急性胰腺炎时EGR1相关ceRNA调控胰蛋白酶原激活的机制研究

The competitive endogenous RNA (ceRNA) theory is that lncRNA and mRNA could regulate each other if they have the same microRNA binding sites, and the interaction of RNA is related to the occurrence of various diseases. So far, there is no report about the relationship between ceRNA and acute pancreatitis (AP) in literature. The results of our pre-experiment suggested that trypsinogen activation was regulated by a complex ceRNA network. In the network, EGR1, a gene caused AP, was over expression. However, it needs to depth study to answer the question that whether the 4 lncRNA which have the same microRNA binding sites with EGR1 could cause trypsinogen activation as ceRNAs. Therefore, we propose the hypothesis: the EGR1-related candidate lncRNA may regulate trypsinogen activation as ceRNAs, which causes and promotes the development of AP. To verify this hypothesis, we will research the mechanism of trypsinogen activation caused by the EGR1-related candidate lncRNA through ceRNA effect in the experiment levels of molecular, cellular and animal. This study will make the ceRNA effect of lncRNA be the entry point to research the mechanism of trypsinogen activation, which will provide new ideas to the study of the pathogenesis of AP.

基于竞争性内源性RNA(ceRNA)理论，拥有相同microRNA结合位点的lncRNA和mRNA通过竞争性结合相同的microRNA进行相互调控，进而影响疾病发生发展。迄今ceRNA与急性胰腺炎(AP)时胰蛋白酶原激活的关系尚无文献报道。我们的预实验结果提示胰蛋白酶原激活受到复杂的ceRNA网络调控，其中显著高表达的EGR1与AP关系最为密切，但与其拥有相同microRNA结合位点的4个lncRNA能否发挥ceRNA作用来调控胰蛋白酶原激活还有待进一步探讨。为此，我们提出假说：EGR1相关候选lncRNA可能通过ceRNA作用调控胰蛋白酶原激活，促进AP发生发展。为验证这一假说，我们将分别从分子、细胞及动物水平探究EGR1相关候选lncRNA作为ceRNA调控胰蛋白酶原激活的机制。本课题将以lncRNA的ceRNA作用作为胰蛋白酶原激活机制研究的切入点，为AP的发病机制研究提供新的思路。

基于竞争性内源性RNA(ceRNA)理论，拥有相同microRNA结合位点的lncRNA和mRNA通过竞争性结合相同的microRNA进行相互调控，进而影响疾病发生发展。本课题将以lncRNA的ceRNA作用作为胰蛋白酶原激活机制研究的切入点，为AP的发病机制研究提供新的思路。. 主要研究内容及重要成果如下：. ①本课题组通过ncRNA与mRNA共表达关系分析得到共表达网络，并对共表达的LNCRNA进行功能及通路分析。对其中13个EGR1相关LNCRNA进行了Real-time PCR实验，胰蛋白酶原激活组中LNCRNA NONRATT03 1103.2、NONRATT012585和NONRATT031002的表达明显升高。②对胰蛋白酶原细胞内激活过程中起关键作用的mRNA和microRNA进行微阵列分析，共发现129个mRNA和64个microRNA有差异表达。生物信息学构建上调mRNA和下调microRNA的调控网络。③通过文献发掘，筛选的mRNA 转染细胞后，分别进行Western Blot及PCR表达验证，并通过激光共聚焦显微镜，流式细胞仪检测细胞内胰蛋白酶原激活情况，本课题组发现Egr1与AP关系最为密切。④本课题结合以前调控网络绘制Egr1相关调控网络，利用生物信息学筛选出miR-92a-3p,细胞水平证实miR-92a-3p下调了AR42J细胞中Egr1的表达，流式细胞仪验证miR-92a-3p通过Egr1调节胰蛋白酶原的激活。⑤动物实验构建大鼠胰腺炎模型，并验证Egr1在大鼠胰腺炎模型中差异表达。. 综上所述本课题通过芯片和生物信息学的筛选，筛出差异表达mRNA和miRNA，筛选出miRNA-92a-3p和EGR1，并研究其在AR42J细胞中的作用；并验证了miR - 92a - 3p可能通过Egr1调节胰蛋白酶原激活的细胞内激活的作用，并成功建立大鼠急性胰腺炎模型。. 本课题执行过程中，在本课题的资助下，发表SCI收录论文4篇，已录用1篇（未见刊）。培养博士研究生3人，硕士研究生2人，皆有标注本课题号的署名SCI收录文章发表 。