遗传变异相关LncRNA通过激活TLR4通路促进大肠癌发生发展的机制研究

More than 50 Colorectal Cancer(CRC) susceptible Single Nucleotide Polymorphisms(SNPs) have been identified by Genome-Wide Association Study. However, most of those SNPs locate in “non-function” area of human genome. Thus, it is necessary to perform the functional verification in order to dissect the mechanism of those loci in the development of CRC. Variant Set Enrichment(VSE) analysis was performed to annotate all the CRC susceptible SNPs. Interestingly, we found one locus, rs6687758, locates on the histone modification sites of H3K4me1 and H3K27AC, with the downstream enrichment of H3K4me3. All of those evidences indicated that rs6687758 may locate an enhancer area, probably of which contains a novel transcript. Indeed, we discovered and verified a novel transcript named Colorectal Cancer LncRNA 1(CCLnc1) locating at upstream of rs6687758.GTEx data show CCLnc1 mRNA expression was affected by the genotype of rs6687758. RNA-seq analysis demonstrated that epitopic expression of CClnc1 could active TLR4-MYD88 pathway. Further study revealed that CCLnc1 might promote F. nucleatum enrichment in mucous of intestinal tract. This study will then further explore the biologic mechanism of CCLnc1 in the development of CRC by the means of RNA co-immunoprecipitation assay, RNA pull-down, mass array and microbiom analysis. Also, this study will establish the analysis process of discovering tumor-associated LncRNAs by means of functional SNPs.

遗传关联分析已发现约50个大肠癌相关基因多态性(SNP)位点，然而多数SNP位于基因组的非功能区，使得对这部分SNP的功能验证成为功能基因组学的研究重点和难点。前期通过遗传变异组富集分析对目前已知大肠癌相关SNP研究发现，SNP rs6687758所在的区域恰好在组蛋白H3K4me1和H3K27AC的修饰位点上，并且在其下游存在转录激活标志物H3K4me3的富集，利用GTEx数据分析发现一条lncRNA(CCLnc1)的表达量和rs6687758各基因型呈比例相关。RNA-seq分析发现CCLnc1可以激活TLR4-MYD88通路，同时，CCLnc1可以影响与大肠癌发生相关的具核梭杆菌在肠黏膜富集。本研究将通过RNA免疫共沉淀，RNA Pull-Down，肠道菌群分析等技术明确CCLnc1在大肠癌中发生中的生物学功能，同时也为利用遗传易感位点发现疾病相关LncRNA提供研究样板。

全基因组关联分析(Genome-wide association studies，GWAS)已经发现了多个与大肠癌风险相关的单核苷酸多态性(Single nucleotide polymorphism, SNP)位点;然而，SNP等遗传因素影响散发性大肠癌发生发展的具体机制仍有待探究。大肠癌高危型SNP rs6687758附近区域为增强子元件序列。其中，包含Lead SNP rs6687758所在区域的碱基变异可能通过改变增强子活性等作用方式，远端调控影响长非编码 RNA RP11-400N13.2 的表达，且在正常人群中分析发现，rs6687758 高危基因型与 RP11- 400N13.2 表达呈正相关。据此，我们确认将rs6695584作为1q41位点的致病SNP。rs6695584的A>G突变产生了BATF的蛋白结合基序，改变了增强子活性，随后激活了RP11- 400N13.2的表达，在此我们将该lncRNA命名为lncSLCC1。两个独立CRC队列的进一步验证证实了CRC组织中lncSLCC1的上调，并揭示了lncSLCC1表达的增加与CRC患者的生存率低有关。从机理上讲，lncRNA-SLCC1与AHR相互作用，并转录激活糖代谢中的关键酶HK2表达，从而驱动糖酵解途径并加速CRC肿瘤生长。功能测定显示lncSLCC1在HK2介导的CRC中诱导糖酵解激活和肿瘤生长。此外，HK2在结直肠癌组织中上调，并与lncSLCC1表达和患者生存率呈正相关。总之，我们的发现揭示了SNP介导的癌基因lncRNA-SLCC1通过激活糖酵解途径促进CRC的风险。