长链非编码RNA基因甲基化在结直肠癌中的诊断价值及应用研究

It has been revealed that greater numbers of ncRNAs promoters were aberrantly methylated, including lncRNAs and miRNAs. Aberrant methylation of lncRNAs genes could widely disturb processes associated with the development and progression of cancer by dysregulation of signaling and metabolic pathways and might act as the upstream link of tumorigenesis. Moreover, aberrant methylation changes in lncRNAs genes existed earlier and were more frequent than those of protein-coding genes. Therefore, it has significant advantages for methylation of lncRNAs genes as potential new tumor markers. Up to now, the diagnostic value and function of methylation of lncRNAs genes in colorectal cancer (CRC) has not yet been elucidated. .To define associations between methylation of lncRNAs genes and CRC, we sough to identify the specific biomarkers of methylation of lncRNAs genes to evaluate the function and diagnostic value. In our previous studies, high-throughput lncRNAs array analysis was performed in tissue samples of CRC and paired-adjacent mucosa. Sixty-two lncRNAs were found significantly downregulated. Two CRC cell lines (SW480 and HT29) were treated with 5-aza-2’-deoxycytidine (5-aza-dC), after which their lncRNAs expression profiles were also analyzed using lncRNAs array. Seventy and eighty-four lncRNAs were significantly upregulated by 5-aza-dC treated, respectively. Finally，twenty-one of those were chosen as candidate biomarkers which were not only downregulated but also high methylated. .In the present study, we will verify the expression and gene methylation levels of such 21 lncRNAs CRC tissues and adjacent mucosa by two-way authentication. Then we can identify CRC candidate biomarkers of lncRNAs genes methylation by detecting their methylation levels in plasma of large cohorts of CRC and controls. Then a specific panel of methylation of lncRNAs genes will be established by logistic regression. Meanwhile, the vivo experiments will be used to demonstrate the role of lncRNAs which are regulated by genes methylation in the tumorigenesis of CRC. The present study can not only provide new method for early diagnosis of CRC, but also help to further reveal the mechanism of lncRNAs in tumorigenesis and development of CRC.

长链非编码RNA（lncRNA）在肿瘤中的异常表达受甲基化调控，且lncRNAs基因的甲基化比编码蛋白基因出现更早、更频繁，是肿瘤发生更为上游的始动环节，作为新型肿瘤标志物具有显著优势。目前，结直肠癌中lncRNAs基因甲基化的诊断价值及受甲基化调控lncRNAs的功能尚未明确。本课题前期对结直肠癌细胞去甲基化前后、结直肠癌和癌旁组织lncRNAs表达谱分析，初步筛选出21个候选lncRNAs分子。现拟进行候选lncRNAs表达量及基因甲基化水平的双向验证，并检测结直肠癌和对照组血浆lncRNAs基因甲基化水平，寻找lncRNAs基因甲基化标志物，构建血浆lncRNAs基因甲基化诊断panel。同时，验证甲基化对lncRNAs表达的调控，通过细胞实验探讨受甲基化调控lncRNAs在结直肠癌中的功能。本研究有望为结直肠癌早期诊断提供新的手段，并进一步揭示lncRNAs在结直肠癌发生中的作用。

结直肠癌是常见的消化道恶性肿瘤，其发病率和死亡率仍呈上升趋势，严重危害人类健康。结直肠癌的发生是一个涉及遗传学、表观遗传学异常的复杂过程。长链非编码RNA（long non-coding RNA，lncRNA）是继microRNA后新近发现的一种ncRNA，在多种肿瘤中表达异常。研究表明，肿瘤中表达异常的lncRNAs依赖甲基化的调节机制，基因甲基化的lncRNAs可通过调节信号通路和代谢途径参与肿瘤的发生发展。lncRNAs基因甲基化有望成为一种有效的新型肿瘤标志物，且可作为肿瘤发生机制中更为上游的始动环节。lncRNAs基因甲基化为研究结直肠癌发生机制提供了新的思路，并为实现其早期诊断提供了可能性。.本研究采用MethylRad技术对5例结肠癌组织和配对癌旁组织进行了全基因组甲基化测序；筛选结直肠癌和癌旁组织两组间lncRNA差异甲基化位点和差异甲基化基因；共检测到773,956 CCGG和59,968 CCWGG位点。其中42,175 CCGG和2,794 CCWGG lncRNA 差异甲基化位点，合并CCGG和CCWGG，去除重复位点后共得到44,877 lncRNA 差异甲基化位点；筛选出1359 CCGG和 1052 CCWGG差异甲基化基因；与TCGA数据库中的lncRNA表达数据联合分析，共发现了15个结肠癌相关lncRNA，并进行后续的功能分析。. 本研究所筛选出的差异甲基化位点可能成为结肠癌的潜在生物标志物，结肠癌相关lncRNA为其机制研究提供新的数据，也为寻找新型结肠癌治疗靶点提供理论依据。