lncRNA SNHG16的可变剪接促进上皮-间质转化和在胃癌转移中的作用

Epithelial-mesenchymal transition (EMT) is required for gastric cancer metastasis, while the regulatory mechanism remains not clear. Our preliminary results showed that the variant 1 and variant 4 of alternatively spliced lncRNA SNHG 16 could promote the EMT in gastric cancer cell metastasis resulted from the splicing factor 1 (SF1) regulated the alternative splicing of lncRNA SNHG 16.In the present study, we will further elucidate that lncRNA SNHG16 alternative splicing promotes EMT and gastric cancer cell migration in the cell level, animal models and gastric cancer tissues by immunohistochemistry and qRT-PCR etc. RNA immunoprecipitation, RNA pull-down and other techniques will be utilized to clarify the direct binding of SF1 and precursor lncRNA SNHG16 and regulatory mechanism of SF1. We will further clarify the role of lncRNA SNHG16 alternative splicing in the regulation of EMT through rescue experiment. This project will focus on the hypothesis that SF1 directly binds precursor lncRNA SNHG16 and regulates the process of its alternative splicing that contributes to the EMT and migration of gastric cancer cells, and explore the novel mechanism of lncRNA SNHG16 alternative splicing and thus reveal the mechanistic nature of it in promoting gastric cancer metastasis. Success of this study will provide original scientific basis for lncRNA SNHG16 acting as novel biomarker and candidate therapeutic target for gastric cancer in the future.

上皮-间质转化（EMT）是胃癌获得转移能力的重要途径，但调控机制尚未阐明。我们的前期研究发现：lncRNA SNHG16的剪接体1和4可促进胃癌细胞EMT，机制可能是通过剪接因子1（SF1）结合到前体lncRNA SNHG16调控可变剪接。本课题拟进一步：应用免疫印迹、原位杂交等方法在细胞模型、动物模型和病理组织中证明lncRNA SNHG16的可变剪接促进EMT和胃癌转移，应用RNA免疫沉淀、RNA pull-down等阐明SF1与前体lncRNA SNHG16的直接结合及其机理，并应用挽救实验证明其可变剪接体对EMT的调控机制。本项目紧紧围绕“SF1直接结合前体lncRNA SNHG16进而调控其可变剪接促进EMT和胃癌转移”这一科学假说，从长链非编码RNA的全新调控机制入手，揭示其促进胃癌转移的本质，为确立lncRNA SNHG16作为胃癌分子标记物和治疗新靶点提供原创性的科学依据。

胃癌发病率一直高居我国各种恶性肿瘤前列，五年生存率低，目前尚无有效的早期诊断手段和治疗方案减缓其发生发展和恶性转化。故发现一种新的早期标志物用于胃癌有效诊断和治疗迫在眉睫。我们前期研究发现核仁RNA宿主基因16 (small nucleolar RNA host gene 16，SNHG16) 与胃癌(gastric cancer，GC)的不良预后和恶性表型相关且首次在胃癌中发现了LncRNA SNHG16的4个变体，但目前4个LncRNA变体在生理和病理情况下的功能尚不清楚。为了进一步研究LncRNA SNHG16的4个变体在GC中的表达和功能，我们在60对GC组织样本和多个细胞系中进行RT-qPCR测定这4个变体的表达。研究发现，在胃癌组织和胃癌细胞系中LncRNA SNHG16的可变剪接体1和可变剪接体4表达上调。进一步的表型研究发现，siRNA敲低LncRNA SNHG16会影响GC细胞的增殖、凋亡、细胞周期进程、侵袭和迁移。机制研究表明，LncRNA SNHG16可通过下调GC细胞Dickkopf WNT信号通路抑制剂3 (DKK3)促进EMT。临床样本检测表明LncRNA SNHG16在肿瘤组织中表达上调，而DKK3表达下调，说明LncRNA SNHG16和DKK3在临床GC组织中的表达呈负相关。这些结果表明，LncRNA SNHG16通过调控DKK3直接或间接地促进GC进展，其有被用作转移预测生物标志物的可能。