miR-532-5p调控RUNX3表达介导胃癌发生发展的分子机制

RUNX3 functions as an important transcription factor regulating the growth of gastric epithelia cell. RUNX3 is downregulated or absent in 50-70% human primary gastric cancer and gastric cancer cell lines, partly due to loss of allele heterozygosity and hypermethylation in RUNX3 gene promoter. So, further studies are necessary to interpret the mechanism of RUNX3 inactivation. MiRNA produces a marked effect by inhibiting protein synthesis at post-transcription level. Bioinformatics analysis showed that miRNA-532-5p binds to the RUNX3 3' untranslated regions perfectly. During previous studies, we first found that miR-532-5p was highly expressed in several human gastric cancer cell lines, and was correlated negatively with the level of RUNX3 protein. Moreover, RUNX3 protein level was downregulated by transfection of miR-532-5p eukaryotic expressing vector or specific miR-532-5p mimics, but upregulated by specific miR-532-5p inhibitor. In addition, the miR-532-5p mimics could apparently decrease the apoptosis of the gastric cancer cell detected by flowcytometry. Based on these results, further studies include: 1)explore the negative regulation mechanism of miR-532-5p on RUNX3 expression; 2)interpret the biological functions and related mechanism of miR-532-5p on the gastric cancer cells in vitro; 3)assess the functions of miR-532-5p and its regulation on RUNX3 in primary gastric cancer specimens and nude mouse transplantation tumor.

RUNX3是调节胃上皮细胞生长的重要转录因子，50-70%的人类原发性胃癌及多种胃癌细胞系中Runx3表达下调或缺失。等位基因杂合性缺失和启动子过度甲基化尚不能完全解释RUNX3失活。miRNA在转录后基因表达调控中发挥重要作用。生物信息学分析显示miR-532-5p可以结合在RUNX3 3'非翻译区。申请者首次发现，miR-532-5p在多株人胃癌细胞中高表达，与RUNX3蛋白表达呈负相关。转染miR-532-5p表达质粒或mimics可以下调RUNX3蛋白表达，而inhibitor则上调RUNX3表达。流式分析结果表明mimics可以减少胃癌细胞的凋亡率。本课题拟在前期研究基础上1）解析miR-532-5p负性调节RUNX3表达的分子机制；2）阐明miR-532-5p对胃癌细胞生物学功能的影响及其分子机制；3）临床标本及裸鼠成瘤实验评价miR-532-5p的功能及对RUNX3的调控。

深入研究胃癌发生、发展及其转归的分子机制是寻找有效的诊断标志和改进治疗方案的根本。近年来，miRNA的发现及其研究领域的兴起开启了胃癌发生发展机制研究的新篇章。.miR-532-5p位于人类染色体Xp11.23，成熟序列由22个核苷酸构成。生物信息学软件ClustalW serve分析不同物种miR-532-5p 序列显示，物种间成熟序列极其保守，表明miR-532-5p在物种进化中发挥重要作用。miR-532-5p与人类胃癌的关系在本课题之前尚未见报道，因此我们的研究旨在阐明miR-532-5p对人类胃癌的影响及其相关的调控机制。.瞬时转染miR-532-5p特异性mimics和稳定过表达miR-532-5p在体外能够明显增强胃癌细胞的克隆形成和迁移能力，减少G1期细胞的比例和凋亡细胞比例，在体内能够促进裸鼠肺脏转移瘤的形成，使肺组织重量增加，瘤结节数目增多。功能获得实验、功能缺失实验、细胞表达谱分析和裸鼠转移瘤实验均显示miR-532-5p能负性调节转录因子RUNX3及其靶基因的表达，荧光素酶活性分析结果证明RUNX3是miR-532-5p调控的直接靶分子。临床标本检测显示miR-532-5p在近一半的原发性胃癌组织中过表达。这些研究结果说明，miR-532-5p能够增强胃癌细胞的生长、迁移和侵袭能力而发挥致癌性miRNA的作用，为解析胃癌发生和侵袭转移的分子机制提供了必要的理论支持。