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UBC12mRNA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-rankχ2=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。
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-rankχ²=4.188,P=0.0407).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.
作者
鲜敬荣
朱晶
邵文琦
熊赢
潘柏申
王蓓丽
郭玮
XIAN Jingrong;ZHU Jing;SHAO Wenqi;XIONG Ying;PAN Baishen;WANG Beili;GUO Wei(Department of Clinical Laboratory,Zhongshan Hospital,Fudan University,Shanghai 200032,China)
出处
《检验医学》
2026年第3期223-232,共10页
Laboratory Medicine
基金
国家自然科学基金青年项目(82402682)
上海市“科技创新行动计划”自然科学基金面上项目(23ZR1411100)
国家重点研发计划项目(2022YFC3602301)。
