Division of Genetics, Cancer Research Institute, Kanazawa University, Kanazawa, Japan; AMED-CREST, AMED, Japan Agency for Medical Research and Development, Tokyo, Japan; Biotherapeutics Translational Research Center, Division of Biomedical Science, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea.
Division of Genetics, Cancer Research Institute, Kanazawa University, Kanazawa, Japan; Innovative Cancer Model Research Unit, Institute for Frontier Science Initiative, Kanazawa University, Kanazawa, Japan.
Gastroenterology. 2019 Mar;156(4):1140-1155.e4. doi: 10.1053/j.gastro.2018.11.059. Epub 2018 Nov 30.
BACKGROUND & AIMS: Gastritis is associated with development of stomach cancer, but little is known about changes in microRNA expression patterns during gastric inflammation. Specific changes in gene expression in epithelial cells are difficult to monitor because of the heterogeneity of the tissue. We investigated epithelial cell-specific changes in microRNA expression during gastric inflammation and gastritis-associated carcinogenesis in mice.
We used laser microdissection to enrich epithelial cells from K19-C2mE transgenic mice, which spontaneously develop gastritis-associated hyperplasia, and Gan mice, which express activated prostaglandin E2 and Wnt in the gastric mucosa and develop gastric tumors. We measured expression of epithelial cell-enriched microRNAs and used bioinformatics analyses to integrate data from different systems to identify inflammation-associated microRNAs. We validated our findings in gastric tissues from mice and evaluated protein functions in gastric cell lines (SNU-719, SNU-601, SNU-638, AGS, and GIF-14) and knockout mice. Organoids were cultured from gastric corpus tissues of wild-type and miR-135b-knockout C57BL/6 mice. We measured levels of microRNAs in pairs of gastric tumors and nontumor mucosa from 28 patients in Japan.
We found microRNA 135b (miR-135B) to be the most overexpressed microRNA in gastric tissues from K19-C2mE and Gan mice: levels increased during the early stages of gastritis-associated carcinogenesis. Levels of miR-135B were also increased in gastric tumor tissues from gp130 mice and patients compared with nontumor tissues. In gastric organoids and immortalized cell lines, expression of miR-135B was induced by interleukin 1 signaling. K19-C2mE mice with disruption of Mir-135b developed hyperplastic lesions that were 50% smaller than mice without Mir-135b disruption and had significant reductions in cell proliferation. Expression of miR-135B in gastric cancer cell lines increased their colony formation, migration, and sphere formation. We identified FOXN3 and RECK messenger RNAs (mRNAs) as targets of miR-135B; their knockdown reduced migration of gastric cancer cell lines. Levels of FOXN3 and RECK mRNAs correlated inversely with levels of miR-135B in human gastric tumors and in inflamed mucosa from K19-C2mE mice.
We found expression of miR-135B to be up-regulated by interleukin L1 signaling in gastric cancer cells and organoids. miR-135B promotes invasiveness and stem-cell features of gastric cancer cells in culture by reducing FOXN3 and RECK messenger RNAs. Levels of these messenger RNA targets, which encode tumor suppressor, are reduced in human gastric tumors.
胃炎与胃癌的发生发展有关，但人们对胃黏膜炎症过程中 microRNA 表达谱的变化知之甚少。由于组织的异质性，上皮细胞中特定基因表达的变化很难监测。我们研究了在小鼠胃黏膜炎症和胃炎相关癌发生过程中上皮细胞特异性 microRNA 表达的变化。
我们使用激光显微切割技术从 K19-C2mE 转基因小鼠（自发发生胃炎相关增生）和 Gan 小鼠（胃黏膜表达激活的前列腺素 E2 和 Wnt 并发生胃肿瘤）中富集上皮细胞。我们测量了上皮细胞富集 microRNA 的表达，并使用生物信息学分析整合来自不同系统的数据，以确定与炎症相关的 microRNA。我们在小鼠的胃组织中验证了我们的发现，并在胃细胞系（SNU-719、SNU-601、SNU-638、AGS 和 GIF-14）和基因敲除小鼠中评估了蛋白质功能。我们从野生型和 miR-135b 基因敲除 C57BL/6 小鼠的胃体组织中培养类器官。我们测量了来自日本 28 名患者的成对胃肿瘤和非肿瘤黏膜中 microRNA 的水平。
我们发现 microRNA 135b（miR-135B）在 K19-C2mE 和 Gan 小鼠的胃组织中表达最上调：在胃炎相关癌发生的早期阶段增加。与非肿瘤组织相比，gp130 小鼠和患者的胃肿瘤组织中 miR-135B 的水平也升高。在胃类器官和永生化细胞系中，白细胞介素 1 信号诱导 miR-135B 的表达。缺乏 Mir-135b 的 K19-C2mE 小鼠形成的增生性病变比缺乏 Mir-135b 的小鼠小 50%，细胞增殖显著减少。在胃癌细胞系中表达 miR-135B 增加了它们的集落形成、迁移和球体形成。我们确定 FOXN3 和 RECK 信使 RNA（mRNA）为 miR-135B 的靶标；它们的敲低减少了胃癌细胞系的迁移。人胃癌组织和 K19-C2mE 小鼠炎症黏膜中 miR-135B 的水平与 FOXN3 和 RECK mRNA 的水平呈负相关。
我们发现白细胞介素 L1 信号在胃癌细胞和类器官中上调 miR-135B 的表达。miR-135B 通过降低 FOXN3 和 RECK 信使 RNA 促进胃癌细胞在培养中的侵袭性和干细胞特征。这些编码肿瘤抑制因子的信使 RNA 靶标的水平在人胃癌肿瘤中降低。
