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High Content Imaging of Barrett's-Associated High-Grade Dysplasia Cells After siRNA Library Screening Reveals Acid-Responsive Regulators of Cellular Transitions.
Cellular and Molecular Gastroenterology and Hepatology ( IF 7.2 ) Pub Date : 2020-05-13 , DOI: 10.1016/j.jcmgh.2020.05.002
Sinead M Phipps 1 , Catherine E Garry 1 , Sepehr Kamal 2 , James D Johnson 2 , John Gilmer 3 , Aideen Long 1 , Dermot Kelleher 4 , Shane P Duggan 5
Affiliation  

Background & Aims

Esophageal adenocarcinoma (EAC) develops from within Barrett’s esophagus (BE) concomitant with gastroesophageal reflux disease (GERD). Wound healing processes and cellular transitions, such as epithelial–mesenchymal transitions, may contribute to the development of BE and the eventual migratory escape of metastatic cancer cells. Herein, we attempt to identify the genes underlying esophageal cellular transitions and their potential regulation by the low pH environments observed in GERD and commonly encountered by escaping cancer cells.

Methods

Small interfering RNA library screening and high-content imaging analysis outlined changes in BE high-grade dysplasia (HGD) and EAC cell morphologies after gene silencing. Gene expression microarray data and low pH exposures studies modeling GERD-associated pulses (pH 4.0, 10 min) and tumor microenvironments (pH 6.0, constant) were used.

Results

Statistical analysis of small interfering RNA screening data defined 207 genes (Z-score >2.0), in 12 distinct morphologic clusters, whose suppression significantly altered BE-HGD cell morphology. The most significant genes in this list included KIF11, RRM2, NUBP2, P66BETA, DUX1, UBE3A, ITGB8, GAS1, GPS1, and PRC1. Guided by gene expression microarray study data, both pulsatile and constant low pH exposures were observed to suppress the expression of GPS1 and RRM2 in a nonoverlapping temporal manner in both BE-HGD and EAC cells, with no changes observed in squamous esophageal cells. Functional studies uncovered that GPS1 and RRM2 contributed to amoeboid and mesenchymal cellular transitions, respectively, as characterized by differential rates of cell motility, pseudopodia formation, and altered expression of the mesenchymal markers vimentin and E-cadherin.

Conclusions

Collectively, we have shown that low pH microenvironments associated with GERD, and tumor invasive edges, can modulate the expression of genes that triggered esophageal cellular transitions potentially critical to colonization and invasion.



中文翻译:

siRNA 文库筛选后 Barrett 相关的高级发育不良细胞的高内涵成像揭示了细胞转变的酸响应调节剂。

背景与目标

食管腺癌 (EAC) 从 Barrett 食管 (BE) 内发展而来,并伴有胃食管反流病 (GERD)。伤口愈合过程和细胞转变,如上皮-间充质转变,可能有助于 BE 的发展和转移性癌细胞的最终迁移逃逸。在此,我们试图通过在 GERD 中观察到的低 pH 环境以及逃逸癌细胞经常遇到的低 pH 环境来确定食管细胞转变的潜在基因及其潜在调节。

方法

小干扰 RNA 文库筛选和高内涵成像分析概述了基因沉默后 BE 高度发育不良 (HGD) 和 EAC 细胞形态的变化。使用基因表达微阵列数据和低 pH 暴露研究建模 GERD 相关脉冲(pH 4.0,10 分钟)和肿瘤微环境(pH 6.0,恒定)。

结果

小干扰 RNA 筛选数据的统计分析定义了 207 个基因(Z-score > 2.0),在 12 个不同的形态簇中,其抑制显着改变了 BE-HGD 细胞形态。该列表中最重要的基因包括KIF11RRM2NUBP2P66BETADUX1UBE3AITGB8GAS1GPS1PRC1。在基因表达微阵列研究数据的指导下,观察到脉动和恒定的低 pH 暴露抑制GPS1RRM2的表达在 BE-HGD 和 EAC 细胞中以非重叠的时间方式,在鳞状食管细胞中没有观察到变化。功能研究发现,GPS1RRM2 分别有助于变形虫和间充质细胞转变,其特征是细胞运动、伪足形成的不同速率以及间充质标志物波形蛋白和 E-钙粘蛋白的表达改变。

结论

总的来说,我们已经证明与 GERD 相关的低 pH 微环境和肿瘤侵袭边缘可以调节基因的表达,这些基因触发对定植和侵袭可能至关重要的食管细胞转变。

更新日期:2020-05-13
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