胰腺癌中调控K-RAS基因的microRNA组(miRNAome)筛选平台的建立

The change of K-RAS gene is one of the most important molecular events in carcinogenesis of pancreatic cancer. At present, very few studies have investigated the miRNAome that regulates K-RAS gene expression in pancreatic cancer systematically and comprehensively. This research is to establish a platform to identify and analyze the miRNAome which directly regulates K-RAS gene in pancreatic cancer by affinity chromatography and deep sequencing technique. Firstly, we integrate the coding sequence of K-RAS and non-coding sequence with MS2 aptamer sequence, and construct a vector expressing RNA molecules. Next, we transfect this vector into pancreatic ductal/ cancer cells, then gather all miRNA molecules that combined with the K-RAS mRNA using affinity chromatography. At last，by high-throughput sequencing platform, deep sequencing could provide the information about the sequences and copy numbers of these miRNAs. Our previous studies have already indentified that two miRNAs (miR-96，miR-193b and miR-217) could bind to 3'UTR of K-RAS mRNA via partial complementarity. Through down-regulating K-RAS expression, these miRNAs cause a great depression of malignant biological behaviors of pancreatic cancer. This research is designed to define the miRNAome that regulates K-RAS gene and its regulatory networks, as well as the pathogenic mechanism underlying miRNA's regulation of K-RAS. The result would provide the evidence for miRNAs' application in pancreatic cancer gene therapy.

K-RAS基因改变是胰腺癌的重要分子事件，本课题拟采用亲和层析结合二代测序技术建立一个对胰腺癌中调控K-RAS基因的miRNAome（miRNA组）进行全面系统的筛选和鉴定的平台。首先构建表达K-RAS及MS2适配子序列的载体，导入胰腺导管/癌细胞，然后采用亲和层析法获取与K-RAS基因的mRNA相结合的所有miRNAs，最后进行二代测序。我们前期已发现miR-96，193b，217能靶向调控K-RAS基因，本课题在原基金课题的基础上进一步对胰腺癌中参与调控K-RAS基因的miRNAome进行全面分析，研究结果将为miRNA用于胰腺癌的基因治疗提供必要的理论基础和实验依据。

本课题采用了亲和层析结合二代测序技术建立一个对胰腺癌中调控KRAS基因的miRNAome（miRNA组）进行全面系统的筛选和鉴定的平台。噬菌体MS2衣壳蛋白是MS2噬菌体的包膜蛋白，它与噬菌体复制酶5’端由19个碱基组成的茎环结构RNA序列-MS2bs有特异性相互作用。首先构建表达KRAS及MS2bs 12个重复序列的载体pcDNA3.1，导入胰腺导管腺癌细胞，使其表达KRAS-12×MS2bs，提取总RNA。构建重组载体pMAL-c5x MBP-MS2，导入大肠杆菌DE3，诱导表达，使其表达带有MBP麦芽糖标签的融合蛋白MBP-MS2，融合蛋白经直链淀粉纯化。利用MS2衣壳蛋白与MS2bsRNA特异性结合的原理，将纯化后的MBP-MS2融合蛋白与KRAS-MS2bs RNA在含有树脂的层析柱中结合，然后利用含有麦芽糖的洗脱液冲洗层析柱，得到与KRAS相关的RNA-蛋白复合体。之后再利用相关方法将RNA与蛋白质分离，分离后的RNA进行质量和纯度的检测，合格后进行二代测序Hiseq深度分析。二代测序分析后，得到了与KRAS基因的mRNA相结合的所有miRNAs。 通过与miRNA数据库(miRBase)中指定范围的miRNA进行比对，鉴定样品中的已知miRNA，对已知miRNA进行家族分析，和已知miRNA的碱基编辑。使用华大自主开发的软件Mireap预测novel miRNA，绘制新的miRNA的二级结构图。本课题进一步对胰腺癌中参与调控KRAS基因的miRNAome进行了全面分析。