miR-133a-1/USH1G轴促进胃癌转移的分子机制研究

Gastric cancer （GC） has a serious impact on the prognosis. In metastatic GC, outcomes are poor, with median survival being around 1 year. Our previous research has found that high USH1G expression was significantly associated with primary TNM stage and 5-year survival rates of GC patients. Besides，USH1G affected the GC cell migration. There are no reports about the relationship between USH1G and cancer. The underlying mechanism is unknown. We found that USH1G is a predicted target of miR-133a-1 by using target prediction programs and could be repressed by miR-133a-1 mimics. A prevailing feature observed in human cancers is the global decrease in miRNA expression compared to the corresponding normal tissue. This miRNA suppression has a causative role in tumorigenesis, implying its potential as a therapeutic target but the underlying mechanism is unknown. Hu antigen R (HuR) is an inhibitory regulator of Microprocessor that recognizes and cleaves pri-miRNA to generate pre-miRNA. Our previous research has demonstrated that depletion of HuR overrode the repressive effect on miR-133a-1 expression. Meanwhile, we found that USH1G interacted with histone deacetylase 2 (HDAC2). It has reported that overexpressed HDAC2 can promote gastric cancer metastasis progress. In this study, we want to demonstrate the hypothesis that RNA-binding protein HuR specifically binds to primary miR-133a-1 transcript to block miR-133a-1 maturation. MiR-133a-1 plays a decisive role in GC metastasis progress by targeting USH1G. USH1G recruits HDAC2 to affect gastric cancer metastasis progress. The study will help us to suggest a new molecular mechanism of gastric cancers tumorigenesis, and provide a new theoretical basis for clinical treatment.

胃癌一旦发生转移会严重影响患者的预后。本课题组前期研究发现USH1G与临床TNM分期晚、预后差密切相关，并能影响胃癌细胞侵袭。USH1G与人类恶性肿瘤的关系未见文献报道，其影响胃癌转移的机制尚不明了。生物学信息学预测miR-133a-1能与USH1G mRNA 3’-非翻译区结合，同时实验验证miR-133a-1可抑制USH1G表达。我们利用患者二代测序的结果和TCGA的数据一致发现miR-133a-1在胃癌中低表达。HuR是miRNA成熟过程中的重要抑制因子，预实验结果表明HuR能抑制miR-133a-1表达。我们进一步使用Co-IP实验提示USH1G能与HDAC2相互作用，且文献报道HDAC2可促进胃癌转移。故本课题组拟阐述HuR抑制miR-133a-1成熟，从而上调USH1G表达；高表达的USH1G通过富集HDAC2影响胃癌转移。本项目可为研究胃癌转移机制提供一个新的切入点。

胃癌转移是胃癌呈现预后差的主要原因，大部分患者在诊断时已经处于胃癌晚期或者转移性胃癌。因此寻找敏感性和特异性俱佳的生物标志物，对实现胃癌的早期筛查、早期诊断以及患者预后评价均具有十分重要的临床意义。近两年，由于对肿瘤与骨架蛋白关系研究的深入，越来越多的骨架蛋白被定义为肿瘤的靶标。USH1G蛋白是一种含锚蛋白重复序列和SAM（Sterile alpha motif）域的蛋白。目前仅有研究显示此基因突变能够引发1G型Usher综合症。Usher综合征是一种常染色体隐性遗传疾病，临床表现为感音神经性听力损害和进行性视力丧失。USH1G与人类恶性肿瘤的关系未见文献报道。.本课题组发现USH1G蛋白在胃癌中表达明显高于非癌症组，USH1G与胃癌Lauran分型和HER-2阳性密切相关，是5年预后的独立危险因素。细胞和动物水平发现USH1G促进平板克隆成团数及细胞增殖；USH1G抑制胃癌细胞的迁移、侵袭和粘附能力。生物信息学预测和ChIP、luciferase报告基因手段证明miR-133a-1通过结合USH1G mRNA 3’非翻译区抑制其表达。miR-133a-1在胃癌组织中低表达，其原因是肿瘤低氧微环境促进RNA结合蛋白HuR高表达，进而抑制miR-133a-1成熟进程。通过Co-IP和质谱分析，我们发现组蛋白脱乙酰基酶2(HDAC2)与USH1G的ANK结构域互作，并抑制p53的转录活性。肌球蛋白重链9（MYH9）能与USH1G的SAM结构域互作，并下调MYH9蛋白水平，减少胃癌细胞丝状伪足形成数量。这一过程泛素——蛋白酶体和自噬参与其中。.综上所述，本课题以支架蛋白USH1G为研究目标，探索USH1G蛋白影响胃癌增殖的机制。探讨miRNA对USH1G表达调控的机制，探寻USH1G在及癌旁组织表达差异的可能原因。寻找与USH1G互作的功能蛋白质，揭示USH1G影响胃癌增殖的可能机制。本课题从上下游两条线对USH1G进行研究，可帮助我们更好理解胃癌发生发展的机制，并为USH1G今后成为胃癌预后标记物的相关临床应用提供部分理论依据。