卡波氏肉瘤组织中差异表达miRNA靶基因预测及生物信息学分析

Prediction and Bioinformatic Analysis of Differentially Expressed miRNA Target Genes in Kaposi’s Sarcoma

卡波氏肉瘤(Kaposi sarcoma,KS)在全球范围内是人免疫缺陷病毒感染者第二易患的肿瘤。本研究旨在确定KS中差异表达的关键微小RNA(microRNA,miRNA)及其靶基因和相关途径,为深入了解该疾病的分子机制提供理论基础。本研究通过测序获得KS组织及瘤旁组织中的差异miRNA,借助starBase数据库预测差异表达miRNA的靶基因,同时用基因本体论(Gene Ontology,GO)数据库、京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)、Reactome通路富集和转录因子注释分析差异miRNA的靶基因,最后用实时荧光定量PCR(Quantitative Real⁃time PCR,qRT⁃PCR)验证下调最明显的三个miRNA。结果显示,上、下调差异miRNA分别为39个和67个。GO和KEGG富集分析显示,上调的差异miRNA靶基因主要富集在组蛋白修饰、核染色质、DNA−结合转录激活因子活性等生物学功能,主要通过cAMP信号通路和长时程增效等通路参与KS的疾病进程;下调的差异miRNA主要与组蛋白修饰、细胞前沿和蛋白丝氨酸/苏氨酸激酶的活性有关,主要通过FoxO及Hippo等信号通路参与KS的疾病进程。Reactome富集显示,差异miRNA多集中在受体酪氨酸激酶信号、信号转导疾病和PI3K⁃Akt等信号通路。转录因子注释结果显示,肿瘤蛋白P53(Tumor Protein P53,TP53)、叉头框转录因子O亚族1(Forkhead Box Protein O1,FOXO1)和叉头框转录因子O亚族3(Forkhead Box Protein O3,FOXO3)等转录因子可能和KS的发生发展密切相关。qRT⁃PCR结果显示,测序中下调最明显的三个miRNA在KSHV感染细胞中均显著下调。总之,本研究筛选出了KS组织中差异表达的miRNA,对其靶基因进行了一系列生物信息学分析,并初步发现miR⁃651⁃3p,miR⁃223⁃3p及miR⁃186⁃5p通过Hippo、FoxO及PI3K⁃AKT信号通路可能在KS的疾病进程中发挥重要作用。这些研究均为探寻KS的发病机制和靶向干预提供了有力的理论基础。

Worldwide,Kaposi's sarcoma(KS)is the second most common tumor in patients infected with the human immunodeficiency virus.We aimed to determine the differentially expressed microRNAs(miRNAs),their target genes,and related pathways in KS.In this way,we wished to provide a theoretical basis for further understanding of the molecular mechanism of KS.Differentially expressed miRNAs in KS tissues and adjacent tissues were obtained by sequencing technology.The target genes of differentially expressed miRNAs were predicted by the starBase database.Simultaneously,enrichment of signaling pathways and transcription⁃factor annotation using the Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG),and Reactome databases were undertaken to analyze the target genes of differentially expressed miRNAs.Finally,real⁃time reverse transcription⁃quantitative polymerase chain reaction(RT⁃qPCR)was done to verify the three most significantly downregulated miRNAs.We discovered that 39 miRNAs had upregulated expression and 67 had downregulated expression.Enrichment analysis using GO and KEGG databases showed that the target genes of miRNAs with upregulated expression were mainly enriched in biological functions such as histone modification,nuclear chromatin,DNA⁃binding transcription activator activity,and mainly involved in the disease process of KS through the cAMP signaling pathway and long⁃term potentiation.The target genes of miRNAs with downregulated expression were mainly related to histone modification,cell leading edge and protein serine/threonine kinase activity,and were involved primarily in the disease process of KS through FOXO and Hippo signaling pathways.Enrichment analysis using the Reactome database showed that differentially expressed miRNAs were mostly concentrated in signaling by receptor tyrosine kinase,diseases of signal transduction and PI3K⁃Akt signaling pathway.The results of transcription⁃factor annotation showed that transcription factors such as tumor protein P53,forkhead box protein O1(FOXO1)and FOXO3 may be closely related to the occurrence and development of KS.RT⁃qPCR showed that three miRNAs with the most downregulation were verified in KSHV⁃infected cells.In conclusion,we screened the differentially expressed miRNAs in KS tissue,carried out a series of bioinformatics analysis of their target genes,and found that miR⁃651⁃3p,miR⁃223⁃3p and miR⁃186⁃5p may have important roles in the disease process of KS through Hippo,FOXO and PI3K⁃Akt signaling pathways.This study provides a strong theoretical basis for exploring the pathogenesis and targeted intervention of KS.