组蛋白甲基转移酶 G9a 通过表观遗传沉默肿瘤抑制基因 RARRES3 促进肝癌的发展。

Histone methyltransferase G9a promotes liver cancer development by epigenetic silencing of tumor suppressor gene RARRES3.

机构信息

State Key Laboratory for Liver Research, The University of Hong Kong, Hong Kong; Department of Pathology, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong; The University of Hong Kong Shenzhen Institute of Research and Innovation, Shenzhen, China.

State Key Laboratory for Liver Research, The University of Hong Kong, Hong Kong; Department of Pathology, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong.

出版信息

J Hepatol. 2017 Oct;67(4):758-769. doi: 10.1016/j.jhep.2017.05.015. Epub 2017 May 19.

DOI:10.1016/j.jhep.2017.05.015

PMID:28532996

Abstract

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is a major leading cause of cancer mortality worldwide. Epigenetic deregulation is a common trait of human HCC. G9s is an important epigenetics regulator however, its role in liver carcinogenesis remains to be investigated.

METHODS

Gene expressions were determined by RNA-Seq and qRT-PCR. G9a knockdown and knockout cell lines were established by lentiviral-based shRNA and CRISPR/Cas9 gene editing system. Tumor-promoting functions of G9a was studied in both HCC cell lines and nude mice model. The downstream targets of G9a were identified by RNA-Seq and confirmed by ChIP assay. The therapeutic value of G9a inhibitors was evaluated both in vitro and in vivo.

RESULTS

We identified G9a as a frequently upregulated histone methyltransferase in human HCCs. Upregulation of G9a was significantly associated with HCC progression and aggressive clinicopathological features. Functionally, we demonstrated that inactivation of G9a by RNAi knockdown, CRISPR/Cas9 knockout, and pharmacological inhibition remarkably abolished H3K9 di-methylation and suppressed HCC cell proliferation and metastasis in both in vitro and in vivo models. Mechanistically, we showed that the frequent upregulation of G9a in human HCCs was attributed to gene copy number gain at chromosome 6p21. In addition, we identified miR-1 as a negative regulator of G9a. Loss of miR-1 relieved the post-transcriptional repression on G9a and contributed to its upregulation in human HCC. Utilizing RNA sequencing, we identified the tumor suppressor RARRES3 as a critical target of G9a. Epigenetic silencing of RARRES3 contributed to the tumor-promoting function of G9a.

CONCLUSION

This study shows a frequent deregulation of miR-1/G9a/RARRES3 axis in liver carcinogenesis, highlighting the pathological significance of G9a and its therapeutic potential in HCC treatment. Lay summary: In this study, we identified G9a histone methyltransferase was frequently upregulated in human HCC and contributes to epigenetic silencing of tumor suppressor gene RARRES3 in liver cancer. Targeting G9a may be a novel approach for HCC treatment.

摘要

背景与目的

肝细胞癌（HCC）是全球癌症死亡的主要原因。表观遗传失调是人类 HCC 的共同特征。G9s 是一种重要的表观遗传学调节剂，但其在肝癌发生中的作用仍有待研究。

方法

通过 RNA-Seq 和 qRT-PCR 确定基因表达。通过基于慢病毒的 shRNA 和 CRISPR/Cas9 基因编辑系统建立 G9a 敲低和敲除细胞系。在 HCC 细胞系和裸鼠模型中研究 G9a 的促肿瘤功能。通过 RNA-Seq 鉴定 G9a 的下游靶标，并通过 ChIP 检测进行验证。在体外和体内评估 G9a 抑制剂的治疗价值。

结果

我们发现 G9a 是人类 HCC 中经常上调的组蛋白甲基转移酶。G9a 的上调与 HCC 进展和侵袭性临床病理特征显著相关。功能上，我们证明通过 RNAi 敲低、CRISPR/Cas9 敲除和药理学抑制失活 G9a，可显著消除 H3K9 二甲基化，并抑制 HCC 细胞在体外和体内模型中的增殖和转移。机制上，我们表明 G9a 在人类 HCC 中的频繁上调归因于染色体 6p21 上的基因拷贝数增加。此外，我们发现 miR-1 是 G9a 的负调节因子。miR-1 的缺失解除了对 G9a 的转录后抑制作用，并导致其在人类 HCC 中的上调。利用 RNA 测序，我们确定了肿瘤抑制因子 RARRES3 是 G9a 的关键靶标。RARRES3 的表观遗传沉默有助于 G9a 的促肿瘤功能。