类泛素结合酶UBC12通过调控多梳蛋白家族成员RING1促进食管癌细胞增殖

doi:10.3969/j.issn.1673-8640.2026.03.003

摘要/Abstract

摘要：

目的探讨类泛素结合酶UBC12调控食管癌细胞增殖的分子机制。方法采用蛋白质组学分析筛选UBC12下游靶点。基于癌症基因组图谱（TCGA）数据库分析食管癌患者RING1蛋白的表达情况。构建敲低UBC12人食管癌细胞（UBC12敲低组）和阴性对照细胞（NC组）、过表达RING1人食管癌细胞（OE组）和空白对照细胞（MOCK组）。采用CCK-8实验和克隆形成实验评估RING1对食管癌细胞增殖的影响。采用高通量测序分析OE组和MOCK组的差异表达基因，并进行生物信息学分析。检测食管癌细胞RING1和UBC12 mRNA的表达情况。采用蛋白降解实验分析UBC12对RING1蛋白的调控机制。选取2017年1月—2018年12月复旦大学附属中山医院食管癌患者30例，收集癌组织和癌旁组织（距肿瘤边缘2 cm）样本，采用免疫组化染色检测RING1蛋白的表达情况，随访所有患者至2025年3月。采用Kaplan-Meier生存曲线分析食管癌患者的生存情况。结果 TCGA数据库分析结果显示食管癌患者癌组织RING1表达低于癌旁组织（P<0.001）。RING1高表达食管癌患者的总生存期和无病生存期均显著长于RING1低表达患者。OE组细胞增殖活力和细胞克隆形成数均显著低于MOCK组（P<0.05）。OE组和MOCK组的差异表达基因显著富集于轴突发育和轴突发生等生物学过程，纤毛基部和染色质重塑复合物等细胞组分，FAD结合和SMAD结合等分子功能，OE组氨酰-tRNA生物合成通路、氧化磷酸化、RNA转运、蛋白质输出、真核生物核糖体生物合成受抑制。与NC组比较，UBC12敲低组RING1蛋白表达增加，RING1 mRNA相对表达量差异无统计学意义（P>0.05）。5种食管癌细胞系（EC1、EC109、K30、K450和K510）UBC12 mRNA相对表达量均显著高于RING1 mRNA（P<0.05），且UBC12蛋白表达与RING1蛋白表达呈负相关（r=-0.7085，P=0.18）。随着放线菌酮（CHX）处理时间的延长，UBC12敲低组RING1蛋白表达逐渐高于NC组，即RING1蛋白半衰期延长。食管癌患者癌组织RING1蛋白表达显著低于癌旁组织（P<0.05）。RING1高表达食管癌患者总生存期显著长于低表达患者（Log-rank χ²=4.188，P=0.040 7）。结论 UBC12通过调控RING1促进食管癌细胞增殖，可为食管癌的靶向治疗提供新靶点。

关键词: 类泛素结合酶UBC12, RING1, 食管癌

Abstract:

Objective To investigate the molecular mechanism of neddylation UBC12 regulating esophageal cancer cell proliferation. Methods Proteomic analysis was used to screen for downstream targets of UBC12. The expression of RING1 in esophageal cancer patients was analyzed based on the Cancer Genome Atlas（TCGA）database. Esophageal cancer cells with UBC12 knockdown（UBC12 knockdown group）and negative control（NC）cells（NC group），as well as esophageal cancer cells overexpressing（OE）RING1（OE group）and mock（MOCK）-transfected control cells（MOCK group），were established. The effects of RING1 on esophageal cancer cell proliferation were assessed using CCK-8 and colony formation assays. High-throughput sequencing was performed to analyze differentially expressed genes between OE and MOCK groups，followed by bioinformatic analysis. The expression levels of RING1 and UBC12 mRNA in esophageal cancer cells were determined. Protein degradation assays were conducted to analyze the regulatory mechanism of UBC12 on RING1 protein. Cancer tissues and adjacent normal tissues（2 cm from tumor margins） were obtained from 30 esophageal cancer patients at Zhongshan Hospital of Fudan University between January 2017 and December 2018. Immunohistochemical staining was used to determine RING1 protein expression，and all the patients were followed up until March 2025. Kaplan-Meier survival curves were used to analyze patient survival. Results TCGA analysis results showed that RING1 expression was lower in cancer tissues compared to adjacent normal tissues（P<0.001）. Esophageal cancer patients with high RING1 expression exhibited longer overall survival and disease-free survival than those with low RING1 expression. Both cell proliferation viability and the number of cell colonies formed were lower in OE group than those in MOCK group（P<0.05）. Differentially expressed genes between OE and MOCK groups were enriched in biological processes such as axon development and axonogenesis，cellular components including ciliary base and chromatin-remodeling complex，molecular functions such as FAD binding and SMAD binding，pathways including aminoacyl-tRNA biosynthesis，oxidative phosphorylation，RNA transport，protein export and eukaryotic ribosome biogenesis were suppressed in OE group. Compared to NC group，RING1 protein expression was increased in UBC12 knockdown group，while no statistically significance was observed in RING1 mRNA relative expression（P>0.05）. The relative expression of UBC12 mRNA was higher than that of RING1 mRNA in 5 esophageal cancer cell lines（EC1，EC109，K30，K450 and K510）（P<0.05），and UBC12 protein expression was negatively correlated with RING1 protein expression（r=-0.7085，P=0.18）. With prolonged cycloheximide（CHX） treatment，RING1 protein expression in UBC12 knockdown group gradually became higher than in NC group，indicating an extended RING1 protein half-life. RING1 protein expression was lower in cancer tissues than in adjacent normal tissues from esophageal cancer patients（P<0.05）. Esophageal cancer patients with high RING1 expression had a longer overall survival than those with low RING1 expression（Log-rank χ²=4.188，P=0.040 7）. Conclusions UBC12 promotes the proliferation of esophageal cancer cells by regulating RING1，suggesting that it may serve as a novel therapeutic target for esophageal cancer.

