长链非编码RNA IRAIN抑制肝癌干细胞增殖的机制研究

In today's society, Liver cancer is a serious threat to human health. With the goal of "providing a new target for the prevention and treatment of liver cancer", this project focuses on the key scientific issue of " the molecular mechanism of IRAIN regulating the expression of SLIT2, and inhibiting the proliferation of liver cancer stem cell". The study chain of "screening and research of liver cancer stem cells - Correlation between IRAIN and hepatocellular carcinoma stem cells - Reveal the molecular mechanism of IRAIN apparent regulation of SLIT2 on CD90+ CD45-hepatoma stem cell proliferation - Revalidation of animal and clinical samples with IRAIN-DNMT1-SLIT2 pathway inhibiting hepatoma stem cell proliferation " is taken as the main research chain. And then to develop IGF-1R as new target for tumor prevention and treatment, and study the molecular mechanisms of proliferation inhibition of liver cancer stem cell. This project intends to propose a hypothesis that IRAIN recruits DNMT1 to down-regulate the SLIT2, and inhibits the proliferation of HCC stem cells. CD90+CD45-HCC stem cells are screened out from clinical liver cancer specimens, and combined with the overexpression / silencing strategy, to clarify the biological function of IRAIN in inhibiting HCC stem cell proliferation, by using Cas9, ChIP, RIP and other technologies to clarify the molecular mechanism of IRAIN’s proliferation inhibition of liver cancer stem cell, and strive to achieve a breakthrough in the study of new targets of prevention and treatment of liver cancer.

肝癌严重威胁人类生命健康。本项目以“为肝癌防治提供新靶标”为目标，围绕“IRAIN表观调控SLIT2表达，抑制肝癌干细胞增殖的分子机制”这一关键科学问题，以“肝癌干细胞的筛选及干性研究―揭示IRAIN 与肝癌干细胞的相关性―揭示IRAIN表观调控SLIT2抑制CD90+CD45-肝癌干细胞增殖的分子机制―对IRAIN-DNMT1-SLIT2通路抑制肝癌干细胞增殖的动物及临床样本再验证”研究链条为主线，发展IGF-1R这一肿瘤防治新靶标，深入研究抑制肝癌干细胞增殖的分子机制。本项目拟提出IRAIN招募DNMT1表观下调SLIT2抑制肝癌干细胞增殖的科学假说，从肝癌标本中筛选出CD90+CD45-肝癌干细胞，结合过表达/沉默策略，明确IRAIN抑制肝癌干细胞增殖的生物学功能，采用Cas9、ChIP、RIP等技术，阐明IRAIN抑制肝癌干细胞增殖的分子机制,力争在肝癌防治新靶标研究方面取得突破。

肝癌干细胞是一种可自我更新的肿瘤起始细胞，具有很强的致瘤性和耐药性。因此，探究肝癌干细胞在肝癌中功能至关重要，从而促进对于肝癌治疗的发展。.我们发现长链非编RNA IRAIN在肝癌干细胞中低表达，抑制肝癌细胞的干性。接着，我们进一步探究IRAIN的潜在抑制肿瘤细胞干性的机制。对过表达IRAIN的肝癌细胞进行转录组测序，发现SLIT2与IRAIN表达成正相关，验证发现SLIT2在肿瘤干细胞中低表达。之后，通过Fish、RNA pull down、ChIP及RIP等实验进一步证明，DNMT1调节SLIT2启动子区域的DNA甲基化水平，在肿瘤干细胞中DNMT1高表达，导致SLIT2启动子区域的DNA甲基化水平升高，从而导致SLIT2表达水平降低。但是当肿瘤干细胞中IRAIN高表达时，IRAIN与DNMT1结合，阻断了DNMT1结合到SLIT2的启动子区域调节其DNA甲基化水平，导致其启动子区域的甲基化水平降低，表观调控 SLIT2 表达水平升高，抑制了肿瘤细胞干性。总之，我们的发现表明IRAIN/DNMT1/SLIT2轴在调节肝癌肿瘤细胞干性方面发挥了至关重要的作用，为肝癌治疗提供了潜在的治疗靶点。