大鼠肝再生的肝细胞周期启动及终止中骨髓瘤基因mRNA与其他非编码RNA的表达变化与作用

Expression change and role of myeloma cancer gene mRNA and the non⁃coding RNA in the hepatocyte cycle initiation and termination during the rat liver regeneration

目的 了解大鼠肝再生(LR)0 h、6 h和72 h时骨髓瘤基因(MYC)及其mRNA相互作用的微小RNA(miRNA)和环状RNA(circRNA)调节肝细胞周期启动及终止的途径与方式。方法 按Higgins等方法制备大鼠2/3肝切除(PH)模型,按Smedsrod等方法分离肝细胞,用大规模定量检测技术测定大鼠肝再生中肝细胞的mRNA、miRNA和circRNA[合称内源竞争RNA(ceRNA)]表达变化,用Cytoscape 3.2软件构建ceRNA的相互作用网,用ceRNA综合分析等方法解析它们的表达相关性和作用相关性。结果 在PH后肝细胞周期启动0~6 h间的0 h和6 h时,MYC mRNA的比值为0.15±0.03和2.36±0.2,miR-134-5p为3.22±0.61和0.08±0.02,circRNA_12112为0.68±0.21和13.35±3.53。同时,PH后0 h时,MYC促进的细胞周期启动相关基因Ras关联域家族成员1(RASSF1)、细胞周期蛋白依赖性激酶2(CDK2)、超氧化物歧化酶2(SOD2)表达下调,抑制的细胞周期启动相关基因嵌套蛋白(NES)、雷达21黏附复合物成分(RAD21)、CUE域包含2(CUEDC2)未发生有意义表达变化。PH后6 h时,MYC促进的细胞周期启动相关基因SOD2表达上调,抑制的细胞周期启动相关基因NES、RAD21、CUEDC2表达下调。相反,在细胞周期终止,即PH后72 h时,MYC mRNA的比值为0.30±0.10,miR-880-3p为0.54±0.01,circRNA_09599为0.54±0.16。同时,MYC促进的细胞周期终止相关基因肝细胞生长因子(HGF)表达上调,抑制的细胞周期终止相关基因MET原癌基因受体酪氨酸激酶(MET)和细胞周期蛋白依赖性激酶抑制剂1A(CDKN1A)表达下调。结论 上述circRNA抑制的miRNA、miRNA抑制的MYC mRNA以及MYC调节的细胞周期启动及终止相关基因的表达相关性和作用相关性有利于PH后6 h时肝细胞处于细胞周期启动状态和PH后72 h时肝细胞处于细胞周期终止状态。

Objective To explore the role pathway and pattern of the myeloma cancer gene(MYC)and its mRNA interaction with the microRNAs(miRNAs)and circular RNA(circRNAs)at hour 0,hour 6 and hour 72 in the rat liver regeneration.Methods The rat 2/3 hepatectomy(PH)model was prepared as described by Higgins,the hepatocytes were isolated according to the method of Smedsrod et al.The expression changes of mRNA,miRNA and circRNA[together named as competing endogenous RNA(ceRNA)]were detected by the large⁃scale quantitative detection technology,the interaction network of ceRNA was constructed by Cytoscape 3.2 software,and their correlation in expression and role were analyzed by ceRNA comprehensive analysis.Results It was found that at hour 0 and hour 6 after PH,the ratio value of MYC mRNA showed 0.15±0.03 and 2.36±0.20,miR⁃134⁃5p indicated 3.22±0.61 and 0.08±0.02,circRNA_12112 displayed 0.68±0.21 and 13.35±3.53.At the same time,the cell cycle initiation⁃related genes ras association domain family member 1(RASSF1),cyclin dependent kinase 2(CDK2),superoxide dismutase 2(SOD2),which were promoted in expression by MYC,were down⁃regulated at hour 0 after PH,but the cell cycle initiation⁃related genes nestin(NES),RAD21 cohesin complex component(RAD21),CUE domain containing 2(CUEDC2),which are inhibieted in expression by MYC,had no meaningful express changes at hour 0 after PH.On the other hand,the cell cycle initiation⁃related gene SOD2,which was promoted in expression by MYC,was up⁃regulated at hour 6 after PH,but the cell cycle initiation⁃related genes NES,RAD21,CUEDC2,which are inhibieted in expression by MYC,were down⁃regulated at hour 6 after PH.In contrary,at hour 72 after PH,the ratio value of MYC mRNA showed 2.36±0.20,miR⁃880⁃3p indicated 0.54±0.01,circRNA_09599 displayd 0.54±0.16.At the same time,the cell cycle termination⁃related gene hepatocyte growth factor(HGF),which is promoted in expression by MYC,was up⁃regulated 72 hours after PH,the cell cycle termination⁃related genes MET proto⁃oncogene receptor tyrosine kinase(MET)and cyclin dependent kinase inhibitor 1A(CDKN1A),which are inhibieted in expression by MYC,were down⁃regulated 72 hours after PH.Conclusion The correlation in expression and role of the miRNAs,which are inhibited by circRNAs,MYC,its mRNA is inhibited by miRNAs,and the cell cycle initiation⁃related and cell cycle termination⁃related genes,which are regulated by MYC,are helpful for the hepatocyte to be in cell cycle initiation state at hour 6 after PH and to be in cell cycle termination state at hour 72 after PH.