三阴性乳腺癌与癌旁组织中差异表达环状RNA筛选及生物信息学分析

doi:10.3969/j.issn.1005-6483.2021.03.008

摘要/Abstract

摘要： 目的筛选三阴性乳腺癌（TNBC）与癌旁组织中差异表达的环状RNA（circRNA），并对其下游可能结合的miRNA及靶基因进行生物信息学分析，预测circRNA在TNBC中可能的调控途径。
方法收集到1例于北京大学深圳医院甲状腺与乳腺外科接受TNBC手术治疗的病人手术样本，行转录组测序分析，并获得TNBC癌组织与癌旁组织的circRNA差异表达谱，分别选择表达上调、下调最显著的5个共10个circRNA。使用RegRNA 2.0网站预测circRNA可能结合的下游miRNA，进一步通过三个生物信息学网站（miRDB、TargetScan、RNAInter）联合预测miRNA可能结合的下游的靶基因，并对所得mRNA进行基因本体（GO）和京都基因与基因组百科全书（KEGG）分析。应用STRING在线工具分析预测出的靶基因PPI网络，将数据输出并导入Cytoscape软件，通过MCODE插件筛选出circRNA相关的TNBC的关键基因。用Kaplan-Meier Plotter在线数据库，分析关键基因表达水平与TNBC预后的关系。
结果 TNBC与癌旁组织中差异表达的circRNA共6547个，其中表达上调2311个，下调4326个；表达显著上调及下调的5个circRNA下游共预测出1161个mRNA；GO分析及KEGG分析结果显示，这些mRNA主要参与RNA聚合酶Ⅱ启动子的转录负调控、染色质的共价修饰、转录调控等生物学进程；细胞信号通路方面主要参与到细胞内吞、缩宫素信号通路、Wnt信号通路及cGMP-PKG信号通路等；共筛选出5个关键基因（UBOX5、UBE2G2、DTX3L、FZR1、RNF19A），其中UBE2G2和DTX3L低表达提示TNBC病人总生存率不良；UBE2G2对应的上游miRNA和circRNA分别是hsa_miR-93-3p和hsa_circ_0001361；DTX3L对应的上游miRNA和circRNA分别是hsa_miR-2115-5p和hsa_circ_0002874。
结论 TNBC细胞中差异表达circRNA所结合miRNA的下游靶基因中，可能的关键基因是UBOX5、UBE2G2、DTX3L、FZR1、RNF19A，其中UBE2G2、DTX3L与病人预后有关，它们对应的调控轴分别为hsa_circ_0001361/miR-93-3p/ UBE2G2和hsa_circ_0002874/miR-2115-5p/DTX3L，提示二者可能参与到TNBC的发生发展。

关键词: 三阴性乳腺癌, 环状RNA, 生物信息学

Abstract: Objective Screening the differential expression of circular RNA (circRNA) in the triple negative breast cancer (TNBC) with its tumor-adjacent tissues and to analyze the potentially downstream miRNAs and target genes associated circRNA candidates by bioinformatics analysis,then predict the potential regulatory pathways of circRNA in TNBC.
Methods One paired triple negative breast cancer sample and adjacent non-cancerous tissues for RNA-Seq was collected from a patient received surgery at department of the thyroid and galactophore in Peking University Shenzhen Hospital.We obtained differential expression of circRNA and a total of ten circRNAs the five most significant up-regulated and down-regulated circRNA were selected.The RegRNA 2.0 website was used to predict the potential miRNAs corresponding to circRNA candidates,and three bioinformatics websites (miRDB,TargetScan,and RNAInter) were further utilized to predict the downstream mRNA of miRNAs.Gene ontology (GO) and Kyoto Encylopedia of Genes and Genomes (KEGG) analysis were performed for these target genes.The protein-protein interaction (PPI) network of the predicted target genes was analyzed by STRING online tools,and the data were exported before we imported them into the Cytoscape software.The key genes were screened out by MCODE plug-ins.Kaplan-Meier Plotter online database was used to analyze the relationship between the expression level of key genes and the prognosis of TNBC.
Results There were 6547 differentially expressed circRNAs in the triple negative breast cancer tissues with its tumor-adjacent tissues,2311 of which were up-regulated and 4326 were down-regulated；a total of 1161 mRNAs were predicted in the downstream of 5 up-regulated circRNAs and 5 down-regulated circRNAs most significantly;the results of GO and KEGG analysis showed that these mRNA were mainly involved in negative regulation of transcription from RNA polymerase Ⅱ promoter,covalent chromatin modification,regulation of transcription and they are mainly involved in endocytosis,oxytocin signaling pathway,Wnt signaling pathway and cGMP-PKG signaling pathway;five key genes were screened out (UBOX5,UBE2G2,DTX3L,FZR1,RNF19A) among which the low expression of UBE2G2 and DTX3L indicated the poor overall survival of triple negative breast cancer patients;the corresponding miRNA and circRNA for UBE2G2 are hsa_miR-93-3p and hsa_circ_0001361,and hsa_miR-2115-5p and hsa_circ_0002874 for DTX3L respectively.
Conclusion Among the downstream target genes of miRNA probably binding to circRNA candidates expressed differentially in triple negative breast cancer,the key genes are may be UBOX5,UBE2G2,DTX3L,FZR1 and RNF19A.UBE2G2 and DTX3L are related to the prognosis of TNBC patients,and their corresponding regulatory axes in TNBC are hsa_circ_0001361/miR-93-3p/ UBE2G2 and hsa_circ_0002874/miR-2115-5p/DTX3L respectively,which suggest that these two axes may participate in the occurrence and development of triple negative breast cancer.

Key words: triple negative breast cancer, circRNA, bioinformatics

戴嘉婧韩馨乐钟福波林哲广江淑琪韦伟方征宇. 三阴性乳腺癌与癌旁组织中差异表达环状RNA筛选及生物信息学分析[J]. 临床外科杂志, 2021, 29(3): 227-230.

DAI Jiajing, HAN Xinle, ZHONG Fubo, et al.. Screening and bioinformatics of differential expression of circular RNAs in triple negative breast cancer[J]. JOURNAL OF CLINICAL SURGERY, 2021, 29(3): 227-230.

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