基于全转录组测序的小鼠HCC中miR-122-5p和miR-27b-3p调控Top2a异常表达的机制分析

Mechanism analysis of miR-122-5p and miR-27b-3p regulating Top2a abnormal expression in rats with HCC based on whole transcriptome sequencing

目的基于全转录组测序探讨小鼠肝细胞癌(hepatocellular carcinoma,HCC)组织中miR-122-5p和miR-27b-3p调控DNA拓扑异构酶2-a(DNA topoisomerase 2-alpha,Top2a)异常表达的可能调控机制。方法通过构建小鼠HCC模型、实时荧光定量PCR(RT-qPCR)和全转录组测序等生物信息学手段,以及结合The Human Protein Atlas(人类蛋白质图谱数据库),分析Top2a在瘤体组织和正常肝组织中的差异表达,并进一步筛选与Top2a或其编码蛋白有PPI和靶标关系的差异(q<0.05,q为校正的P)表达基因和miRNA,分析其功能。构建Top2a编码蛋白的蛋白质-蛋白质互作图(PPI)和分析Top2a及相关基因参与的生物学功能。结果与对照组相比,瘤体组织中Top2a差异过表达(P<0.05),且存在87个与Top2a编码蛋白互作、差异表达的蛋白编码基因。GO功能富集显示,Top2a及相关基因主要存在于细胞核,且多数具有蛋白结合和DNA结合活性,参与RNA聚合酶Ⅱ启动子转录的正向调节、细胞对DNA损伤刺激的反应和染色体分离等生物学过程。同时,存在miR-27b-3p和miR-122-5p这2种靶向于Top2a的miRNA在瘤体组织中表达显著低于对照组织。Top2a基因在肝癌组织中RNA和蛋白质表达水平均高于正常肝组织,此外,Top2a基因低表达的肝癌患者的生存率明显高于高表达的患者。结论Top2a基因在HCC中过表达是其细胞抑制了miR-122-5p和miR-27b-3p表达的结果。而Top2a在细胞核内通过蛋白结合和DNA结合等活性,影响转录起始、DNA损伤刺激反应和染色体分离等生物学过程,则是其在HCC中发挥作用的分子机制。Top2a基因可能是预测肝癌预后的重要生物标志物,有一定的临床诊断价值。

Objective To explore the potential mechanisms of miR-122-5p and miR-27b-3p in regulating the abnormal expression of DNA topoisomerase 2-alpha(Top2a)in hepatocellular carcinoma(HCC)tissues of mice using whole transcriptome sequencing.Methods By constructing a mouse HCC model and employing real-time fluorescence quantitative PCR(RT-qPCR)and whole transcriptome sequencing,combined with the analysis of The Human Protein Atlas database,differential expression of Top2a in tumor tissues and normal liver tissues was analyzed.Differentially expressed genes and miRNAs with protein-protein interaction(PPI)and target relationships with Top2a or its encoded proteins(q<0.05,q is the corrected P value)were further screened,and their functions were analyzed.A protein-protein interaction(PPI)network of Top2a-encoded protein was constructed,and the biological functions of Top2a and its related genes were analyzed.Results Compared with the control group,Top2a showed overexpression in tumor tissues(P<0.05),and 87 protein-coding genes that interacted with Top2a and exhibited differential expression were identified.GO functional enrichment analysis showed that Top2a and its related genes were mainly located in the nucleus and had protein binding and DNA binding activities,participating in biological processes such as positive regulation of RNA polymeraseⅡpromoter transcription,cellular response to DNA damage stimulation,and chromosome segregation.Moreover,in tumor tissues,the expression of miR-27b-3p and miR-122-5p,which target Top2a,was significantly lower than in control tissues.The RNA and protein expression levels of the Top2a gene were higher in liver cancer tissues than in normal liver tissues.Additionally,HCC patients with low expression of the Top2a gene exhibited significantly higher survival rates compared to those with high expression.Conclusion The overexpression of the Top2a gene in HCC is a result of cellular inhibition of miR-122-5p and miR-27b-3p expression.Top2a affects biological processes such as transcription initiation,cellular response to DNA damage stimulation,and chromosome segregation through its activities of protein binding and DNA binding in the nucleus,which represents the molecular mechanism of its role in HCC.The Top2a gene may serve as an important biomarker for predicting the prognosis of HCC and has clinical diagnostic value to a certain extent.