miRNA423-5p调控三叶因子表达及在胃肠道粘膜修复中的作用机制

The TFF peptides are a family of small regulatory proteins consisting of three members, including TFF1 (pS2), TFF2 (SP) and TFF3 (ITF). TFF functions include mucus stabilization and repair, apoptosis, and inflammatory responses. According to the important roles of TFF peptides, the expression regulation of the TFF peptides seem to have great significance. Previously, we have identified the markedly higher expression of TFF1 in nomal gastric tissues, and we also found the significant direct correlation between miRNA423-5p and TFF1. Furthermore, over-expression of miRNA423-5p caused a decrease in mRNA expression and protein level of TFF1. So we presume that the TFF peptides are important target genes for miRNA to take part in gastric mocusal repair. In this project, we will perform detecting the regulation of TFF1 by miRNA423-5p and the potential target sites within the sequences of 3' UTR of TFF1 genome, and we also focus on the effect of miRNA423-5p through TFFs mediation on cell proliferation and migration. This study will illustrate the potential mechanism of the TFF peptides on gastric mocusal repair, and further offers evidence that miRNA through TFFs mediation represent a novel class of gastric mocusal injuries drugs.

三叶因子参与胃肠道黏膜保护及相关病变，其表达与调控具重要意义。我们前期研究明确三叶因子对细胞生物学行为的影响，探讨信号通路，筛选鉴定结合蛋白，检测表达规律。预实验中qRT-PCR检测发现miRNA423-5p与TFF1表达负相关，miRNA423-5p模拟物转染正常胃黏膜细胞GES-1后下调TFF1表达，并促进细胞增殖与集落形成而抑制细胞迁移。本课题拟探讨miRNA423-5p调控三叶因子表达及在其介导下参与黏膜修复的作用及机制，并进一步开展如下研究：1）miRNA423-5p调控TFF1表达及作用模式；2）TFF1介导miRNA423-5p参与黏膜修复的相关机制及信号通路；3）miRNA423-5p参与TFFs协同表达调控。本课题有助于阐明三叶因子表达调控模式，阐明其在胃肠道黏膜病变中的作用及分子机制，为以TFF1为治疗靶点进行以miRNA为新型治疗工具的临床应用提供理论依据。

三叶因子TFF1具有黏膜保护与修复作用，其过程与黏膜上皮细胞的增殖、凋亡以及迁移能力都密切相关，并且TFF1与胃肠道恶性肿瘤发生发展也具有密切关系。microRNA是基因表达调控网络中关键一环，影响着多条信号传导通路。许多microRNA与胃癌密切相关，可以通过抑制癌基因的表达或负性调节抑癌基因从而影响肿瘤的发生发展，并且还可以直接以甲基化酶为靶蛋白通过影响甲基化水平参与下游基因的表达调控。本课题拟检测microRNA对TFF1表达的调控作用，并进一步阐明TFF1因子在胃肠道粘膜病变及胃癌发生发展中的作用。. 本课题应用软件及生物信息平台，对调控TFF1表达的microRNA进行筛选，预测了可能结合在TFF1的5’UTR、3’UTR或CDS区域的microRNA信息。筛选出特异性结合在TFF1的3’UTR 区域的miRNA423-5p，首先应用荧光定量PCR明确TFF1与miRNA423-5p在肿瘤组织和正常组织中的差异性表达；其次，通过转染miRNA423-5p模拟物或抑制物明确miRNA423-5p对TFF1表达的负调控作用；再次，构建含TFF1的3’UTR区域的报告基因质粒，应用荧光素酶活性实验检测证实TFF1是miRNA-423-5p下游靶基因；最后，细胞功能学检测表明miRNA-423-5p通过负调控TFF1表达诱导细胞周期蛋白Cyclin D1和Cyclin D3及细胞粘附蛋白β-catenin的表达，胃癌细胞中促进增殖并抑制侵袭，胃黏膜细胞中促进细胞增殖，抑制细胞凋亡与迁移。.其次，筛选出特异性结合在TFF1 CDS区的miR218-5p，首先应用荧光定量PCR及免疫组化检测明确胃癌组织中miR218-5p与TFF1表达呈负相关；其次，验证胃癌细胞中miR218-5p以TFF1为下游靶基因并负调控其表达；再次，检测明确miR218-5p下调TFF1的表达及分泌，体内外水平促进胃癌细胞增殖；最后，开展信号通路研究明确miR218-5p依赖Erk1/2通路发挥功能。.综上，本项目明确了microRNA对TFF1表达的调控作用，即探讨了miRNA423-5p与miRNA218-5p对TFF1表达的调控模式及负调控作用；同时，通过体内外水平研究探讨TFF1表达变化对胃癌细胞及胃黏膜细胞生物学行为的影响，进一步阐明TFF1因子在胃肠道肿瘤进展以及胃肠道黏膜