泛素/ISG15结合酶E2L6在胰腺癌中的作用及其临床价值分析

Analysis of the function of ubiquitin/ISG15-conjugating enzyme E2L6 in pancreatic cancer and its clinical value

背景与目的:胰腺癌具有高度迁移性和侵袭性的特点,预后不良,但癌基因的突变及分子调控关系等的改变会影响胰腺癌的恶性生物学行为。泛素/ISG15结合酶E2L6(UBE2L6)在泛癌中的表达及胰腺癌中的生物学功能依然不清楚。本研究拟通过生物信息学分析及实验验证,探讨UBE2L6对胰腺癌增殖、迁移侵袭能力的影响及可能的分子机制。方法:从UCSC Xena下载TCGA和GTEx的RNA-seq数据,用R(4.0.2)软件的“limma”包分析UBE2L6在泛癌中的差异表达。利用从GEO数据集下载数据及qRT-PCR分别验证UBE2L6在胰腺癌组织及细胞中的差异表达。胰腺癌细胞转染靶向UBE2L6的siRNA后用CCK-8细胞增殖、克隆形成、Transwell迁移和侵袭实验分析UBE2L6对胰腺癌细胞生物学功能的影响。利用UBE2L6的分子相关性、蛋白相互作用及基因富集分析(GSEA)探讨其可能的作用机制。进一步利用TCGA数据库的胰腺癌临床数据,分析UBE2L6的表达与其临床病理特征的关系及临床应用价值。结果:表达差异分析提示UBE2L6在泛癌中表达增高,在胰腺癌组织及BxPC-3(t=33.82,P<0.0001)、PANC-1(t=7.36,P=0.0018)、AsPC-1(t=9.61,P=0.0007)、SW1990(t=10.26,P=0.0005)及MIA PaCa-2(t=12.65,P=0.0002)等胰腺癌细胞系中亦高表达。细胞功能实验显示,UBE2L6可促进胰腺癌PANC-1和AsPC-1细胞的增殖、迁移及侵袭。Spearman相关性分析提示BCL2A1蛋白与UBE2L6的相关性最大(r=0.442)。蛋白相互作用分析显示,其可能与HERC6、RPS27A、HERC5、UBA52、ISG15、UBA7、DDX58、UBC、UBB、ARIH1等蛋白有相互作用。同时,GSEA富集分析提示UBE2L6与肿瘤免疫微环境等密切相关。而临床病理相关性分析显示,UBE2L6高表达与胰腺癌组织病理学分级密切相关(χ^(2)=6.966,P=0.031)。单因素及多因素Cox回归分析表明,年龄及淋巴结转移是胰腺癌患者的独立危险因素(P<0.05),UBE2L6的表达水平与胰腺癌患者的预后明显相关(P<0.05),但不是其独立预后因素(P>0.05)。ROC曲线及Kaplan-Meier生存分析提示,UBE2L6对胰腺癌具有较高的临床诊断价值(AUC=0.970,95%CI=0.950~0.989)且与胰腺癌患者的预后相关(P<0.05)。结论:UBE2L6在胰腺癌中表达增高并促进胰腺癌细胞的增殖、迁移及侵袭,其分子机制可能与细胞凋亡、泛素化修饰、MAPK信号通路及肿瘤免疫微环境等有关,且UBE2L6表达水平与胰腺癌的诊断及预后密切相关。

Background and Aims:Pancreatic cancer is characterized by high metastatic potential and invasiveness,and dismal prognosis.However,Mutations in oncogenes and changes in molecular regulatory relationships can affect the malignant biological behaviors of pancreatic cancer.The expression of ubiquitin/ISG15-conjugating enzyme E2L6(UBE2L6)in pan-cancer and its biological function in pancreatic cancer remain unclear.Therefore,this study was performed to explore the effect of UBE2L6 on the proliferation,migration,and invasion abilities of pancreatic cancer and its underlying molecular mechanism through bioinformatic analysis and experimental verification.Methods:The RNA-seq data of TCGA and GTEx were downloaded from UCSC Xena,and the differential expressions of UBE2L6 in pan-cancer were analyzed using the“limma”package of R(4.0.2).The differential expression of UBE2L6 in pancreatic cancer tissue and cell lines was verified by the Gene Expression Omnibus(GEO)datasets and qRT-PCR,respectively.In pancreatic cancer cells after transfection with siRNA targeting UBE2L6,the influences of UBE2L6 on the biological functions of pancreatic cells were analyzed by CCK-8 proliferation,colony formation,Transwell migration and invasion assays.The potential action mechanism of UBE2L6 were explored by the molecular correlation,protein interaction,and gene set enrichment analysis(GSEA).Then,the clinical data of pancreatic cancer were downloaded from the TCGA database to analyze the association of UBE2L6 expression with the clinicopathologic features and its clinical application value.Results:Differential expression analysis suggested that UBE2L6 expression was up-regulated in pancancer as well as in pancreatic cancer tissue and a variety of pancreatic cancer cell lines such as BxPC-3(t=33.82,P<0.0001),PANC-1(t=7.36,P=0.0018),AsPC-1(t=9.61,P=0.0007),SW1990(t=10.26,P=0.0005),and MIA PaCa-2(t=12.65,P=0.0002).Cell function assays showed that UBE2L6 promoted the proliferation,migration,and invasion of PANC-1 and AsPC-1 cells.Spearman correlation analysis indicated that BCL2 A1 protein had the highest correlation with UBE2L6(r=0.442).The protein interaction analysis showed that it potentially interacted with HERC6,RPS27 A,HERC5,UBA52,ISG15,UBA7,DDX58,UBC,UBB,and ARIH1.Meanwhile,GSEA enrichment analysis suggested that UBE2L6 was closely related to the tumor immune microenvironment.While clinicopathologic correlation analysis showed that the high expression of UBE2L6 was closely correlated with the histopathological grade of pancreatic cancer(χ^(2)=6.966,P=0.031).Next,univariate and multivariate Cox regression analysis showed that age and lymph node metastasis were independent risk factors for pancreatic cancer patients(both P<0.05),and the expression of UBE2L6 was significantly correlated with the prognosis of pancreatic cancer patients(P<0.05),but it was not an independent prognostic factor(P>0.05).Furthermore,ROC curve and Kaplan-Meier survival analysis suggested that UBE2L6 had a high clinical diagnostic value for pancreatic cancer(AUC=0.970,95%CI=0.950-0.989)and was correlated with the prognosis of pancreatic cancer patients(P<0.05).Conclusion:UBE2L6 is highly expressed in pancreatic cancer,and it promotes the proliferation,migration,and invasion of pancreatic cancer cells.Its molecular mechanism may be related to cell apoptosis,ubiquitination,the MAPK signaling pathway,and tumor immune microenvironment.In addition,UBE2L6 is closely associated with the diagnosis and prognosis of pancreatic cancer.