Table_1_MicroRNA-1224 Inhibits Tumor Metastasis in Intestinal-Type Gastric Cancer by Directly Targeting FAK.DOCX

Intestinal-type gastric cancer (GC) of the Lauren classification system has specific epidemiological characteristics and carcinogenesis patterns. MicroRNAs (miRNAs) have prognostic significance, and some can be used as prognostic biomarkers in GC. In this study, we identified miR-1224 as a potential survival-related miRNA in intestinal-type GC patients by The Cancer Genome Atlas (TCGA) analysis. Using quantitative real-time PCR (qRT-PCR), we showed that the relative expression of miR-1224 was significantly decreased in intestinal-type GC tissues compared to matched adjacent normal mucosa tissues (p < 0.01). We found that high miR-1224 expression was associated with no lymph-node metastasis (p < 0.05) and good prognosis (p = 0.028) in 90 intestinal-type GC tissues. Transfection of intestinal-type GC cells with miR-1224 mimics showed that miR-1224 suppressed cell migration in vitro (wound healing assay and Transwell migration assay), whereas the transfection of cells with miR-1224 inhibitor promoted cell migration in vitro. miR-1224 also suppressed intestinal-type GC cell metastasis in a xenograft mouse model. Furthermore, bioinformatics, luciferase reporter, Western blotting, and immunohistochemistry (IHC) studies demonstrated that miR-1224 directly bound to the focal adhesion kinase (FAK) gene, and downregulated its expression, which decreased STAT3 and NF-κB signaling and subsequent the epithelial-to-mesenchymal transition (EMT). Repression of FAK is required for the miR-1224-mediated inhibition of cell migration in intestinal-type GC. The present study demonstrated that miR-1224 is downregulated in intestinal-type GC. miR-1224 inhibits the metastasis of intestinal-type GC by suppressing FAK-mediated activation of the STAT3 and NF-κB pathways, and subsequent EMT. miR-1224 could represent an important prognostic factor in intestinal-type GC.

劳伦分型系统界定的肠型胃癌（GC）具备特异性流行病学特征与致癌模式。微小RNA（miRNAs）具有预后价值，部分可作为胃癌的预后生物标志物。本研究依托癌症基因组图谱（TCGA）分析，将miR-1224鉴定为肠型胃癌患者中潜在的生存相关miRNA。采用实时定量聚合酶链反应（qRT-PCR）检测发现，相较于配对癌旁正常黏膜组织，miR-1224在肠型胃癌组织中的相对表达水平显著降低（p < 0.01）。对90例肠型胃癌组织的分析显示，miR-1224高表达与无淋巴结转移（p < 0.05）及良好预后（p = 0.028）显著相关。向肠型胃癌细胞中转染miR-1224模拟物后，体外实验（划痕愈合实验与Transwell迁移实验）证实miR-1224可抑制细胞迁移；而转染miR-1224抑制剂则可促进细胞体外迁移。此外，miR-1224在异种移植小鼠模型中可抑制肠型胃癌细胞的转移能力。进一步通过生物信息学分析、荧光素酶报告基因实验、蛋白质印迹法及免疫组织化学（IHC）研究证实，miR-1224可直接结合黏着斑激酶（FAK）基因并下调其表达，进而抑制STAT3与NF-κB信号通路活化及后续的上皮间质转化（EMT）。抑制FAK表达是miR-1224介导的肠型胃癌细胞迁移抑制的必要环节。本研究表明，miR-1224在肠型胃癌中表达下调；其通过抑制FAK介导的STAT3、NF-κB通路活化及后续上皮间质转化，进而阻碍肠型胃癌的转移，有望成为肠型胃癌的重要预后因子。