LncRNA H19与miR200a构建ceRNA网络介导胰腺癌干细胞失干性的机制

Long non-coding RNA(lncRNA) and microRNA can play an important role in cancer stem cells, they can accurately regulate the expression of target gene through a new regulatory model: competing endogenous RNA(ceRNA). LncRNA H19 and miR200a can be ceRNAs each other. In the previous research, we found that miR200a could regulate the stemness characteristics of pancreatic cancer stem cells (pCSCs) by TGFβ2 signaling pathway and the expression of H19 in pCSCs was corresponding with miR200a. Based on this basis, we intend to complete the following projects:1. We observe the competitive expression of H19 and miR200a by regulation the activity of of them in pCSCs respectively, then detect the expression of its downstream target genes, observe the changes of expression of stemness genes and biological stemness characteristics. 2.Study the mechanism of H19 and miR200a controlling the expression of downstream target gene precisely through ceRNA network, mediating the mesenchymal epithelial transformation (MET) and the loss of stemness of pCSCs.3.Construct the three dimensional culture system consisting of pancreatic decellularized scaffold, bioreactor and medium to provide the simulated microenvironment for pCSCs.Then explore new targets for the treatment of pancreatic cancer.

LncRNA与miRNA在肿瘤干细胞中发挥重要调控作用，它们通过一种全新调控模式：竞争性内源性RNA (ceRNA)调控网络，精准调控靶基因表达，LncRNA H19与miR200a可互为ceRNA；前期研究发现miR200a通过TGFβ2信号通道调控胰腺癌干细胞（pCSCs）干性特征，而H19在pCSCs中与miR200a对应表达。在此基础上，本项目拟完成：1、通过分别调控pCSCs中H19和miR200a活性，观测其相互竞争性表达，检测其下游靶基因的表达变化，观察干性基因的表达和干性生物学特性的变化；2、研究H19与miR200a通过构建ceRNA网络对话精准靶控下游基因表达，介导pCSCs发生间质-上皮转化(MET)并失去干性的机制；3、构建由胰腺脱细胞支架、生物反应器和灌注培养液为主体的肿瘤细胞三维培养体系，为pCSCs提供模拟肿瘤微环境的研究平台。以此为胰腺癌的治疗探寻新靶点。

胰腺癌是消化系统常见的恶性肿瘤之一，其恶性程度高、进展迅速、5年生存率低，预后极差。LncRNA与miRNA在肿瘤干细胞中发挥重要调控作用，它们通过一种全新调控模式：竞争性内源性RNA (ceRNA)调控网络，精准调控靶基因表达。我们通过RTFQ-PCR分析TP73-AS1在胰腺癌及癌旁组织、正常人胰腺上皮细胞和胰腺癌细胞系中的表达水平；通过组织芯片分析TP73-AS1在58对PC组织中的表达水平与临床病理因素之间关系；通过慢病毒转染TP73-AS1，检测TP73-AS1表达对胰腺癌细胞增殖、细胞周期、凋亡、侵袭、迁移的影响及其迁移、侵袭相关分子的表达的影响。同时，通过双荧光素酶活性报告基因及RTFQ-PCR检测和验证TP73-AS1与miR-200a的结合和调节关系；通过干预TP73-AS1和miR-200a 表达分析TP73-AS1通过miR-200a调控PC细胞迁移、侵袭及转移相关蛋白MMP14的表达。最后，通过构建小鼠腹腔转移模型，进一步验证TP73-AS1对胰腺癌转移功能的影响。我们研究发现TP73-AS1在PC组织及细胞中呈高表达，同时TP73-AS1的表达水平与PC的淋巴结转移及胰腺癌临床分期显著相关；干扰TP73-AS1后，PC细胞的迁移、侵袭能力均降低；TP73-AS1可能通过调节miR-200a从而调节MMP14表达来影响胰腺癌侵袭转移能力，TP73-AS1/miR-200a/MMP14轴可能为胰腺癌治疗提供一种新思路。