ZG16通过CTNNA3抑制肝癌EMT及其对PD-L1的负调控作用研究

doi:10.19586/j.2095-2341.2025.0152

生物技术进展 ›› 2026, Vol. 16 ›› Issue (2): 422-431.DOI: 10.19586/j.2095-2341.2025.0152

• 研究论文 • 上一篇

ZG16通过CTNNA3抑制肝癌EMT及其对PD-L1的负调控作用研究

江策¹(), 刘昳², 赵亚磊³, 张海云⁴, 常香荣⁴()

^1.陕西省康复医院医学检验科，西安 710065
^2.西安交通大学第一附属医院中医科，西安 710061
^3.西安交通大学第一附属医院感染科，西安 710061
^4.陕西省康复医院消化内科，西安 710065

收稿日期:2025-10-31 接受日期:2026-01-06 出版日期:2026-03-25 发布日期:2026-04-27
通讯作者: 常香荣
作者简介:江策 E-mail: 441404487@qq.com；
基金资助:
陕西省自然科学基础研究计划项目(2022JQ-833)

ZG16 Inhibits EMT in Hepatocellular Carcinoma via CTNNA3 and Negatively Regulates of PD-L1

Ce JIANG¹(), Yi LIU², Yalei ZHAO³, Haiyun ZHANG⁴, Xiangrong CHANG⁴()

^1.Department of Medical Laboratory，Shaanxi Rehabilitation Hospital，Xi'an 710065，China
^2.Department of Traditional Chinese Medicine，the First Affiliated Hospital of Xi'an Jiaotong University，Xi'an 710061，China
^3.Department of Infection，the First Affiliated Hospital of Xi'an Jiaotong University，Xi'an 710061，China
^4.Department of Gastroenterology，Shaanxi Rehabilitation Hospital，Xi'an 710065，China

Received:2025-10-31 Accepted:2026-01-06 Online:2026-03-25 Published:2026-04-27
Contact: Xiangrong CHANG

摘要/Abstract

摘要：

探讨了酶原颗粒蛋白16（zymogen granule protein 16，ZG16）对肝癌细胞上皮间质转化（epithelial-mesenchymal transition, EMT）的影响及其与免疫检查点分子程序性死亡-配体1（programmed death-ligand 1，PD-L1）的潜在关联。基于GEO数据库和KMplot网站分析ZG16在肝癌（hepatocellular carcinoma，HCC）与正常组织中的表达差异及其与患者预后的关系。利用Huh7和HepG2细胞转染pcDNATM3.1-ZG16过表达质粒以过表达ZG16；利用Western blot检测ZG16、CTNNA3、E-cadherin、N-cadherin、Vimentin和PD-L1的蛋白表达水平；免疫荧光技术检测ZG16、CTNNA3的定位及蛋白表达水平；免疫共沉淀实验检测ZG16与CTNNA3的相互作用；通过划痕实验检测细胞迁移能力；通过平板克隆实验检测细胞克隆形成能力。生物信息学分析显示，ZG16在肝癌组织中表达显著下调，且其高表达的患者生存率显著性升高（P<0.05）。体外实验证实，相较于正常组，OE-ZG16组肝癌细胞的E-cadherin表达升高，N-cadherin、Vimentin的表达降低（P<0.05），Huh7和HepG2细胞迁移能力、克隆能力显著降低（P<0.05）。肝癌患者ZG16表达水平与PD-L1呈负相关，过表达ZG16显著降低PD-L1的表达水平。机制方面，过表达ZG16显著提高CTNNA3的蛋白表达水平（P<0.05）；免疫共沉淀及荧光共定位实验证实两者共定位于细胞质并相互作用。功能回复实验表明，与过表达ZG16的Huh7细胞相比，同时过表达ZG16并下调CTNNA3可促进EMT进程，增强细胞迁移能力和克隆形成能力（P<0.05）。ZG16上调CTNNA3抑制肝癌细胞的上皮间质转化进程。此外，ZG16还负调控PD-L1的蛋白表达。研究结果可为肝癌的临床治疗提供新的分子靶点和理论依据。

关键词: 肝细胞癌, 酶原颗粒蛋白16, 上皮间质转化, CTNNA3, PD-L1

Abstract:

To investigate the effect of zymogen granule protein 16 （ZG16） on epithelial mesenchymal transition （EMT） in hepatocellular carcinoma cells and its potential association with the immune checkpoint molecule programmed death-ligand 1 （PD-L1）. The expression differences of ZG16 between hepatocellular carcinoma （HCC） and normal liver tissues， as well as its relationship with patient prognosis， were analyzed based on the GEO database and the KM-Plot website. Huh7 and HepG2 cells were transfected with the pcDNATM3.1-ZG16 overexpression plasmid to overexpress ZG16. Protein expression levels of ZG16， CTNNA3， E-cadherin， N-cadherin， Vimentin and PD-L1 were detected by Western blot. The localization and protein expression levels of ZG16 and CTNNA3 were examined using immunofluorescence. The interaction between ZG16 and CTNNA3 was detected by co-immunoprecipitation. Cell migration ability was assessed through scratch wound healing assays， and cell colony formation ability was evaluated using colony formation assays.Bioinformatic analysis revealed that ZG16 expression was significantly downregulated in HCC tissues， and higher ZG16 expression was associated with significantly improved patient survival （P<0.05）. In vitro experiments showed that compared to the control group， ZG16 overexpression significantly increased E-cadherin expression and decreased N-cadherin and Vimentin expression （P<0.05）， along with significantly reduced cell migration and colony formation abilities in Huh7 and HepG2 cells （P<0.05）. In HCC patients， ZG16 expression levels were negatively correlated with PD-L1， and ZG16 overexpression significantly reduced PD-L1 protein levels. Mechanistically， ZG16 overexpression significantly increased CTNNA3 protein levels （P<0.05）. Co-immunoprecipitation and co-localization immunofluorescence confirmed that ZG16 and CTNNA3 interact and co-localize in the cytoplasm. Functional rescue experiments demonstrated that， compared to Huh7 cells overexpressing ZG16 alone， simultaneous overexpression of ZG16 and knockdown of CTNNA3 promoted the epithelial-mesenchymal transition process and enhanced cell migration and colony formation abilities （P<0.05）. ZG16 upregulates CTNNA3 to inhibit the EMT process in hepatocellular carcinoma cells. Additionally， ZG16 negatively regulates PD-L1 protein expression. The research results can provide new molecular targets and theoretical basis for the clinical treatment of liver cancer.

