Abstract

Glycosylation is a common posttranslational modification of proteins, which plays a role in the malignant transformation, growth, progression, chemoresistance, and immune response of tumors. Disulfide isomerase family A3 (PDIA3) specifically acts on newly synthesized glycoproteins to promote the correct folding of sugar chains. Studies have shown that PDIA3 participates in multidrug-resistant gastric cancer (MDR-GC). In this study, we performed western blot analysis and immunohistochemistry to identify PDIA3 expression. Cell proliferation was assessed by CCK-8 assay. Transwell assays were used to detect the migration and invasion abilities of cells. Immunoprecipitation coupled to mass spectrometry (IP-MS) analysis was employed to identify PDIA3-interacting proteins and the associated pathways in MDR-GC cells. Glycoprotein interactions and translocation were detected by immunofluorescence assay. The results showed that PDIA3 knockdown significantly inhibited the proliferation, invasion, and migration abilities of MDR-GC cells. Kyoto Encyclopedia of Genes and Genomes analysis of the IP-MS results showed that PDIA3 was closely associated with focal adhesion pathways in MDR-GC cells. Additionally, important components of focal adhesion pathways, including fibronectin-1 (FN1) and integrin α5 (ITGA5), were identified as pivotal PDIA3-binding glycoproteins. Knockdown of PDIA3 altered the cellular locations of FN1 and ITGA5, leading to abnormal accumulation. In conclusion, our results suggest that knockdown of PDIA3 inhibited the malignant behaviors of MDR-GC cells and influenced the translocation of FN1 and ITGA5.

中文翻译：

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内质网驻留氧化还原酶PDIA3的沉默抑制耐多药胃癌的恶性生物学行为

摘要

糖基化是一种常见的蛋白质翻译后修饰，在肿瘤的恶性转化、生长、进展、化疗耐药和免疫反应中发挥作用。二硫键异构酶家族 A3 (PDIA3) 专门作用于新合成的糖蛋白以促进糖链的正确折叠。研究表明，PDIA3参与了耐多药胃癌（MDR-GC）。在这项研究中，我们进行了蛋白质印迹分析和免疫组织化学来鉴定 PDIA3 的表达。通过CCK-8测定评估细胞增殖。Transwell实验用于检测细胞的迁移和侵袭能力。免疫沉淀结合质谱 (IP-MS) 分析用于鉴定 MDR-GC 细胞中的 PDIA3 相互作用蛋白和相关通路。通过免疫荧光测定检测糖蛋白相互作用和易位。结果表明，PDIA3敲低显着抑制了MDR-GC细胞的增殖、侵袭和迁移能力。京都基因和基因组百科全书对 IP-MS 结果的分析表明，PDIA3 与 MDR-GC 细胞中的粘着斑通路密切相关。此外，粘着斑通路的重要成分，包括纤连蛋白-1 (FN1) 和整合素 α5 (ITGA5)，被确定为关键的 PDIA3 结合糖蛋白。PDIA3 的敲低改变了 FN1 和 ITGA5 的细胞位置，导致异常积累。总之，我们的结果表明 PDIA3 的敲低抑制了 MDR-GC 细胞的恶性行为并影响了 FN1 和 ITGA5 的易位。