miR-551参与胃癌发生发展的作用及机制研究

According to the papers published recently, microRNA is really important for the tumor formation, metastasis and drug resistance. Our preliminary data demonstrated that the expression of miR-551 were very low in gastric cancer tissues and related cell lines, also which is closely associated to the clinicopathological stages. Based on the above studying, we will construct the relevant stable gastric cancer cell lines. They are miR-551 co-overexpression stable cell lines and co-knockdown stable cell lines, respectively. Moreover, we are going to use the nude mice to construct the relevant animal models. We will clarify the function of miR-551 in gastric cancer after several related experiments: tumor growth curve analysis, colony formation assay, flow cytometry and invasion assay. Furthermore, we will clarify the mechanism that how the two genes regulate their downstream genes and how they effect their transcriptional ways in gastric cancer. Apparently, the Bioinformatics technology, protein microarray, Dual-Luciferase Reporter Assay, Chromatin Immunoprecipitation Assay and Gel shift technique are necessary for our research. Hopefully, some new and useful approaches can be revealed via this studying to cambat gastric cancer. We also try to find a more-specific ideal molecular marker which could be valuable and helpful for the early diagnosis and prognosis evaluation of gastric cancer.

研究表明 miRNA 与肿瘤的发生、转移、耐药等病理进程密切相关。我们前期发现miR-551在胃癌组织和细胞中，其表达均明显下调，且与胃癌的临床病理分期关系密切。本项目在前期研究基础上，拟建立miR-551稳定过表达及沉默的胃癌细胞系，并构建裸鼠模型，通过生长曲线分析、集落形成、流式细胞分析及侵袭转移相关实验等，探讨miR-551参与胃癌发生发展的作用；通过生物信息学方法、蛋白芯片技术、双荧光素酶报告基因分析、染色质免疫共沉淀及凝胶滞留实验等技术，深入探讨miR-551调控下游靶基因及上游转录调控的机制，阐明miR-551参与胃癌发生发展的分子机制。通过我们的研究，将为胃癌发病机制以及未来的靶向治疗提供新的思路和理论基础。并有望提供新一代特异性高的理想分子标记物应用于胃癌的早期诊断及预后判效。

项目背景：MicroRNA芯片显示miR-551b-3p在胃癌组织中表达下调，但是其在胃癌发生发展中的作用及机制尚不明确。.主要研究内容：检测miR-551b-3p在胃癌细胞和GES-1细胞，胃癌和癌旁组织中的表达。建立miR-551b-3p过表达胃癌细胞模型。通过细胞生长曲线测定、平板集落形成实验、细胞凋亡测定、划痕实验、Transwell细胞迁移和侵袭实验、裸鼠移植瘤实验等分析上调miR-551b-3p表达后对胃癌细胞增殖、迁移、侵袭等生物学行为的影响。筛选miR-551b-3p的靶基因及上游lncRNA。通过双荧光素酶报告基因实验验证miR-551b-3p和靶基因、上游lncRNA和miR-551b-3p的相互作用。采用Western blot技术检测胃癌细胞过表达miR-551b-3p后靶基因的表达变化。通过细胞生长曲线测定、Transwell细胞迁移实验检测上游lncRNA对胃癌细胞增殖、迁移的影响，以及改变上游lncRNA表达水平对miR-551b-3p及其靶基因表达的影响。.重要结果：miR-551b-3p在胃癌细胞株中的表达低于其在GES-1中的表达，在胃癌组织中的表达水平低于癌旁组织，且与胃癌患者肿瘤分化程度呈正相关，与局部浸润深度及淋巴结转移呈负相关，miR-551b-3p高表达胃癌患者的生存期高于miR-551b-3p低表达的患者。胃癌细胞过表达miR-551b-3p可以明显抑制细胞的增殖、抑制细胞的克隆形成、促进细胞的凋亡、抑制细胞迁移和侵袭能力、体内生长能力。TMPRSS4是miR-551b-3p的直接靶基因，lncRNA SMARCC2可以通过自身的miR-551b-3p结合位点与miR-551b-3p相互作用，并调控miR-551b-3p在胃癌细胞中的表达水平。LncRNA SMARCC2 促进胃癌细胞体外增殖、迁移能力。LncRNA SMARCC2通过ceRNA机制调控胃癌细胞TMPRSS4的表达。.科学意义：本项目首次发现LncRNA SMARCC2/miR-551b-3p/TMPRSS4轴在胃癌发生发展中扮演重要角色。为寻找胃癌早期诊断、评估患者分级、分期及预后的标记物提供了一个可能的选择，并有助于了解 miR-551b-3p 在胃癌分子调控网络中所处的位置及扮演的角色，为进一步探索 miR-551b-3p 应用于胃癌临床治疗提供