胃肠道间质瘤中Slug调控相关miRNAs的鉴定及其抑制肿瘤转移机制的研究

The recurrence and metastasis rate of gastrointestinal stromal tumors (GISTs) is up to 50%. Our previous studies have shown that Slug correlated closely with metastasis of GISTs,silencing of Slug gene impaired invasiveness of GISTs cells in vitro. However, it's not clear that how Slug takes role in regulating metastasis of GISTs. In this project, the miRNAs which might take a role in regulation of Slug expression will be predicted by bioinformatics technique, screened by miRNA microarray tests, and verified by dual luciferase reporter assay and regulation of gene expression experiment. Subsequently, the corressions of miRNAs with clinicopathologic characteristics of GIST patients will be analyzed. The influence of Slug and miRNAs on invasive and metastasis ability of GIST cells, and the related mechanism will be detected. Finally, the influence of miRNAs and Slug on growth and metastasis of GIST xenograft tumor will be detected by whole-body fluorescence imaging. This project aims to identify miRNAs that regulate slug expression and to investigate the molecular mechanisms of their influence on GISTs metastasis. Furthermore, this project aims to assess the feasibility of using exogenous miRNAs to inhibit invasion and metastasis of GIST cells, which will provide a new idea for the treatment of GISTs.

胃肠道间质瘤(GIST)术后5年内的复发转移率高达50%。我们在前期研究中发现转录因子Slug的表达与GIST的转移密切相关，靶向沉默Slug基因能有效抑制GIST细胞的侵袭转移，但在机体内Slug如何参与GIST的转移并不清楚。本项目拟利用生物信息学技术和miRNA芯片筛选出GIST中Slug调控相关的miRNAs；通过基因表达调控实验、双荧光素酶报告系统进行验证。然后分析miRNAs与GIST临床病理特征的关系；检测 miRNAs对GIST细胞侵袭转移能力的影响及并探讨其中的分子机制。最后利用整体荧光成像技术在活体内实时监测miRNAs对GIST原位移植瘤生长转移的影响。本项目旨在鉴定Slug调控相关的miRNAs，探讨miRNAs/Slug影响GIST转移的分子机制，并评估应用外源性miRNAs抑制GIST细胞侵袭转移的可行性，为GIST的治疗提供新的思路。

胃肠道间质瘤（gastrointestinal stromal tumors, GIST）是最常见的胃肠道间叶源性肿瘤，主要源自于c-Kit和PDGFRA基因的突变。据报道约有50%的患者在术后5年内复发转移并最终因此死亡，复发转移已经成为GIST治疗的瓶颈。.我们在前期研究中发现转录因子Slug的表达与GIST的转移密切相关，但在机体内Slug参与GIST转移的机制并不明确。本项目通过TargetScan等软件计算出425个备选的Slug调控相关的miRNA，miRNA芯片检测出在Slug高表达的GIST组织中，339个miRNA上调， 85个miRNA下调，二者的交集共有12个miRNA。利用生物信息学技术筛选出8个与转移相关的miRNA，Real time PCR进一步验证N/P比值＞2的miRNA有5个：miRNA-1-3P、miRNA-200b-3p、miRNA-32-3P、miRNA-30c-1-3P、miRNA-363-3P。通过转染miRNA mimics和inhibitor，Real time PCR证实能下调Slug表达的，Transwell侵袭实验验证能抑制GIST细胞侵袭转移的miRNA共3个：miR-200b-3p，miR-30c-1-3P、miR-363-3P，被鉴定为在GIST细胞中Slug调控相关的miRNA。我们通过基因表达谱芯片、生物信息学技术、GIST细胞中Slug基因的基因沉默和过表达实验、Transwell侵袭转移实验，证实了Slug下游与转移相关的靶基因，包括：CDH1（负调控）、TSPAN9（正调控）、FGF3（正调控）。然后我们通过荧光素酶实验发现miR-200b-3p，miR-30c-1-3P、miR-363-3P均能与Slug-3UTR端结合，负向调控荧光强度的表达。最后通过ChIP实验证实Slug能够与CDH1、TSPAN9、FGF3基因的启动子区域靶向结合，且Slug基因沉默组CDH1的表达上调，而TSPAN9、FGF3的表达下调，提示Slug能负向调控CDH1的表达，正向调控TSPAN9、FGF3的表达，影响GIST的侵袭转移。.通过本研究，我们鉴定了在GIST细胞中Slug调控相关的miRNA，探讨了miRNAs/Slug影响GIST转移的分子机制，希望为GIST的治疗提供新的思路。