环状 RNA circRHOT1 通过启动 NR2F6 表达促进肝细胞癌进展。
Circular RNA circRHOT1 promotes hepatocellular carcinoma progression by initiation of NR2F6 expression.
机构信息
Gastrointestinal Department, Affiliated Hospital of Guilin Medical University, Lequn road No.15, Xiufeng district, Guilin, 541001, China.
出版信息
Mol Cancer. 2019 Jul 19;18(1):119. doi: 10.1186/s12943-019-1046-7.
BACKGROUND
Increasing evidence has revealed a close relationship between non-coding RNAs and cancer progression. Circular RNAs (circRNAs), a recently identified new member of non-coding RNAs, are demonstrated to participate in diverse biological processes, such as development, homeostatic maintenance and pathological responses. The functions of circRNAs in cancer have drawn wide attention recently. Until now, the expression patterns and roles of circRNAs in hepatocellular carcinoma (HCC) have remained largely unknown.
METHODS
Bioinformatics method was used to screen differentially expressed novel circRNAs in HCC. Northern blotting, qRT-PCR, in situ hybridization (ISH) and RNA-FISH were utilized to analyzed the expression of circRHOT1 in HCC tisues.CCK8, colony formation, EdU assays were used to analyze proliferation of HCC cells. Transwell assay was utilized to analyze HCC cell migration and invasion. FACS was used for apoptosis analysis. Xenograft experiments were used to analyze tumor growth in vivo. Mass spectrum, RNA pulldown, RIP and EMSA was utilized to test the interaction between circRHOT1 and TIP60. RNA-sequencing method was used to analyze the downstream target gene of circRHOT1.
RESULTS
We identified circRHOT1 (hsa_circRNA_102034) as a conserved and dramatically upregulated circRNA in HCC tissues. HCC patients displaying high circRHOT1 level possessed poor prognosis. Through in vitro and in vivo experiments, we demonstrated circRHOT1 significantly promoted HCC growth and metastasis. Regarding the mechanism, we conducted a RNA pulldown with a biotin-labeled circRHOT1-specific probe and found that circRHOT1 recruited TIP60 to the NR2F6 promoter and initiated NR2F6 transcription. Moreover, NR2F6 knockout inhibited growth, migration and invasion, whereas rescuing NR2F6 in circRHOT1-knockout HCC cells rescued the proliferation and metastasis abilities of HCC cells.
CONCLUSION
Taken together, circRHOT1 inhibits HCC development and progression via recruiting TIP60 to initiate NR2F6 expression, indicating that circRHOT1 and NR2F6 may be potential biomarkers for HCC prognosis.
背景
越来越多的证据表明，非编码 RNA 与癌症的发生发展密切相关。circRNA（环状 RNA）是最近发现的非编码 RNA 的新成员，参与多种生物学过程，如发育、内稳态维持和病理反应。circRNA 在癌症中的功能引起了广泛关注。到目前为止，circRNA 在肝细胞癌（HCC）中的表达模式和作用仍知之甚少。
方法
采用生物信息学方法筛选 HCC 中差异表达的新型 circRNA。应用Northern blot、qRT-PCR、原位杂交（ISH）和 RNA-FISH 分析 HCC 组织中 circRHOT1 的表达。CCK8、集落形成、EdU 检测分析 HCC 细胞的增殖。Transwell 检测分析 HCC 细胞的迁移和侵袭。FACS 进行凋亡分析。异种移植实验分析体内肿瘤生长。质谱、RNA 下拉、RIP 和 EMSA 检测 circRHOT1 与 TIP60 的相互作用。RNA 测序方法分析 circRHOT1 的下游靶基因。
结果
我们发现 circRHOT1（hsa_circRNA_102034）是 HCC 组织中一种保守且显著上调的 circRNA。circRHOT1 高表达的 HCC 患者预后不良。通过体外和体内实验，我们证实 circRHOT1 显著促进 HCC 的生长和转移。关于其机制，我们用生物素标记的 circRHOT1 特异性探针进行 RNA 下拉，发现 circRHOT1 将 TIP60 募集到 NR2F6 启动子并启动 NR2F6 转录。此外，NR2F6 敲除抑制了生长、迁移和侵袭，而在 circRHOT1 敲除 HCC 细胞中恢复 NR2F6 的表达则恢复了 HCC 细胞的增殖和转移能力。
结论
总之，circRHOT1 通过募集 TIP60 启动 NR2F6 表达抑制 HCC 的发生发展，表明 circRHOT1 和 NR2F6 可能是 HCC 预后的潜在标志物。
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