miR-10b通过下调Tiam1蛋白降低Rac1活性抑制胃癌细胞侵袭转移的机制及检验应用研究

Nowadays, the positive rate of tumor markers for gastric cancer screening is less than 20%, whereas lack of tumor markers in assessing tumor metastasis and recurrence. It is confirmed that abnormal activation of Tiam1/Rac1 can promote polar change, inflammatory cell infiltration, invasion and metastasis in gastric cancer cells. However, the up stream factor regulating Tiam1 and the relative mechanism is still unclear. Based on bioinformatics software query, it is speculated that as an upstream factor, miR-10b downregulates activation of Tiam1/Rac1 signal pathway by targeting Tiam1, consequently inhibits Tiam1/Rac1 mediated tumorigenesis and metastasis. Hence, in this study, detection of miR-10b and Tiam1 expression levels in different TNM stages was carried out using in vitro experiments such as lentiviral packaging plasmid and GST-pull down, in order to confirm the hypothesis that miR-10b inhibits Tiam1/Rac1 mediated tumorigenesis and metastasis. This study will promote miR-10b and Tiam1 to become gastric tumor markers for clinical early diagnosis and evaluation of metastasis, which is hoped in the future miR-10b and Tiam1 will transfer into domestic test reagents, and provide theoretical and experimental bases for target therapy.

目前检验应用的肿瘤标志物筛查早期胃癌阳性率不足20%，并缺少评价转移及复发的标志物。国内外的研究及我们的前期工作已证实Tiam1/Rac1信号通路异常活化促进胃癌细胞的极性改变、炎性浸润、侵袭和转移，但上游因子对 Tiam1的调控及其机制尚不清楚。我们基于生物信息学软件推测：miR-10b作为上游因子通过抑制Tiam1下调Tiam1/Rac1信号通路的活化，从而抑制Tiam1/Rac1介导的肿瘤细胞的发生及转移。为此，本研究应用慢病毒转染和GST-pull down等胃癌细胞体外实验、裸鼠腹膜转移模型构建、检测裸鼠转移瘤组织和胃癌患者中miR-10b、Tiam1表达，以证实miR-10b通过下调Tiam1蛋白表达抑制Tiam1/Rac1通路介导的胃癌侵袭转移的假说，为miR-10b和Tiam1成为临床早期胃癌诊断与评估转移的肿瘤标志物、进而转化成为国产检验试剂及靶向治疗提供理论和实验依据。

胃癌(Gastric cancer, GC)是消化系统最常见的恶性肿瘤之一，T淋巴细胞侵袭诱导因子1 (invasion and metastasis 1, TIAM1)在胃癌的侵袭和转移中起重要作用。本研究通过实验，在分子、细胞和组织水平上证明miR-10b通过下调TIAM1通路抑制胃癌侵袭转移，以深入研究胃癌增殖和转移的机制，发现用于实验室诊断胃癌的肿瘤标志物，并探索可以用于靶向治疗的药物。研究方法及结果主要包括：通过QPCR和Western Blot技术确定在胃癌组织和细胞中TIAM1高表达且miR-10b-5p低表达，通过双荧光素酶实验确定miR-10b-5p靶向调控TIAM1，通过QPCR证实肿瘤抑制因子runt相关转录因子3（RUNX3）在核心结合因子亚基β(CBFβ)的辅助下调控miR-10b-5p表达，通过CCK-8、Transwell等证实了CBFβ/RUNX3-miR-10b-TIAM1分子轴对胃癌细胞增殖、迁移和侵袭的调控作用，通过检测人胃癌组织miR-10b-5p和TIAM1的表达并分析患者临床病理特征证实miR-10b-5p和TIAM1mRNA表达水平与胃癌患者临床病理特征相关且可以反映胃癌进展，通过构建胃癌裸鼠移植瘤模型进一步证实miR-10b-5p抑癌作用并在表观遗传层面筛选靶向抑瘤药物，此外，体外实验还发现TIAM1的配体integrin b1还能够增强胃癌以外细胞如乳腺上皮细胞的干细胞样特性，通过筛选胃癌患者和健康者血清发现了胃癌相关炎症因子以及多种其他miRNAs。本研究完成miR-10b调控Tiam1/Rac1信号通路的机制研究，并构建裸鼠移植瘤模型，进一步证实miR-10b通过靶向下调Tiam1抑制胃癌进展，为转化成国产检验试剂及靶向治疗提供理论和实验依据。