长链非编码RNA Linc504928参与KRT19磷酸化促进肝癌增殖的机制研究

Keratin 19 has been proved to play a key role in the invasion of human hepatocellular carcinoma (HCC), however the detailed mechanism of the regulation of KRT19 still remained unclear. Based on our published results and gene chip, we aimed to investigate the function of lncRNA in regulating HCC cell proliferation by binding KRT19. Preliminary study and forecast results indicated Linc504928 could bind KRT19 directly and promoting tumor cell proliferation by declined the KRT19 phosphorylation level (serine/ threonine), we speculate the Ser35, a phosphorylation site on KRT19, may be the target. In this research, the function of Linc504928 will be first explored by clinical follow up, the gain/loss assay in vitro (liver cell lines) and in vivo (subcutaneously transplanted tumor). Secondly the point mutation, RNA pull-down assay and cell proliferative assay will be employed to determine whether Linc504928 could competitive inhibition the KRT19 phosphorylation on Ser35 site. Finally we plan to seek the signaling pathways downstream of Linc504928/p-KRT19 by TGF-β1 and (or) NOTCH1. Since Linc504928 and the KRT19 phosphorylation has not been reported in HCC, our study is an original source of innovation and may provide new idea for the research and treatment of HCC.

KRT19与肝细胞肝癌预后相关，但其参与肝癌发展的机制尚不明确。本研究结合前期成果进一步探索与KRT19相结合的非编码RNA在肝癌中的作用及机制。结合RIP及芯片发现，Linc504928与KRT19存在直接结合，细胞实验发现其能通过抑制KRT19丝/苏氨酸磷酸化促进肝癌细胞增殖。研究推测Linc504928可能通过抑制KRT19 Ser35磷酸化从而促进肝癌发展。研究拟通过沉默/过表达Linc504928细胞实验、动物模型及临床随访验证其作用；其次，通过磷酸化位点突变等鉴定Linc504928是否可结合并抑制KRT19 Ser35位点磷酸化；最后探明磷酸化KRT19下游可能的TGF-β1或NOTCH1信号通路。鉴于Linc504928及KRT19磷酸化在肝癌增殖中作用机制未有报道，本研究创新性的探究了Linc504928/p-KRT19调控网络，也为肝癌增殖机制研究提供了新的思路。

近期研究表明非编码RNA与肝癌（HCC）的发生、转移及复发密切相关。本研究延续角蛋白19（Keratin，KRT19）相关成果进一步挖掘其在肝癌中的调控网络，并聚焦于长链非编码RNA (LncRNA)在肝细胞癌中的调控机制进行深入研究。通过构建肝癌患者临床数据库， RIP-Seq技术，生物信息学等方法筛选得到与KRT19密切相关的长链非编码RNA Linc-KILH (ENST00000504928.1，即Linc504928)。利用临床标本验证其表达与肿瘤大小、预后等临床特征的相关性；接着改变Linc-KILH表达水平观察其对肝癌细胞功能的影响；最后通过细胞实验及动物模型，阐明了Linc-KILH是通过结合并抑制KRT19 Ser35位点磷酸化，从而增强肝癌细胞中KRT19与β-catenin之间的相互作用，β-catenin入核内并激活下游Notch1信号通路，最终调控肝癌细胞增殖转移功能变化。本研究首次完整阐明Linc-KILH在肝癌发展中的作用机制，基本完成了原定目标。与此同时，本研究团队在临床实践、胰腺肿瘤及环状RNA领域进行了相关平行研究。在此青年基金的资助下，本人发表论文多篇，其中位列第一作者2篇，正在投稿2篇；获得国家实用新型专利2 项；协助培养研究生2名。本研究初步探究鉴定出多个参与调控肿瘤增殖及转移相关的重要非编码RNA，对进一步开发肿瘤早期诊断标志物以及潜在治疗靶标具有重要的指导意义。