[摘 要] 目的：探究双特异性磷酸酶26（DUSP26）在肺腺癌（LUAD）A549细胞增殖、迁移和侵袭中的作用及其分子机制。 方法：检索肿瘤数据库GEPIA2网站DUSP26表达数据，分析DUSP26在LUAD患者和正常人肺组织中的表达差异。收集2022 年10月至2023年10月期间萍乡市人民医院手术切除的12例LUAD组织和癌旁组织标本，通过免疫组织化学（IHC）和WB法检 测DUSP26在LUAD组织和癌旁组织之间的表达差异；通过WB法检测DUSP26在4种LUAD细胞（A549、SK-LU-1、Calu-3、 H1299）和2种正常支气管上皮细胞（BEAS-2B、HBEC）中的表达差异。利用慢病毒转染细胞的方法构建稳定过表达DUSP26 （DUSP26-OE）及阴性对照（DUSP26-OENC）的A549细胞，通过克隆形成、划痕愈合实验、Transwell实验分别检测DUSP26过表达 对细胞增殖、迁移及侵袭能力的影响，WB法检测各组细胞中TGF-β1/SMAD2/3通路、EMT相关蛋白的表达水平，细胞免疫荧光 法检测细胞中Ki-67、cyclin D1表达水平。加入TGF-β1重组蛋白进行回复实验。构建A549细胞裸鼠荷瘤模型，观察DUSP26过 表达对移植瘤体内生长的影响，WB法检测移植瘤组织中TGF-β1/SMAD2/3通路、EMT相关蛋白表达水平，免疫荧光染色法检测 移植瘤组织中Ki-67、cyclin D1表达水平。结果：DUSP26在LUAD组织和细胞中均呈低表达（P < 0.05或P < 0.01或P < 0.001 或P < 0.000 1）。与DUSP26-OENC组相比，DUSP26-OE组A549细胞的增殖、迁移和侵袭能力均显著降低（P < 0.01或P < 0.001）， TGF-β1、p-SMAD2/3、vimentin、N-cadherin、snail、Ki-67、cyclin D1表达均降低（P < 0.01或P < 0.001或P < 0.000 1），E-cadherin表 达升高（P < 0.000 1）。加入5 ng/mL TGF-β1重组蛋白后，可部分逆转在体外实验中由DUSP26过表达导致的结果。成功构建裸 鼠A549细胞荷瘤模型，DUSP26-OE组裸鼠移植瘤生长速度缓慢，体积和质量均减小（均P < 0.001），移植瘤组织中TGF-β1、 p-SMAD2/3、vimentin、N-cadherin、snail、Ki-67、cyclin D1表达均降低（P < 0.01或P < 0.001），E-cadherin表达升高（P < 0.000 1）。 结论：DUSP26在LUAD组织和细胞中均呈低表达状态，上调DUSP26的表达水平能够通过抑制TGF-β1/SMAD2/3信号通路抑 制A549细胞的增殖、迁移和侵袭。

[Abstract] Objective: To investigate the role and molecular mechanism of dual-specificity phosphatase 26 (DUSP26) in the proliferation, migration and invasion of lung adenocarcinoma (LUAD) A549 cells. Methods: The expression profile of DUSP26 was retrieved from the GEPIA2 tumor database, and its differential expression in LUAD tissues and normal human lung tissues were analyzed. Twelve pairs of LUAD tissue and paracancerous tissue surgically resected at Pingxiang People's Hospital between October 2022 and October 2023 were collected. The difference in DUSP26 expression between LUAD tissues and paracancerous tissues was analyzed using immunohistochemical (IHC) staining and Western blotting (WB). Additionally, the expression of DUSP26 in four LUAD cells (A549, SK-LU-1, Calu-3, H1299) and two normal bronchial epithelial cells (BEAS-2B, HBEC) was detected using WB method. A549 cells stably overexpressing DUSP26 (DUSP26-OE) or corresponding negative control (DUSP26-OENC) were constructed via lentiviral transfection. The effects of DUSP26 overexpression on cell proliferation, migration and invasion were detected using colony formation, scratch assay, and Transwell chamber assay, respectively. The expression levels of TGF-β1/SMAD2/3 pathway-and epithelial-mesenchymal transition (EMT)-related proteins were detected using WB method, and the expression levels of Ki-67 and cyclin D1 in cells were detected by immunofluorescence staining. Rescue experiments were conducted by adding 5 ng/mL recombinant TGF-β1. A nude mouse xenograft model was established using A549 cells to observe the effect of DUSP26 overexpression on the in vivo growth of transplanted tumors. The expression levels of TGF- β1/SMAD2/3 pathway-and EMT-related proteins in transplanted tumor tissues were assessed using WB method, and the expression levels of Ki-67 and cyclin D1 in transplanted tumor tissues were detected using immunofluorescence staining. Results: DUSP26 expression was downregulated in both LUAD tissues and cells (P < 0.05, P < 0.01, P < 0.001 or P < 0.000 1). Compared with the DUSP26-OENC group, the DUSP26-OE group showed significantly reduced proliferation, migration and invasion of A549 cells (P < 0.01 or P < 0.001). Furthermore, the protein levels of TGF-β1, p-SMAD2/3, vimentin, N-cadherin, snail, Ki-67, and cyclin D1 were significantly reduced (P < 0.01, P < 0.001 or P < 0.000 1), while E-cadherin level was increased in the DUSP26-OE group (P < 0.000 1). The addition of 5 ng/mL TGF-β1 recombinant protein partially reversed the effects of DUSP26 overexpression in vitro experiments. The nude mice A549 cell xenograft model was successfully constructed. The growth rate of transplanted tumors was significantly slower in the DUSP26-OE group, with reduced volume and mass (all P < 0.001). The protein levels of TGF-β1, p-SMAD2/3, vimentin, N-cadherin, snail, Ki-67, and cyclin D1 in the transplanted tumor tissues were all reduced (P < 0.01 or P < 0.001), while E-cadherin level was increased (P < 0.000 1). Conclusion: DUSP26 is downregulated in both LUAD tissues and cells. Upregulation of DUSP26 suppresses the proliferation, migration and invasion of A549 cells by inhibiting the TGF-β1/SMAD2/3 signaling pathway.

[基金项目] 江西省萍乡市科技计划项目（No. 2021PY078）