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

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。

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.