A novel ETV6-miR-429-CRKL regulatory circuitry contributes to aggressiveness of hepatocellular carcinoma
A novel ETV6-miR-429-CRKL regulatory circuitry contributes to aggressiveness of hepatocellular carcinoma
复制标题
一种新型 ETV6-miR-429-CRKL 调节电路有助于肝细胞癌的侵袭性
DOI：
10.1186/s13046-020-01559-1
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发表时间：
2020-04-23
影响因子：
11.3
通讯作者：
Sun, Ming-Zhong
中科院分区：
文献类型：
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作者：
Guo, Chunmei;Gao, Chao;Sun, Ming-Zhong
背景肿瘤转移是肝细胞癌（HCC）高死亡率的主要原因之一。E-Twenty Six variant gene 6（ETV 6）是一种强转录抑制因子，与肿瘤的发生、发展密切相关。方法采用Western blotting、实时荧光定量PCR和免疫组织化学方法检测ETV 6、CRKL在HCC中的表达水平（v-crk肉瘤病毒CT 10癌基因同源物（禽）样）和miR-429; Transwell小室和F-actin细胞骨架染色检测ETV 6和CRKL表达下调对肝癌细胞迁移、侵袭和细胞骨架的影响;免疫共沉淀法检测CRKL与ETV 6之间的相互作用;染色质免疫沉淀法检测ETV 6与miR-429之间的相互作用。在肝癌组织和肝癌细胞中ETV 6和CRKL表达频繁增加，而miR-429表达下调。此外，在肝癌患者的肿瘤组织和肝癌细胞中，ETV 6上调与CRKL上调呈正相关，并且ETV 6和CRKL上调与miR-429下调也建立了两种负相关。功能研究表明，ETV 6的过表达和敲低对HCC细胞的迁移、侵袭、细胞骨架F-actin的表达和排列有明显的促进和抑制作用，而CRKL的过表达与ETV 6的过表达具有相似的作用。ETV 6通过直接与miR-429的启动子区结合负调控miR-429的表达，miR-429通过选择性靶向CRKL-3′-UTR负调控CRKL的表达，ETV 6通过与miR-429的启动子区结合负调控CRKL的表达，ETV 6通过与miR-429的启动子区结合负调控CRKL的表达。ETV 6直接结合CRKL并正向调节其表达，CRKL反过来正调控ETV 6的表达。结论我们的数据表明，ETV 6通过直接结合CRKL促进HCC细胞的迁移和侵袭。miR-429启动子区通过调节CRKL表达。新发现的ETV 6-miR-429-CRKL调控通路有助于HCC的侵袭性，这为HCC诊断和治疗参数的基础研究提供了新的线索。
BackgroundTumor metastasis is one of the main causes of the high mortality of hepatocellular carcinoma (HCC). E-Twenty Six variant gene 6 (ETV6) is a strong transcriptional repressor, associated with the development and progression of tumors. However, the exact role and underlying mechanism of ETV6 in HCC remain unclear.MethodsWestern blotting, quantitative real-time PCR and immunohistochemistrywere used todetect the expression levels of ETV6, CRKL (v-crk sarcoma virus CT10 oncogene homologue (avian)-like) and miR-429 in HCC tissues and cells; Transwell chamber and F-actin cytoskeleton staining assay to examine the effects of ETV6 and CRKL deregulation on the migration, invasion and cytoskeleton of HCC cells; Co-immunoprecipitation assay to determine the interaction between CRKL and ETV6; Chromatin immunoprecipitation assay to investigate the interaction between ETV6 and miR-429.ResultsWe established a novel ETV6-miR-429-CRKL regulatory circuitry contributes to HCC metastasis. ETV6 and CRKL were frequently increased, while miR-429 was downregulated in both hepatocarcinoma tissues and hepatocarcinoma cells. Moreover, ETV6 upregulation was positively correlated with CRKL upregulation, and two negative correlations were also established for ETV6 and CRKL upregulation with miR-429 downregulation in both hepatocarcinoma patients’ tumorous tissues and hepatocarcinoma cells. Functional investigations revealed that overexpression and knockdown of ETV6 was remarkably effective in promoting and suppressing HCC cell migration, invasion, cytoskeleton F-actin expression and arrangement, whereas, CRKL overexpression exhibited similar effects to the overexpression of ETV6. Mechanistically, ETV6 negatively regulates miR-429 expression by directly binding to the promoter region of miR-429; miR-429 negatively regulates CRKL expression by selectively targetingCRKL-3′-UTR; ETV6 directly binds to CRKL and positively regulates its expression, which in turn CRKL positively regulates ETV6 expression.ConclusionsOur data demonstrated that ETV6 promotes migration and invasion of HCC cells by directly binding to promoter region of miR-429 via modulating CRKL expression. The newly identified ETV6-miR-429-CRKL regulatory circuitry contributes to the aggressiveness of HCC, which provides new clues for fundamental research on diagnosis and treatment parameters for HCC.