senescence and targeted elimination of these senescent tumor cells are new strategies for tumor therapy. However, the role of EZH2 in regulating cellular senescence remains poorly understood.

METHODS

Bioinformatics analyses suggested that EZH2 and DNA topoisomerase II alpha (TOP2A) are coexpressed in tumors, including HCC. Kyoto Encyclopedia of Genes and Genome (KEGG) pathway enrichment analyses and gene set enrichment analyses (GSEA) suggests a correlation of EZH2 and TOP2A expression with cellular senescence in HCC. MicroRNA (miRNA) inhibitor and mimics, siRNA, PLKO-shRNA, and plenti6.3-miR-139 were used to upregulate or downregulate the expression of target genes. CCK8, EdU, clone formation, and senescence-associated β-galactosidase (SA-β-gal) staining assays were performed to assess cell proliferation and cellular senescence phenotypes. Dual-luciferase reporter and chromatin immunoprecipitation assays were performed to investigate the targeted binding and inhibition of TOP2A 3' untranslated region (UTR) by miR-139-5p and the DNA enrichment of miR139-5p by EZH2 and H3K27me3. BALB/c nude mice were used to establish a xenograft tumor model and verify the phenotypes upon EZH2 and TOP2A silencing and miR-139 overexpression in vivo. In addition, tissue microarrays were used to analyze the expression patterns and correlations among EZH2, TOP2A, and miR-139-5p expression in HCC.

RESULTS

Bioinformatics analysis revealed that EZH2 and TOP2A are coexpressed in HCC. In vitro gain- and loss-of-function experiments showed that inhibition of EZH2 and TOP2A induces cellular senescence and inhibits proliferation of HCC cells. In vivo tumorigenesis assays indicated that EZH2 and TOP2A knockdown inhibits tumorigenesis by inducing cellular senescence. Mechanistically, EZH2 promotes TOP2A expression by regulating the H3K27me3-mediated epigenetic silencing of miR-139-5p. TOP2A is a direct target of miR-139-5p, and inhibition of miR-139-5p can reverse the promotion by EZH2 of TOP2A expression. The overexpression of miR-139-5p induces cellular senescence and inhibits proliferation of HCC cells both in vitro and in vivo. Clinically, expression of EZH2 and TOP2A are higher in HCC tissues than in normal tissues, and this high coexpression indicates a worse outcome of patients with HCC. Moreover, expression of EZH2 and TOP2A is significantly correlated with tumor differentiation grade, tumor invasion, and TNM stage in HCC. miR-139-5p expression is lower in HCC tumors than in normal tissues and is correlated with better prognosis of HCC patients.

CONCLUSIONS

Our study revealed the role of the EZH2/miR-139-5p/TOP2A axis in regulating cellular senescence and cell proliferation in HCC, enriching the molecular mechanisms of EZH2-mediated epigenetic regulation in HCC. Therefore, our results provide insight into the therapeutic potential of targeting EZH2 to induce cellular senescence and then destroy senescent cells for HCC.

摘要

背景

表观遗传改变在肝细胞癌（HCC）发展中起着重要作用。EZH2 是一种众所周知的表观遗传修饰物，作为肿瘤中的致癌基因，通过促进肿瘤抑制基因的 H3K27me3 介导的转录抑制来发挥作用。“衰老细胞”已被提议作为癌症概念化的标志性核心组成部分之一。诱导细胞衰老和靶向消除这些衰老肿瘤细胞是肿瘤治疗的新策略。然而，EZH2 在调节细胞衰老中的作用仍知之甚少。

方法

生物信息学分析表明，EZH2 和 DNA 拓扑异构酶 II alpha（TOP2A）在肿瘤中包括 HCC 中共同表达。京都基因与基因组百科全书（KEGG）途径富集分析和基因集富集分析（GSEA）表明，EZH2 和 TOP2A 表达与 HCC 中的细胞衰老相关。使用 microRNA（miRNA）抑制剂和模拟物、siRNA、PLKO-shRNA 和 plenti6.3-miR-139 上调或下调靶基因的表达。CCK8、EdU、克隆形成和衰老相关β-半乳糖苷酶（SA-β-gal）染色测定用于评估细胞增殖和细胞衰老表型。双荧光素酶报告和染色质免疫沉淀测定用于研究 miR-139-5p 对 TOP2A 3'非翻译区（UTR）的靶向结合和抑制以及 miR139-5p 由 EZH2 和 H3K27me3 介导的 DNA 富集。使用 BALB/c 裸鼠建立异种移植肿瘤模型，并在体内验证 EZH2 和 TOP2A 沉默和 miR-139 过表达的表型。此外，组织微阵列用于分析 HCC 中 EZH2、TOP2A 和 miR-139-5p 表达的表达模式和相关性。

结果

生物信息学分析显示 EZH2 和 TOP2A 在 HCC 中共同表达。体外增益和失能实验表明，抑制 EZH2 和 TOP2A 可诱导 HCC 细胞衰老并抑制增殖。体内肿瘤发生实验表明，EZH2 和 TOP2A 敲低通过诱导细胞衰老抑制肿瘤发生。机制上，EZH2 通过调节 miR-139-5p 的 H3K27me3 介导的表观遗传沉默来促进 TOP2A 的表达。TOP2A 是 miR-139-5p 的直接靶标，抑制 miR-139-5p 可以逆转 EZH2 对 TOP2A 表达的促进作用。miR-139-5p 的过表达在体外和体内均诱导 HCC 细胞衰老并抑制增殖。临床上，EZH2 和 TOP2A 的表达在 HCC 组织中高于正常组织，这种高共表达表明 HCC 患者的预后更差。此外，EZH2 和 TOP2A 的表达与 HCC 中的肿瘤分化程度、肿瘤侵袭和 TNM 分期显著相关。miR-139-5p 在 HCC 肿瘤中的表达低于正常组织，与 HCC 患者的较好预后相关。