低氧条件下脯氨酸羟化酶2调控泛素特异性蛋白酶22参与食管鳞状细胞癌侵袭性生物学行为

Under hypoxic conditions, prolyl hydroxylase domain-containing protein 2 regulates ubiquitin-specific protease 22 to participate in the invasive biological behavior of esophageal squamous cell carcinoma

摘要

目的 探究脯氨酸羟化酶2(prolyl hydroxylase domain-containing protein 2, PHD2)在低氧微环境对食管鳞状细胞癌(esophageal squamous cell carcinoma, ESCC)恶性生物学行为的影响,以及其与泛素特异性蛋白酶22(ubi-quitin-specific protease 22, USP22)之间的调控关系。 方法 运用高通量基因表达公共数据库(gene expression omnibus, GEO)分析PHD2 mRNA在ESCC组织和正常组织中的表达。以正常食管鳞状上皮细胞(immortalized human esophageal epithelial cells-SV40, HET-1A)为对照,采用Western blotting法检测食管鳞癌细胞系(KYSE-410、KYSE-30、KYSE-150、EC-9706、TE-1)中PHD2表达;转染构建PHD2沉默及过表达ESCC细胞模型;在常氧和低氧(1%O₂)状态下采用CCK8法检测ESCC细胞增殖能力,采用划痕愈合和Transwell迁移实验检测ESCC细胞迁移能力;比较PHD2沉默及过表达后,对USP22表达水平影响,并验证其调控关系;采用免疫共沉淀实验检测PHD2与USP22之间的相互作用。 结果 基因表达公共数据库分析结果显示,正常组织中PHD2 mRNA表达水平高于食管鳞癌组织;与ESCC细胞相比,正常食管鳞状上皮细胞 HET-1A中PHD2表达较高(P<0.05);低氧促进ESCC细胞增殖、迁移能力;沉默PHD2增加ESCC细胞增殖、迁移能力,而过表达可抑制其增殖、迁移能力(P<0.05);低氧24 h后PHD2表达降低而USP22表达升高(P<0.05);沉默PHD2,USP22表达升高(P<0.05),过表达PHD2,USP22表达降低(P<0.05);免疫共沉淀实验显示,PHD2与USP22之间存在结合。 结论 PHD2在癌组织和ESCC细胞系中表达较低;过表达PHD可阻碍ESCC细胞增殖、迁移;常氧条件下过表达PHD2,USP22表达下降;乏氧条件下过表达PHD2,但对USP22起调控作用,提示实体肿瘤乏氧条件下两者同样存在调控关系,协同参与食管鳞癌的侵袭性生物学行为,PHD2可作为ESCC治疗新靶点。

Abstract

Objective To investigate the effects of prolyl hydroxylase domain-containing protein 2(PHD2)on the malignant biological behaviors of esophageal squamous cell carcinoma(ESCC)in a hypoxic microenvironment, and to elucidate its regulatory relationship with ubiquitin-specific protease 22(USP22). Methods The high-throughput public gene expression database, gene expression omnibus(GEO), was utilized to analyze the expression of PHD2 mRNA in ESCC tissues and normal tissues. Western blotting was performed to evaluate PHD2 expression levels in a panel of esophageal squamous cell carcinoma(ESCC)cell lines(KYSE-410, KYSE-30, KYSE-150, EC-9706 and TE-1), with normal immortalized human esophageal epithelial cells(HET-1A, immortalized by SV40)serving as the control. ESCC cell models with PHD2 silencing(knockdown)and overexpression were established via transfection. Under normoxic and hypoxic(1%O₂)conditions, the proliferative capacity of ESCC cells was evaluated using the CCK-8 assay, while the migratory ability was assessed via wound healing and Transwell migration assays. Furthermore, the effects of PHD2 silencing and overexpression on USP22 expression levels were evaluated to validate their regulatory relationship. Finally, co-immunoprecipitation(Co-IP)assays were performed to demonstrate the interaction between PHD2 and USP22. Results Analysis of public gene expression databases revealed that PHD2 mRNA expression levels were higher in normal tissues than in ESCC tissues. Compared with ESCC cells, the expression of PHD2 was significantly higher in normal esophageal squamous epithelial cells(HET-1A)(P<0.05). Hypoxia promoted the proliferation and migration abilities of ESCC cells. Silencing of PHD2 enhanced the proliferation and migration of ESCC cells, whereas PHD2 overexpression suppressed these abilities(P<0.05). After 24 hours of hypoxia, PHD2 expression decreased while USP22 expression increased(P<0.05). Furthermore, PHD2 silencing upregulated the expression of USP22(P<0.05), while PHD2 overexpression downregulated USP22 expression(P<0.05). Finally, Co-IP assays confirmed the physical interaction between PHD2 and USP22. Conclusion PHD2 is expressed at low levels in both ESCC tissues and cell lines, and its overexpression suppresses the proliferation and migration of ESCC cells. Under normoxic conditions, PHD2 overexpression downregulates USP22 expression; notably, under hypoxic conditions, PHD2 maintains its regulatory effect on USP22. This suggests that a regulatory relationship likewise exists between USP22 and PHD2 under the hypoxic conditions of solid tumors, and they synergistically contribute to the aggressive biological behaviors of ESCC. PHD2 may therefore serve as a novel therapeutic target for ESCC.

关键词

脯氨酸羟化酶2 / 泛素特异性蛋白酶 22 / 食管鳞状细胞癌 / 增殖 / 迁移

Key words

Prolyl hydroxylase domain-containing protein 2 / Ubiquitin-specific protease 22 / Esophageal squamous cell carcinoma / Proliferation / Migration

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基金资助

青海省自然科学基金(2022-ZJ-922)

青海省“昆仑英才·高端创新创业人才”杰出人才计划项目