胆固醇结石差异lncRNA表达基因的分析及其功能研究

Expressed analysis and functional studies of differential expressed lncRNA genes associated with cholesterol gallstone

目的探索胆固醇结石形成相关的差异表达lncRNA基因,并通过生物信息学方法分析差异lncRNA基因的生物学功能。方法将24只C57BL/6小鼠随机分为胆固醇结石组16只和空白对照组8只。胆固醇结石组小鼠饲喂致石饲料5周,空白对照组饲喂常规饲料。5周后,将胆固醇结石组小鼠随机分为模型对照组8只和熊去氧胆酸组8只。之后空白对照组饲喂常规饲料,模型对照组饲喂致石饲料,熊去氧胆酸组饲喂致石饲料并给予熊去氧胆酸灌胃。2周后处死小鼠,收集3组小鼠的肝脏组织及胆汁样本,观察胆囊大体样本并对小鼠胆汁进行脂质分析。同时对3组小鼠的肝脏组织进行lncRNA表达谱测定,并分析差异表达基因,包括Cis-/Trans-靶基因的基因本体(GO)和京都基因与基因组百科全书(KEGG)Pathway分析。结果16只胆固醇成石小鼠模型构建成功。模型对照组、熊脱氧胆酸组及空白对照组胆囊胆汁总胆固醇含量的差异有统计学意义(P<0.05),模型对照组较空白对照组和熊脱氧胆酸组明显升高(P<0.05),但熊脱氧胆酸组与空白对照组比较差异无统计学意义(P=0.59);而3组的胆囊胆汁总胆汁酸、总胆红素及直接胆红素水平比较差异均无统计学意义(P>0.05)。3组小鼠肝脏组织的差异lncRNA基因分析结果显示,模型对照组与熊脱氧胆酸组共有49种差异lncRNA基因。将差异基因分别进行GO和KEGG pathway分析后发现,模型对照组与空白对照组的差异基因富集得到88个GO term和18条pathway,熊脱氧胆酸组与空白组的差异基因富集得到205个GO term和20条pathway。结论熊脱氧胆酸对胆囊结石有治疗作用;差异表达lncRNA基因在胆固醇结石形成及熊脱氧胆酸的预防结石形成中起到重要调控作用,为进一步探讨lncRNA的具体调控机制奠定了基础。

Objective To explore the differential expressed lncRNA genes associated with formation of cholesterol gallstone, and analyze the biological functions of differential expressed lncRNA through bioinformatics. Methods A total of 24 C57BL/6 mice were randomly divided into normal control group (n=8) and lithogenic group (n=16), which were treated with chow diets and lithogenic diets respectively for 5 weeks. After 5 weeks, mice of the lithogenic group were randomly divided into model control group (n=8) and ursodeoxycholic acid treatment group (n=8). Afterwards, mice of the normal control group were still fed with chow diets, mice of the model control group were fed with lithogenic diets, mice of the ursodeoxycholic acid treatment group were fed with ursodeoxycholic acid. After 2 weeks, collected liver tissues and gallbladder bile from the three groups, and observed gallbladder gross sample and analyzed lipids component of gallbladder bile, meanwhile detected the differential expressed lncRNA and analyzed the biological functions of differential expressed lncRNA through bioinformatics, including Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) pathway analysis. Results We successfully constructed the mice model of cholesterol gallstone. Total cholesterol level of gallbladder in the model control group had significantly higher than those of the normal control group and ursodeoxycholic acid treatment group (P<0.05), yet there was no significant difference between the normal control group and ursodeoxycholic acid treatment group (P=0.59). The levels of total bile acid, total bilirubin, and direct bilirubin had no significant difference among the three groups (P>0.05). There were 49 kinds of common overlapped difference lncRNA between the ursodeoxycholic acid treatment group and the model control group through differential expression analysis of lncRNA in liver tissues of the mice in three groups. GO and KEGG path analysis were performed separately by differential expressed lncRNA, and 88 kinds of GO terms and 18 kinds of pathways were significantly enriched from the model control group and the normal control group, 205 kinds of GO terms and 20 kinds of pathways were significantly enriched from the ursodeoxycholic acid treatment group and the normal control group. Conclusions Ursodeoxycholic acid has therapeutic effect for cholesterol gallstone. Differential expressed lncRNAs play an important regulatory role in the formation of cholesterol gallstone and the prevention of gallstone formation by ursodeoxycholic acid treatment, which further lay the foundation in discussing specific mechanism regulated by lncRNA.