长链非编码RNA-CACS2及其启动子区多态性与结直肠癌发生发展的相关性及作用机制研究

The long non-coding RNA (LncRNA) CASC2 involved in cancer development, but the effect and mechanism was unknown. In our previous research we have found LncRNA CASC2 down-regulated in colorectal cancer(CRC) tissue, its low expression associated with promoter hypermethylation. And then we perform a screening to investigate the association polymorphisms in the promoter region of the LncRNA CASC2 with the risk of CRC in a hospital based case-control study. The results shown that some single nucleotide polymorphism(SNP)located within the promoter CpG island were associated with susceptibility toCRC.Based on the previous research results, ,we proposed the scientific hypothesis that CASC2 promoter region polymorphism located CpG island, may modify the state of methylation ,involving in the regulation of CASC2 expression levels, and thus affecting important regulatory network participation by CASC2 , which is closely related to colorectal cancer development. In order to verify this hypothesis. The project intends to confirm the interaction among LncRNA CASC2 promoter region polymorphism, CpG island methylation state, expression levels, and their association with CRC development by further expand sampls. In vitro, the construction of recombinant plasmid, Site-Directed Mutagenesis, double luciferase reporter gene assay, electrophoresis mobility shift assay (EMSA) and Chromatin Immunoprecipitation (ChIP)experiments were performed to demonstrate the exact mechanism that polymorphic change the methylation status of CpG islands, regulating CASC2 expression and its regulation network. Further, gene cloning and siRNA technology were used to get different expression levels of CASC2 colorectal cancer cell model, by a series of cytological experiments to observe the phenotypic changes in cell proliferation, differentiation, invasion, metastasis. Lastly, the animal experiments such as tumor-bearing nude mice, were performed to further confirm that the CASC2 role in colorectal cancer and potential treatment. The project will take full advantage of translational medicine research strategy, through multiple levels of "cells - animals - patients" to demonstrate the mechanism of the LncRNA polymorphism and SNP locus change involved the regulation of LncRNA in the CRC development process. Its completion will provide new candidate markers for CRC diagnosis and treatment, and new potential drug targets for treatment of CRC.

长链非编码RNA(LncRNA) CASC2参与肿瘤发生发展，但作用机制不明。申请者前期工作发现LncRNA CASC2在肠癌组织中表达下调，其低表达与启动子区高甲基化相关，且位于启动子区CpG岛的单核苷酸多态性（SNP）亦与肠癌相关。综合分析以上结果，我们提出"CASC2启动子区的SNPs通过影响CpG岛甲基化调控CASC2表达，调节CASC2的重要调控网络，从而与肠癌发生发展密切相关"的科学假设。本项目拟进一步扩大样本验证该假设，在体外细胞实验中，通过构建重组质粒、荧光素酶报告基因、EMSA/ChIP实验获得SNP位点改变CpG岛甲基化状态参与调控CASC2的具体机制；采用基因克隆、siRNA干扰技术获得CASC2不同表达水平的肠癌细胞模型，通过一系列细胞学实验观察其增殖、分化、侵袭、转移等细胞表型改变。最后采用荷瘤裸鼠等动物体内实验确证CASC2在肠癌中的作用，并探讨其潜在治疗效果。

在该项目的资助下，我们对肠癌发生发展相关的长链非编码RNA进行了系统的研究，取得了以下研究结果：首先，我们采用高通量lncRNA 芯片的方法在结直肠癌及配对癌旁组织中筛查得到1189 条差异表达的lncRNAs 和1812 条差异表达的mRNAs。对筛选得到的mRNA进行GO 分析和Pathway 分析，对筛选得到的 lncRNA 的子类Enhancer lncRNA 和lincRNA 进行分析，并将其与邻近的mRNA 结合分析可为lncRNA 可能的调控机制和功能推断提供依据。其次，我们针对芯片筛选结果中高表达的长链非编码 UCA1进行深入的研究，发现UCA1在结直肠癌组织和肠癌细胞中表达上调，病理参数分析发现 UCA1 表达与肿瘤的分期、远处转移及淋巴结转移有相关性，而生存分析显示 UCA1 高表达的病人有较差的预后，并提示 UCA1 是独立的预后因子；功能分析发现在结直肠癌细胞中沉默UCA1 可能通过导致细胞周期G0G1 期阻滞而抑制细胞增殖，促进细胞凋亡，并明显抑制细胞侵袭转移；通过生物信息学分析及荧光素酶报告分析发现转录因子Ets-2 可以调控UCA1 的活性。再者，我们还针对芯片中另外两个高表达的长链编码LncRNA-CTD903和LncRNA U50535与分别与结直肠癌的侵袭转移进行相关的机制研究。研究发现LncRNA-CTD903过表达抑制通过抑制Wnt介导的EMT活性，抑制结直肠癌的侵袭转移。而LncRNA U50535上调ZEB1基因，下调Ecaderin的表达，促进肿瘤EMT的发生，与肿瘤预后相关。该研究的完成提示lncRNAs 与结直肠癌发生发展有密切关系，为进一步研究肠癌分子机制与更好的进行肠癌临床诊断预后开辟了新的途径。