长链非编码RNA在肝癌中的临床意义及作用机制的研究

Long non-coding RNA (lncRNA) is generally considered as non-protein coding transcripts longer than 200 nucleotides. They are closely related to the initiation and progression of cancer and have become a hot spot of research. In our previous studies, through analyzing the lncRNA expression profile in 50 self-made microarrays of liver cancer tissues, we found that the expression levels of LCRR were markedly higher in liver tumor tissues than in the paired adjacent non-tumor tissues and that LCRR was closely associated with recurrence and survival in liver cancer patients. Based on the above results, this project is aimed at:1) identifying the signatures which can be used to diagnoze and prognose hepatocellular cancer by detecting the lncRNA expression profile in other 300 cases of hepatocellular carcinoma samples; 2) verify the expression levels of LCRR in 300 liver cancer tissues and clarifying its role and significance in clinical practice by combining with clinical data analysis; 3) generating stable cell lines with over-expression and knock-down expression of LCRR so as to study their effects on various biological characteristics, and on tumorigenicity and metastasis of hepatocellular carcinoma in vivo; 4) Microarray is employed to detect gene expression alteration after LCRR is up- or down-regulated and identify the genes and their signal pathways regulated by LCRR using bioinformatics, and then we will confirm the results with all kinds of molecular biological methods. In this way, we will clarify the molecular mechanism of LCRR; 5) providing theoretical and experimental evidence for LCRR application in clinical practice.

长链非编码RNA（lncRNA）是一类长度超过200个核苷酸的非编码RNA,作用广泛,并与肿瘤的发生发展密切相关，已成为一个新的研究热点。申请者前期通过自制芯片对50例肝癌中lncRNA表达谱进行了初步的研究，发现一组表达标签能准确地区分癌和非癌组织，其中LCRR在肝癌中显著上调，并与病人的复发和生存密切相关。在此基础上，本课题拟收集300例肝癌继续采用芯片检测lncRNA表达谱，以鉴定出可用于肝癌诊断和预后评估的标签；RT-PCR验证LCRR表达后，结合临床资料分析，明确其在临床中的作用和意义；构建LCRR稳定上调和下调的细胞株，观察其对肝癌细胞各种生物学特性的影响及其在动物体内对肝癌成瘤和转移的作用；用芯片检测稳定株的基因表达谱、在线数据库预测和RNA-蛋白下拉试验等鉴定出LCRR的结合蛋白及其信号通路，用实验方法证实信号通路基因，从而阐明其分子作用机制，为临床应用提供理论和实验依据。

本项目基本按照计划顺利进行，完成了研究内容和目标，培养了多名研究生，发表了标注本课题资助号的SCI文章7篇，正在投稿Hepatology杂志一篇和申请一个专利。主要研究成果如下：我们收集了616例肝癌病例（原计划为300例），分为发现组、验证组和独立组1（院内）和独立组2（院外）四组。以自制芯片分析了发现组 127对肝癌及癌旁组织的LncRNA表达谱，建立了一个9-LncRNA标签,其区分肝癌和癌旁组织的准确率达了98.8%；生存分析发现17个LncRNA与肝癌患者的预后明显相关，通过风险分数模型筛选出一个4-LncRNA标签，能有效地预测病人生存时间，而且其是一个独立的预后因素；采用RT-PCR方法检测了这4个LncRNA在验证组、独立组1和独立组2中的表达水平，并验证了这一标签的预后预测作用；当4-LncRNA分标签和临床分期系统（TNM和BCLC分期）组成联合预测模型时，其预后评估效率明显优于单独的临床分期或4-LncRNA标签。以上结果曾经投稿Lancet杂志，现在重新修改后准备投稿Hepatology，而且正在将4-LncRNA标签申请专利。接着，我们对标签中的2个lncRNA在肝癌中的生物学功能及其机制进行了深入的研究：RP11-134O21.1在肝癌中表达升高，与病人的预后差相关；体外实验显示，上调此lncRNA的表达可显著提高肝癌细胞系的增殖、克隆形成、迁移、侵袭能力和上皮间质转化（EMT），下调其表达则得到相反的结果；体内实验表明，上调其表达可促进肝癌在裸鼠皮下成瘤和肺部转移灶的形成；在机制方面，表达谱芯片检测发现其主要调控细胞核内DNA转录相关的通路相关；RNA下拉实验与质谱检测相结合，筛选出此RP11-134O21.1与HSPA4(HSP70)蛋白结合，与表达谱芯片结果相印证。第二个LncRNA为XLOC_012786，其在肝癌中表达下降，与病人预后差相关；体外实验显示，其过表达可显著抑制肝癌细胞系的增殖、克隆形成、迁移、侵袭和EMT；体内实验表明，其过表达可抑制裸鼠肝癌移植瘤和肺转移灶的形成；全基因组表达谱芯片检测结果显示其可调控PI3K-Akt信号通路；蛋白免疫印迹检测显示，其过表达导致了磷酸化的AKT、S6与4EBP1下降、敲降则上调它们的磷酸化，这一结果显示此LncRNA通过抑制AKT通路磷酸化来阻抑肝癌的转移。此结果正在撰写论文。