收稿日期: 2025-07-08
网络出版日期: 2025-11-05
基金资助
国家自然科学基金项目(82260120);宁夏自然科学基金项目(2023AAC03200);宁夏回族自治区教育厅高等学校科学研究项目(NYG-2022-043)
The role of GPNMB in hypoxia induced epithelial-mesenchymal transition in human chorionic trophoblast cells
Received date: 2025-07-08
Online published: 2025-11-05
目的 探讨糖蛋白非转移性黑色素瘤蛋白B（glycoprotein nonmetastatic melanoma protein B，GPNMB）在缺氧诱导的人绒毛膜滋养层细胞HTR-8/SVneo上皮-间质转化（epithelial- mesenchymal transition，EMT）中的作用。 方法 体外培养HTR-8/SVneo细胞，观察缺氧对GPNMB表达的影响。转染GPNMB过表达质粒（pcDNA3.1-GPNMB）或干扰片段（si-GPNMB-1/2）及相应的对照片段（pcDNA3.1-NC或si-NC），并使用自噬激动剂雷帕霉素（Rapamycin， Rap）处理，分为Normoxia组、Hypoxia组、Normoxia /Hypoxia + si-NC或si-GPNMB组、Normoxia /Hypoxia + pcDNA3.1-NC或pcDNA3.1-GPNMB组、Normoxia/Hypoxia + Rap、Hypoxia + Rap + pcDNA3.1-NC或pcDNA3.1-GPNMB组。通过qRT-PCR、Western blot、细胞免疫荧光技术检测GPNMB的表达，Western blot检测自噬相关蛋白（LC3B II/I 、p62）及EMT相关蛋白（E-cadherin、N-cadherin）的表达，划痕实验和Transwell实验检测细胞的迁移和侵袭能力。 结果 与Normoxia组相比，Hypoxia组HTR-8/SVneo细胞的GPNMB 的mRNA及蛋白表达水平均降低，p62、N-cadherin蛋白表达量降低，LC3B II/I 、E-cadherin蛋白表达量升高（P < 0.05）；与Hypoxia + si-NC组相比，Hypoxia + si- GPNMB-2组p62、N-cadherin蛋白表达量降低，LC3B II/I 、E-cadherin蛋白表达量升高（P < 0.05）；与Hypoxia + pcDNA3.1-NC组相比，Hypoxia + pcDNA3.1- GPNMB组则发生相反的变化。与Hypoxia组相比，Hypoxia + Rap组LC3B II/I 和E-cadherin蛋白水平增高，而p62和N-cadherin蛋白水平降低（P < 0.05）；与Hypoxia + pcDNA3.1-GPNMB组相比，Hypoxia + Rap + pcDNA3.1-GPNMB组可减弱GPNMB过表达对HTR-8/SVneo细胞EMT的促进作用，表现为p62、N-cadherin蛋白表达量降低，LC3BII/I、E-cadherin蛋白表达量升高（P < 0.05）。 结论 在缺氧诱导的HTR-8/SVneo细胞中，GPNMB能抑制细胞自噬、促进EMT过程、增强细胞的迁移与侵袭能力。
田甜 , 曹萍 , 章旭红 , 马小红 , 李婧睿 , 丁学琴 , 杨晓明 . GPNMB在缺氧诱导的人绒毛膜滋养层细胞上皮-间质转化中的作用[J]. 实用医学杂志, 2025 , 41(20) : 3135 -3144 . DOI: 10.3969/j.issn.1006-5725.2025.20.001
Objective To investigate the role of Glycoprotein non-metastatic melanoma protein B （GPNMB） in hypoxia-induced epithelial–mesenchymal transition （EMT） in human chorionic trophoblast HTR-8/SVneo cells. Methods HTR-8/SVneo cells were cultured in vitro to investigate the effect of hypoxia on GPNMB expression. The cells were transfected with either a GPNMB overexpression plasmid （pcDNA3.1-GPNMB）， small interfering RNA targeting GPNMB （si-GPNMB-1/2）， or their respective negative controls （pcDNA3.1-NC or si-NC）， and were also treated with the autophagy agonist rapamycin （Rap）. The experimental groups were categorized as follows： Normoxia， Hypoxia， Normoxia/Hypoxia + si-NC or si-GPNMB， Normoxia/Hypoxia + pcDNA3.1-NC or pcDNA3.1-GPNMB， Normoxia/Hypoxia + Rap， and Hypoxia + Rap + pcDNA3.1-NC or pcDNA3.1-GPNMB. GPNMB expression levels were evaluated using qRT-PCR， Western blotting， and immunofluorescence staining. The expression of autophagy-related proteins （LC3B Ⅱ/Ⅰ， p62） and epithelial-mesenchymal transition （EMT） markers （E-cadherin， N-cadherin） was analyzed by Western blotting. Cell migration and invasion capacities were assessed using wound healing and Transwell assays. Results Compared with the Normoxia group， the mRNA and protein levels of GPNMB were downregulated in the Hypoxia group. Additionally， the protein levels of p62 and N-cadherin were reduced， while LC3B Ⅱ/Ⅰ and E-cadherin expression levels were increased （P < 0.05）. Compared with the Hypoxia + si-NC group， the Hypoxia + si-GPNMB-2 group showed significantly decreased protein levels of p62 and N-cadherin， along with elevated levels of LC3B Ⅱ/Ⅰ and E-cadherin （P < 0.05）. Compared with the Hypoxia + pcDNA3.1-NC group， the Hypoxia + pcDNA3.1-GPNMB group exhibited opposite trends. Notably， compared with the Hypoxia group， the Hypoxia + Rap group showed increased LC3B Ⅱ/Ⅰ and E-cadherin levels， accompanied by reduced p62 and N-cadherin levels （P < 0.05）. However， compared with the Hypoxia + pcDNA3.1-GPNMB group， the Hypoxia + Rap + pcDNA3.1-GPNMB group attenuated the promoting effect of GPNMB overexpression on EMT in HTR-8/SVneo cells， as evidenced by decreased p62 and N-cadherin protein expression levels and increased LC3B Ⅱ/Ⅰ and E-cadherin protein expression levels （P < 0.05）. Conclusion In hypoxia-induced HTR-8/SVneo cells， GPNMB inhibits autophagy， promotes the epithelial-mesenchymal transition， and enhances cell migration and invasion.
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