异常寡核苷酸结合折叠域蛋白基因对肝细胞癌DNA复制起始的影响

Effect of abnormal oligonucleotide binding folding domain protein gene on the initiation of DNA replication in hepatocellular carcinoma

目的关于异常寡核苷酸结合折叠域蛋白基因(OBGs)通过微小染色体维持(MCM)复合物影响肝细胞癌DNA复制起始的报道较少。文中旨在探讨逆转录相关基因(RTGs)在肝细胞癌中的作用及可变剪接和单核苷酸位点变异(SNV)与基因表达异常的相关性。方法选取150只洁净级昆明小鼠,采用随机数字表法取100只,双前肢腋下注射对数生长期肝细胞癌细胞株H22等渗盐水悬液作为H22组,余50只注射0.2 mL不含H22的等渗盐水作为对照组。从H22组小鼠中选出成瘤小鼠,解剖取出瘤体,从对照组小鼠中取健康肝组织。提取H22组和对照组总RNA,分析差异表达基因。筛选差异表达的逆转录相关DEGs(RDEGs),对RDEGs进行GO和KEGG分析。对RDEGs编码蛋白进行互作分析,对RDEGs多态性与基因表达进行相关性分析。结果肝细胞癌中有193个差异表达的RTGs,共参与2个生物学程序、3个细胞组分、1个分子功能、3个信号通路和3个功能部位;其功能主要集中在DNA复制,尤其复制起始中OBGs参与的MCM复合体及端粒复合体构建。可变剪接分析结果显示,4个RDEGs在基因的3个位点发生了差异表达的可变剪接,且可变剪接与相应基因的表达呈正相关。SNV和INDEL分析显示肝细胞癌组织中共有157个RDEGs,发生1541个SNV和78个INDEL位点的改变;上述基因位点的改变主要发生在基因的6个部位,即外显子区、内含子区、基因间区、基因下游区、基因上游区和拼接区;共有28个基因的SNV改变差异有统计学意义(P<0.05)。基因位点多态性分析结果显示,5个OBGs在肝细胞癌中出现了SNV现象,而健康肝组织中则没有SNV。结论RTGs中的OBGs可能在肝细胞癌中通过自身的基因多态性突变引起MCM复合体及端粒复合体的改变,从而调控DNA复制起始及保护端粒的完整性,进而在癌细胞的增殖过程中发挥重要作用。可变剪接及SNV则可能是一些基因表达的重要调控因素。

Objective There are few reports about abnormal oligonucleotide binding fold domain protein genes(OBGs)affecting the initiation of DNA replication in hepatocellular carcinoma through the microchromosome maintenance(MCM)complex.This study aims to explore the roles of reverse-transcription-related genes(RTGs)in Hepatocellular Carcinoma cells(HCC)and the correlation between gene polymorphisms and abnormal gene expression.Methods We created a mouse model by injecting hepatocellular carcinoma cell line H22(logarithmic growth phase)and dissected the tumor bodies from tumor-forming mice.The control group was treated by isotonic saline without H22.The healthy liver tissue cells were taken from the control mice.The total RNA of the H22 group and control group were extracted,and differentially expressed genes were analyzed.Screening of differentially expressed reverse transcription-related DEGs(RDEGs),GO and KEGG analysis of RDEGs.The interaction analysis of RDEGs encoded proteins,and the correlation analysis of RDEGs polymorphism and gene expression.Results There were 193 differentially expressed RTGs in HCCs,which were involved in two biological procedures,three cell components,one molecular function,three signal pathways,and three functional sites;Its function is mainly concentrated in DNA replication,especially the construction of MCM complex and telomere complex in which OBGs participate in the initiation of replication.Most related genes had OB fold domains.The results also showed that both AS and SNV caused gene polymorphism was positively correlated with gene expression,and most OBGs in HCC had SNV phenomenon,but not occurred in healthy liver tissue.Conclusion Collectively,AS and SNV may be important regulatory factors for gene expression.SNV may particularly affect the function of OBGs in the MCM complex to abnormally initiate DNA replication in HCC.