中国汉民族散发性结直肠癌候选基因3’-UTR功能风险预警遗传标志物系统筛查

MicroRNAs (miRNA) regulate pathogenesis，immunity and other biological processes through specifically binding to the 3' untranslated region（3'-UTR） of target mRNA . It is well reported that the altered expression of oncogenes or tumor suppressor genes caused by their 3'-UTR variant-mediated abnormalities of miRNA binding capability involves in the process of tumor development. To date, there has been no report on systematically screening and identifying 3'-UTR functional variants of sporadic colorectal cancer (sCRC) candidate genes . In this project, we plan to identify the variants in 3'-UTR of 22 candidate genes for sCRC by sequencing, and then systematically screen out the potential functional variants, which affect miRNA binding capability, with the special algorithm of miRanda3.2a. By transfecting Hela cells with wild /mutant-type dual luciferase reporter recombinant expression vectors, we ascertain the functional variants by determining the difference of luciferase activity. Furthermore, we confirm that the variant-mediated changes of miRNA regulation are involved in functional alteration. In addition, we verify the impact of site-specific miRNA on the expression of target gene in colorectal epithelial SW480 cells. Finally, by case-control association studies and detecting difference of target gene expression between different genotype groups in tumor tissues, we could identify the functional genetic risk markers for sCRC in Chinese Han population.

miRNA通过和靶mRNA 3'-UTR特异结合，调控疾病发生等生物学过程。肿瘤发生时，3'-UTR序列变异引发miRNA结合能力异常改变，受miRNA调控的癌/抑癌基因表达发生失调。目前,系统筛查并确定散发性结直肠癌（sCRC）候选基因3'-UTR功能变异位点的工作未见报道。本项目对sCRC 22个候选基因3'-UTR测序，确定变异位点；运用miRanda软件筛查可能影响miRNA结合能力的位点；对可能的功能变异位点构建野生型和突变型双荧光素酶重组表达载体，转染Hela细胞，通过检测荧光素酶活性差异确定功能变异位点，进而确定变异位点介导的特异miRNA调控水平变化在功能改变中的作用；利用肠癌上皮细胞确定位点特异miRNA对肿瘤细胞目的基因表达的影响；结合病例-对照关联研究及检测不同基因型在肿瘤组织中目的基因表达水平的差异，确定中国汉族人群sCRC疾病风险预警的功能遗传标志物。

散发性结直肠癌是常见的致死癌症之一，但是缺乏散发性结直肠癌早期筛查的遗传标志物。miRNA通过和靶基因的mRNA的3'-UTR特异结合调控基因表达，参与多种生物学过程。散发性结直肠癌相关基因的microRNA 结合位点的突变可能改变这种调控过程，影响癌症的发生。本研究采用生物信息手段筛查散发性结直肠癌基因3’-UTR上的可能改变microRNA结合能力的SNP 位点。预测结果显示，rs11466537，rs17026336和rs746978可能改变相应的microRNA与靶基因的结合能力。双荧光素酶报告实验结果显示rs11466537位点的T等位基因与hsa-miR-1193的结合能力强于A等位基因(A是低频率的位点)。rs17026336的C等位基因与hsa-miR-3191的结合能力弱于T等位基因。rs746978的等位基因改变对双荧光素酶报告实验结果未见显著影响。实时定量和Western blot的结果显示，hsa-miR-1193及hsa-miR-3191可以明显抑制TGFBR2基因的表达。并且，过表达hsa-miR-1193的HT-29 细胞系的增殖能力明显增强，hsa-miR-1193的抑制则降低HT-29 细胞系的增殖能力。hsa-miR-3191的过表达使得SW480细胞的迁移侵袭能力明显增强，hsa-miR-3191的抑制则减弱其迁移侵袭能力。最后，基因分型结果显示，rs11466537的A等位基因在正常人血液样本的基因频率是1.5%，而肿瘤病人的正常肠组织样本中则无一例携带A等位基因。rs17026336未在病例对照样本中检测到。结果表明，hsa-miR-1193及hsa-miR-3191可能通过抑制TGFBR2基因的表达参与了sCRC的发生发展， rs11466537位点的A 等位基因干扰了hsa-miR-1193对TGFBR2基因的抑制，A等位基因可能是sCRC的保护性因素。