Key words: hepatocellular carcinoma, zymogen granule protein 16, epithelial-mesenchymal transformation, CTNNA3, PD-L1

中图分类号:

Q786

江策, 刘昳, 赵亚磊, 张海云, 常香荣. ZG16通过CTNNA3抑制肝癌EMT及其对PD-L1的负调控作用研究[J]. 生物技术进展, 2026, 16(2): 422-431.

Ce JIANG, Yi LIU, Yalei ZHAO, Haiyun ZHANG, Xiangrong CHANG. ZG16 Inhibits EMT in Hepatocellular Carcinoma via CTNNA3 and Negatively Regulates of PD-L1[J]. Current Biotechnology, 2026, 16(2): 422-431.

图/表 7

图1 ZG16表达与肝癌患者肿瘤分期及预后A：GSE254461数据集筛选差异表达基因；B：ZG16表达与肝癌临床分期相关；C：ZG16表达与肝细胞癌患者生存率相关性

Fig. 1 ZG16 expression and its association with tumor stage/prognosis in hepatocellular carcinoma

图2 肝癌细胞中ZG16 mRNA及蛋白表达情况A：RT-qPCR检测肝癌细胞系ZG16 mRNA水平；B：Western blot检测肝癌细胞系ZG16蛋白水平。*表示与L-02组相比，在P<0.05水平上具有统计学意义。

Fig. 2 ZG16 mRNA and protein expression in hepatocellular carcinoma cells

图3 过表达 ZG16 对Huh7细胞EMT和转移的影响A：Western blot检测ZG16过表达后Huh7细胞中EMT标志物表达水平；B：划痕实验检测ZG16过表达对Huh7细胞迁移的影响；C：Transwell侵袭实验检测ZG16过表达对Huh7细胞侵袭能力的影响。*表示与OE-NC组相比，在P<0.05水平上具有统计学意义。

Fig. 3 The effect of overexpression of ZG16 on EMT and metastasis of Huh7 cells

图4 过表达 ZG16 对HepG2细胞EMT和转移的影响A：Western blot检测ZG16过表达后HepG2细胞中EMT标志物表达水平；B：划痕实验检测ZG16过表达对HepG2细胞迁移的影响；C：Transwell侵袭实验检测ZG16过表达对HepG2细胞侵袭能力的影响。*表示与OE-NC组相比，在P<0.05水平上具有统计学意义。

Fig. 4 The effect of overexpression of ZG16 on EMT and metastasis of HepG2 cells

图5 过表达 ZG16 对Huh7和HepG2细胞PD-L1水平的影响A：GEPIA2数据库分析肝癌患者ZG16与PD-L1表达水平相关性；B：Western blot检测Huh7细胞中ZG16过表达对PD-L1水平的影响；C：Western blot检测HepG2细胞中ZG16过表达对PD-L1水平的影响。*表示与OE-NC组相比，在P<0.05水平上具有统计学意义。

Fig. 5 The effect of overexpression of ZG16 on PD-L1 level in Huh7 and HepG2 cells

图6 过表达ZG16对CTNNA3表达水平的影响及与其相互作用A：RT-qPCR检测ZG16过表达对CTNNA3 mRNA表达水平的影响；B：Western blot检测ZG16过表达对CTNNA3蛋白表达的影响；C：免疫荧光实验观察ZG16和CTNNA3在细胞中的定位；D：免疫共沉淀实验检测ZG16与CTNNA3蛋白的相互作用。*表示与OE-NC组相比，在P<0.05水平上具有统计学意义。

Fig. 6 ZG16 overexpression affects CTNNA3 expression and protein interaction

图7 CTNNA3敲低逆转过表达ZG16对Huh7细胞的EMT及转移的影响A：Western blot检测CTNNA3敲低对ZG16过表达肝癌细胞EMT标志物表达水平的影响；B：划痕实验检测CTNNA3敲低对ZG16过表达肝癌细胞迁移的影响；C：Transwell侵袭实验检测CTNNA3敲低对ZG16过表达肝癌细胞侵袭能力的影响。*表示与OE-ZG16+si-NC组相比，在P<0.05水平上具有统计学意义。

Fig. 7 CTNNA3 depletion abolishes ZG16 overexpression-induced EMT and metastasis in Huh7 cells

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ZG16通过CTNNA3抑制肝癌EMT及其对PD-L1的负调控作用研究

ZG16 Inhibits EMT in Hepatocellular Carcinoma via CTNNA3 and Negatively Regulates of PD-L1

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