Key words: Neddylation UBC12, RING1, Esophageal cancer

中图分类号:

R446.1

鲜敬荣, 朱晶, 邵文琦, 熊赢, 潘柏申, 王蓓丽, 郭玮. 类泛素结合酶UBC12通过调控多梳蛋白家族成员RING1促进食管癌细胞增殖[J]. 检验医学, 2026, 41(3): 223-232.

XIAN Jingrong, ZHU Jing, SHAO Wenqi, XIONG Ying, PAN Baishen, WANG Beili, GUO Wei. Neddylation UBC12 promotes esophageal cancer cell proliferation by regulating polycomb protein RING1[J]. Laboratory Medicine, 2026, 41(3): 223-232.

图/表 8

表1 引物序列

基因名称	正向引物（5'~3'）	反向引物（5'~3'）
UBC12	ATGAGGGCTTCTACAAGAGTGG	ATTGTCTCACACTTCACCTTGG
RING1	AGAATGCCAGCAAAACGTGG	AGATAGGGCACATGAGTTCTGA
GAPDH	GTCATCCAACGGGAATGCA	TGATCGGTTACCGTGATCAAAA

图1 敲低UBC12后食管癌细胞RING1的表达情况注：（a）敲低UBC12的K450细胞免疫印迹图；#1和#2为2条敲低序列；（b）分子相关性网格图。

图2 肿瘤组织中RING1的表达情况和食管癌患者的生存情况注：（a）TCGA数据库中癌组织和癌旁组织RING1基因表达量比较，左侧无黑框为癌旁组织，右侧加黑框为癌组织；与癌旁组织比较，*P<0.05；（b）食管癌组织和癌旁组织RING1基因表达量；（c）RING1高表达和低表达食管癌患者总生存期的Kaplan-Meier生存曲线[Log-rank χ2=4.90，P=0.009 6；风险比（hazard ratio，HR）=0.55，P=0.01]；（d）RING1高表达和低表达食管癌患者无病生存期的Kaplan-Meier生存曲线[Log-rank χ2=9.83，P=0.004 2；HR=0.50，P=0.047]；—低表达组；—高表达组。

图3 RING1对食管癌细胞增殖的影响注：（a）RING1过表达的免疫印迹图；（b）K30细胞和EC109细胞的增殖活力曲线，与MOCK组比较，*P<0.05、**P<0.01、***P<0.001； MOCK组； OE组；（c）K30和EC109细胞克隆形成情况；（d）OE组和MOCK组细胞克隆形成数比较。

图4 RING1对食管癌细胞关键通路的影响注：（a）K30细胞中OE组和MOCK组基因表达变化的韦恩图；（b）差异表达基因的火山图；表达上调、表达下调、表达无变化；（c）GO功能富集分析；（d）GSEA。

图5 食管癌细胞中UBC12表达与RING1表达的关系注：（a）UBC12敲低组和NC组RING1蛋白表达的免疫印迹图，#1和#2为2条敲低序列；（b）NC组、UBC12敲低组UBC12蛋白和RING1蛋白相对表达量比较； NC组、 UBC12敲低组#1敲低序列、 UBC12敲低组#2敲低序列；与NC组比较，*P<0.05、**P<0.001；（c）UBC12敲低组、NC组UBC12 mRNA和RING1 mRNA相对表达量比较； NC组、 UBC12敲低组#1敲低序列、 UBC12敲低组#2敲低序列；与NC组比较，*P<0.001；（d）5种食管癌细胞系（EC1、EC109、K30、K450和K510）中UBC12 mRNA和RING1 mRNA相对表达量比较； UBC12 mRNA、□RING1 mRNA；与UBC12 mRNA比较，*P<0.05、**P<0.01、***P< 0.001；（e）5种食管癌细胞系中UBC12和RING1蛋白表达的免疫印迹图；（f）UBC12蛋白表达和RING1蛋白表达的相关性，Y=-0.415 1X + 24 735（r=-0.708 5）。

图6 RING1蛋白的泛素化注：（a）抑制剂对RING1蛋白水平的影响；（b）RING1蛋白降解实验；（c）UniBrowser数据库预测RING1的E3连接酶；（d）基于PhosphoSitePlus数据库预测RING1的蛋白修饰位点；磷酸化位点；乙酰化位点；泛素化位点；其他位点；（e）RING1和MDM2蛋白结合实验。

图7 RING1表达的免疫组化结果和不同RING1蛋白表达食管癌患者的Kaplan-Meier生存曲线注：（a）RING1免疫组化染色图；（b）癌组织和癌旁组织RING1表达的比较；（c）不同RING1蛋白表达食管癌患者的Kaplan-Meier生存曲线；高表达组、低表达组。

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