基于小鼠肝细胞癌全转录组测序的Cbx3基因上调表达机制分析

Mechanism Analysis of Cbx3 Gene Upregulated Expression Based on Whole Transcriptome Sequencing in Mouse Hepatocellular Carcinoma

为了探讨染色质盒蛋白3(chromobox 3,Cbx3)基因在肝细胞癌(hepatocellular carcinoma,HCC)中上调表达的可能机制,通过构建小鼠HCC模型,分别取30 d的瘤体组织和癌旁组织及接种0、3、30 d未成瘤小鼠的肝组织,通过全转录组测序和生物信息学手段分析Cbx3基因在瘤体组织及各对照组织中的表达,并进一步筛选与Cbx3或其编码蛋白有蛋白-蛋白互作(protein-protein interaction,PPI)、竞争性内源RNA(competing endogenous RNA,ceRNA)和靶标关系的差异(q<0.05,n=3,下同)表达基因[以下将Cbx3编码蛋白PPI涉及的蛋白的编码基因称Cbx3相关基因(Cbx3-associated gene,CAG)],并分析其功能。结果显示,与对照组相比,瘤体组织中Cbx3上调表达,且存在4个Cbx3转录本及116个CAG。其中转录本NM_007624.3尽管在各组织中皆有大量表达,但在瘤体组织的表达远高于各对照组织的。GO功能富集分析结果显示,Cbx3基因及CAG主要存在于细胞核中,共同参与HCC小鼠中表观遗传学的基因表达正调控。同时,存在mmu-miR-744-5p、mmu-miR-125b-2-3p、mmu-miR-99a-3p和mmu-miR-151-5p等4种靶向于Cbx3基因的小RNA(microRNA,miRNA),其在瘤体组织中的表达显著低于各对照组织的。另外,在瘤体组织的表达远高于各对照组织的,包括2种长链非编码RNA(long non-coding RNA,lncRNA)在内的31种分子与Cbx3基因互为ceRNA。研究结果表明,Cbx3基因的上调表达主要是转录本NM_007624.3的上调表达;Cbx3在HCC小鼠中的功能主要是参与表观遗传学的基因表达正调控;对照组织中大量表达从而抑制Cbx3表达的mmu-miR-744-5p、mmu-miR-125b-2-3p、mmu-miR-99a-3p和mmu-miR-151-5p等4种miRNA,由于在瘤体组织中的低表达而减弱了抑制效应进而促发Cbx3在瘤体组织中上调表达;多种分子通过ceRNA机制与Cbx3基因竞争性结合以上4种miRNA而进一步促进了HCC小鼠瘤体组织中Cbx3基因的表达。

To explore the possible mechanism of upregulated expression of chromobox 3(Cbx3)in hepatocellular carcinoma(HCC),the mouse HCC models were constructed.Both the tumor tissues and the paracancer tissues of 30 d,and the non-tumorous liver tissues of 0 d,3 d,and 30 d were taken respectively.Whole transcriptome sequencing was performed,and bioinformatics was used to analyze the expression of Cbx3 gene in tumors and diversity control tissues.Then we identified differentially expressed genes in protein-protein interaction(PPI),competing endogenous RNA(ceRNA),and target correlations with Cbx3 genes(q<0.05,n=3)(the gene involved in PPI belonged to Cbx3-associated gene,CAG),and analyzed their functions.The results showed that,compared with the controls,expression of Cbx3 gene was upregulated in HCC tissues(q<0.05,n=3),and there were 4 differentially expressed transcripts(q<0.05,n=3)of Cbx3 gene and 116 CAG in the HCC mice.Although the transcript NM_007624.3 had a large amount of expression in various tissues,its expression in the tumor tissues was much higher than that in the controls.The GO analysis showed that the Cbx3 gene and CAG mainly existed in the nucleus and involved in the positively epigenetic regulation of gene expression in the HCC mice.At the same time,there were 4 Cbx3-targeted microRNA(miRNA),including mmu-miR-744-5 p,mmu-miR-125-2-3 p,mmu-miR-99 a-3 p,and mmu-miR-151-5 p,their expression in the tumor tissues was significantly lower than that in the controls.In addition,31 molecules including 2 long non-coding RNA(lncRNA)with Cbx3 gene were ceRNA,which were higher expressed in the tumor tissues than that in the controls.The results indicated that the upregulated expression of Cbx3 gene was mainly that of the transcript NM_007624.3,its function in the HCC mice was mainly involved in the positively epigenetic regulation of gene expression.Especially,the 4 miRNAs,mmu-miR-744-5 p,mmu-miR-125 b-2-3 p,mmu-miR-99 a-3 p and mmu-miR-151-5 p,which were expressed in large quantities in the controls,inhibited the expression of Cbx3 gene since the low expression of them in the tumor tissues weakened the inhibitory effect and promoted the upregulated expression of Cbx3 gene in the tumor tissues.Last but not least,a variety of molecules competed with the Cbx3 gene to combine with the 4 miRNAs mentioned above through ceRNA mechanism to further promote the expression of Cbx3 gene in the tumor tissues in the HCC mice.