Cancer Science Institute of Singapore, National University of Singapore, Singapore.
Cancer Science Institute of Singapore, National University of Singapore, Singapore; Department of Surgery, Graduate School of Medicine, Tohoku University, Miyagi, Japan.
Gastroenterology. 2017 Jan;152(1):218-231.e14. doi: 10.1053/j.gastro.2016.09.018. Epub 2016 Sep 23.
BACKGROUND & AIMS: Little is known about the mechanisms of gastric carcinogenesis, partly because it has been a challenge to identify characterize gastric stem cells. Runx genes regulate development and their products are transcription factors associated with cancer development. A Runx1 enhancer element, eR1, is a marker of hematopoietic stem cells. We studied expression from eR1 in the stomach and the roles of gastric stem cells in gastric carcinogenesis in transgenic mice.
We used in situ hybridization and immunofluorescence analyses to study expression of Runx1 in gastric tissues from C57BL/6 (control) mice. We then created mice that expressed enhanced green fluorescent protein (EGFP) or CreERT2 under the control of eR1 (eR1-CreERT2;Rosa-Lox-Stop-Lox [LSL]-tdTomato, eR1-CreERT2;Rosa-LSL-EYFP mice). Gastric tissues were collected and lineage-tracing experiments were performed. Gastric organoids were cultured from eR1-CreERT2(5-2);Rosa-LSL-tdTomato mice and immunofluorescence analyses were performed. We investigated the effects of expressing oncogenic mutations in stem cells under control of eR1 using eR1-CreERT2;LSL-KrasG12D/+ mice; gastric tissues were collected and analyzed by histology and immunofluorescence.
Most proliferation occurred in the isthmus; 86% of proliferating cells were RUNX1-positive and 76% were MUC5AC-positive. In eR1-EGFP mice, EGFP signals were detected mainly in the upper part of the gastric unit, and 83% of EGFP-positive cells were located in the isthmus/pit region. We found that eR1 marked undifferentiated stem cells in the isthmus and a smaller number of terminally differentiated chief cells at the base. eR1 also marked cells in the pyloric gland in the antrum. Lineage-tracing experiments demonstrated that stem cells in the isthmus and antrum continuously gave rise to mature cells to maintain the gastric unit. eR1-positive cells in the isthmus and pyloric gland generated organoid cultures in vitro. In eR1-CreERT2;LSL-Kras G12D/+ mice, MUC5AC-positive cells rapidly differentiated from stem cells in the isthmus, resulting in distinct metaplastic lesions similar to that observed in human gastric atrophy.
Using lineage-tracing experiments in mice, we found that a Runx1 enhancer element, eR1, promotes its expression in the isthmus stem cells of stomach corpus as well as pyloric gland in the antrum. We were able to use eR1 to express oncogenic mutations in gastric stem cells, proving a new model for studies of gastric carcinogenesis.
胃发生癌变的机制尚不清楚，部分原因是难以鉴定胃干细胞。Runx 基因调控发育，其产物是与癌症发展相关的转录因子。Runx1 增强子元件 eR1 是造血干细胞的标志物。我们研究了 eR1 在胃中的表达以及胃干细胞在转基因小鼠胃发生癌变中的作用。
我们使用原位杂交和免疫荧光分析来研究 C57BL/6（对照）小鼠胃组织中 Runx1 的表达。然后，我们创建了在 eR1 控制下表达增强型绿色荧光蛋白（EGFP）或 CreERT2 的小鼠（eR1-CreERT2；Rosa-Lox-Stop-Lox [LSL]-tdTomato，eR1-CreERT2；Rosa-LSL-EYFP 小鼠）。收集胃组织并进行谱系追踪实验。从 eR1-CreERT2(5-2);Rosa-LSL-tdTomato 小鼠中培养胃类器官并进行免疫荧光分析。我们使用 eR1-CreERT2；LSL-KrasG12D/+ 小鼠研究了 eR1 控制下表达致癌突变对干细胞的影响；收集胃组织进行组织学和免疫荧光分析。
大多数增殖发生在峡部；86%的增殖细胞为 RUNX1 阳性，76%为 MUC5AC 阳性。在 eR1-EGFP 小鼠中，主要在上部胃单位检测到 EGFP 信号，83%的 EGFP 阳性细胞位于峡部/ pit 区。我们发现 eR1 标记了峡部的未分化干细胞和基底处数量较少的终末分化主细胞。eR1 还标记了胃窦中的幽门腺细胞。谱系追踪实验表明，峡部和胃窦的干细胞不断产生成熟细胞以维持胃单位。在体外，eR1 阳性细胞在峡部和幽门腺中产生类器官培养物。在 eR1-CreERT2；LSL-Kras G12D/+ 小鼠中，MUC5AC 阳性细胞迅速从峡部干细胞分化，导致明显的化生病变，类似于人类胃萎缩中观察到的病变。
我们通过在小鼠中的谱系追踪实验发现，Runx1 增强子元件 eR1 促进其在胃体峡部的干细胞以及胃窦幽门腺中的表达。我们能够使用 eR1 在胃干细胞中表达致癌突变，为胃发生癌变的研究提供了一个新模型。
