H2A.Z 是一种高度保守的促癌组蛋白变异体，可分为2种亚型，分别是H2AFZ和H2AFV。这两种亚型的组蛋白变异体虽然在蛋白质水平仅有3个氨基酸的区别，但它们为何在癌症组织表达升高，其机制是什么？这两个亚型是否在肝癌的发生发展发挥着不同的作用呢？这些问题一直困扰众人，而这些问题的解决将可能为诊断或治疗肝癌提供新的靶点。为此，本课题组通过收集116例肝癌病人的癌和癌旁组织，以及利用公共数据库GEO和TCGA，发现H2AFZ和H2AFV在癌组织表达表达水平升高（Figure 1），且均与预后不好有关。随后，利用CHIP-Seq和cBioPortal数据库分析发现H2AFZ和H2AFV在肝癌中发挥着相似作用，都与细胞增殖有关，但这两个亚型也有各自独特功能，H2AFZ可能与RNA加工处理有关，而H2AFV与纺锤体、微管形成有关(Figure 2)。接着，利用CRISPR-Cas9技术构建H2AFZ敲除的细胞模型，通过MTT、细胞周期实验发现H2AFZ与肝癌细胞增殖密切相关(Figure 3)，通过RNA-Seq发现敲除H2AFZ后会造成一系列可变剪切事件的发生(Figure 4)。以上结果说明H2A.Z的两个亚型在肝癌的发生和发展过程中发挥着重要而独特的作用，这些结果有助于推动肝癌诊治的发展，为肝癌诊治提供新思路。

Figure 1. H2AFZ and H2AFV were overexpression in HCC

A, B) Expression of H2AFZ and H2AFV in HCC samples and paired para-carcinoma tissues in Guangxi HCC cohort, respectively; C, D) Expression of H2AFZ and H2AFV in HCC and para-carcinoma tissues based on TCGA RNA-seq data, respectively; E) ROC curve analysis about the diagnostic efficiency of H2AFZ, H2AFV and combined in HCC.

Figure 2. KEGG pathways and GO annotations from co-expressed genes of H2AFV or H2AFZ

A) Overlapped pathways from H2AFV and H2AFZ co-expressed genes. B) Partly specific GO annotations from H2AFV co-expressed genes. C) Partly particular GO annotations from H2AFZ co-expressed genes. The upper represents partly significant Biological Processes (red), Cellular Components were shown in the middle (blue), and Molecular Function in the below (green).

Figure 3. H2AFZ knockout inhibits cell proliferation in HCC cells in vitro.

A) H2AFZ knockout were examined by western blotting in HCC cells lines (HepG2 and 7402). B) MTT assay results for H2AFZ knockout and control cells. C) Colony formation assay results for H2AFZ knockout and control cells. D) Flow cytometry examines cell cycle of H2AFZ knockout and control cells.

Figure 4. Inhibition of H2AFZ may affect RNA Splicing, and then lead to hepatocarcinogenesis.

A, B) UpSet plots in HepG2 cells and BEL-7402 cells, showing the interactions among the five types of significantly altered AS events. One gene may have up to five types of AS events. AS: Alternative Splicing; SE: Skipping Exon; A3SS: Alternative 3' splice Site; A5SS: Alternative 5' splice Site; MXE: Mutually Exclusive Exons; RI: Retention Intron. C) Venn diagram. Overlapped genes from HepG2 cells and BEL-7402 cells. D) Partly significant GO terms from overlapped genes.

       汤绍梅同学、黄晓量同学、王玺同学为文章的共同第一作者，王秋雁教授、陶玉婷老师为文章的通讯作者。本研究由广西重点实验室基因组与个体化研究中心和广西自然科学基金创新团队(2016GXNSFGA38006)，国家自然科学基金（31471271，31560311），广西科学技术基础和人才专项基金（AD17195090）支持。