食管腺癌在美国和西方国家的发病率急速上升。Barrett食管和食管腺癌的发展与慢性胃食管反流病（GERD）有关。在GERD条件下，Barrett食管和食管腺癌细胞暴露于酸性胆盐（ABS）引起高水平的氧化应激和DNA损伤。本研究调查了胰岛素样生长因子结合蛋白2（IGFBP2）在调节ABS诱导的DNA双链断裂中的作用。使用实时RT-PCR、西方印迹、免疫组化、免疫荧光、共免疫沉淀、流式细胞术以及环己酰亚胺（CHX）追踪实验。为了模拟GERD条件，使用酸性胆盐（pH 4）的混合物在2D和3D器官类型培养模型中。建立了IGFBP2过表达和沉默的EAC细胞，以检查IGFBP2在ABS诱导的DNA损伤中的功能和机制作用。我们的结果表明，与癌前Barrett细胞系相比，EAC细胞系中IGFBP2 mRNA和蛋白水平较高，并且IGFBP2在EAC中频繁过表达（31/57）。将EAC细胞用ABS处理，以模拟GERD条件，可引起IGFBP2表达水平的上升。在FLO1细胞中敲低内源性IGFBP2（具有构成高水平的IGFBP2），在短暂暴露于ABS后可导致DNA双链断裂和细胞凋亡显著增加。另一方面，在OE33细胞（内源性IGFBP2水平较低）中外源性IGFBP2的过表达对ABS诱导的DNA双链断裂和细胞死亡具有保护作用。我们发现，IGFBP2对ABS诱导的EGFR和DNA-PKcs的核积累和磷酸化是必要的，这是DNA损伤修复活性所必需的。使用共免疫沉淀实验，在酸性胆盐处理后检测到IGFBP2与EGFR和DNA-PKcs的共定位。我们进一步证明，使用环己酰亚胺追踪实验，IGFBP2促进EGFR蛋白在ABS暴露时的稳定性。IGFBP2通过促进EGFR-DNA-PKcs信号轴的稳定和活化来保护EAC细胞免受ABS诱导的DNA损伤和凋亡的影响。

The incidence of esophageal adenocarcinoma (EAC) is rising rapidly in the US and Western countries. The development of Barrett's esophagus (BE) and its progression to EAC have been linked to chronic gastroesophageal reflux disease (GERD). Exposure of BE and EAC cells to acidic bile salts (ABS) in GERD conditions induces high levels of oxidative stress and DNA damage. In this study, we investigated the role of insulin-like growth factor binding protein 2 (IGFBP2) in regulating ABS-induced DNA double-strand breaks.Real-time RT-PCR, western blot, immunohistochemistry, immunofluorescence, co-immunoprecipitation, flow cytometry, and cycloheximide (CHX) chase assays were used in this study. To mimic GERD conditions, a cocktail of acidic bile salts (pH 4) was used in 2D and 3D organotypic culture models. Overexpression and knockdown of IGFBP2 in EAC cells were established to examine the functional and mechanistic roles of IGFBP2 in ABS-induced DNA damage.Our results demonstrated high levels of IGFBP2 mRNA and protein in EAC cell lines as compared to precancerous Barrett's cell lines, and IGFBP2 is frequently overexpressed in EACs (31/57). Treatment of EAC cells with ABS, to mimic GERD conditions, induced high levels of IGFBP2 expression. Knocking down endogenous IGFBP2 in FLO1 cells (with constitutive high levels of IGFBP2) led to a significant increase in DNA double-strand breaks and apoptosis, following transient exposure to ABS. On the other hand, overexpression of exogenous IGFBP2 in OE33 cells (with low endogenous levels of IGFBP2) had a protective effect against ABS-induced double-strand breaks and apoptosis. We found that IGFBP2 is required for ABS-induced nuclear accumulation and phosphorylation of EGFR and DNA-PKcs, which are necessary for DNA damage repair activity. Using co-immunoprecipitation assay, we detected co-localization of IGFBP2 with EGFR and DNA-PKcs, following acidic bile salts treatment. We further demonstrated, using cycloheximide chase assay, that IGFBP2 promotes EGFR protein stability in response to ABS exposure.IGFBP2 protects EAC cells against ABS-induced DNA damage and apoptosis through stabilization and activation of EGFR - DNA-PKcs signaling axis.