大鼠肝再生启动阶段肝细胞CCAAT增强子结合蛋白α mRNA、微小RNA-144-3p和3种环状RNA的表达和作用

Expression and role of CCAAT enhancer binding protein α mRNA,microRNA-144-3p and three kinds of circular RNAs of hepatocytes during the rat liver regeneration initiation

目的了解大鼠肝再生启动阶段CCAAT增强子结合蛋白α(CEBPα)mRNA、微小RNA(miR)-144-3p、rno-LOC100365958_0001、rno-Usp3_0010和rno-AC241873_0010调节肝细胞处于G_(0)期还是G_(1)期的途径和方法。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,用大规模定量分析技术检测大鼠肝再生中肝细胞竞争性内源RNA(ceRNA)表达的变化,用Cytoscape 3.2软件构建ceRNA的相互作用网络,用ceRNA综合分析等方法解析它们表达的相关性和作用相关性。结果PH后0 h和6 h时,CEBPαmRNA的比值为4.88±0.78和1.12±0.27,miR-144-3p为1.30±0.11和2.97±0.81,rno-LOC100365958_0001为6.99±0.55和0.16±0.05,rnoUsp3_0010为8.48±0.67和0.13±0.01,rno-AC241873_0010为11.85±0.93和0.09±0.02。CEBPα促进的G_(0)期相关基因细胞周期蛋白依赖性激酶2相关蛋白2(CDK2AP2)为2.55±0.42和0.74±0.11,G_(0)/G_(1)开关2(G_(0)S2)为3.85±0.23和0.64±0.08,信号转导子和转录激活因子1(STAT1)为2.57±0.13和1.32±0.13,转化生长因子β受体2(TGFBR2)为2.77±0.20和0.79±0.13,抑制的G_(0)期相关基因细胞周期蛋白依赖性激酸酶抑制剂1a(CDKN1a)为0.39±0.07和0.93±0.15。CEBPα促进的G_(1)期相关基因细胞周期蛋白G2(CCNG2)为1.19±0.09和0.25±0.06,抑制的G_(1)期相关基因ETS变异转录因子6(ETV6)为0.77±0.05和2.22±0.68,血红蛋白加氧酶1(HMOX1)为1.05±0.21和4.57±0.88,MAPK14为1.01±0.15和2.01±0.32,STAT3为0.74±0.15和2.88±0.24,肿瘤坏死因子(TNF)为0.80±0.14和2.29±0.51。结论PH后0 h时,CEBPαmRNA上调,有利于CEBPα促进的G_(0)期相关基因表达和肝细胞处于G_(0)期。相反,PH后6 h时,rno-LOC100365958_0001、rno-Usp3_0010、rno-AC241873_0010和miR-144-3p的相互作用加强了后者对CEBPαmRNA的抑制,不利于CEBPα形成,有利于CEBPα抑制的G_(1)期相关基因表达和肝细胞处于G_(1)期。

Objective To explore the pathways and patterns which CCAAT enhancer binding proteinα(CEBPα)mRNA,miR-144-3 p,rno-LOC100365958_0001,rno-Usp3_0010 and rno-AC241873_0010 regulate the hepatocytes in G_(0) phase and G_(1) phase during rat liver regeneration(LR).Methods The rat 2/3 hepatectomy(partial hepatectomy,PH)model was prepared as described by Higgins,the hepatocytes of rat liver right lobes were isolated in 9∶00-11∶00 am according to the method of Smedsrod,the large-scale quantitative detection of competitive endogenous RNAs(ceRNAs)was processed by the high-throughput biotechnology,the interaction network of ceRNAs was constructed by Cytoscape 3.2 software,and the correlation in expression and role of ceRNAs was analyzed by ceRNA comprehensive analysis.Results It was found that at 0 hour and 6 hours after PH,the ratio value of CEBPαmRNA showed 4.88±0.78 and 1.12±0.27,miR-144-3 p displays 1.30±0.11 and 2.97±0.81,rno-LOC100365958_0001 indicateed 6.99±0.55 and 0.16±0.05,rnoUsp3_0010 shows 8.48±0.67 and 0.13±0.01,rno-AC241873_0010 indicates 11.85±0.93 and 0.09±0.02.The G_(0) phase-related genes promoted by CEBPαwere following,cyclin dependent kinase 2 associated protein 2(CDK2 AP2)2.55±0.42 and 0.74±0.11,G_(0)/G_(1) swich 2(G_(0)S2)3.85±0.23 and 0.64±0.08,the signal transducer and activator of transcription 1(STAT1)2.57±0.13 and 1.32±0.13,transforming growth factor beta receptor 2(TGFBR2)2.77±0.20 and 0.79±0.13,but the G_(0) phase-related gene cyclin dependent kinase inhibitor 1 a(CDKN1a)inbibited by CEBPα0.39±0.07 and 0.93±0.15.The G_(1) phase-related gene cyclin G2(CCNG2)promoted by CEBPα1.19±0.09 and 0.25±0.06,but the G_(1) phase-related genes inbibited by CEBPαwere following,ETS variant transcription factor 6(ETV6)0.77±0.05 and 2.22±0.68,hemoglobin oxygenase 1(HMOX1)1.05±0.21 and 4.57±0.88,MAPK141.01±0.15 and 2.01±0.32,STAT30.74±0.15 and 2.88±0.24,tumor necrosis factor(TNF)0.80±0.14 and 2.29±0.51.Conclusion CEBPαmRNA is up-regulated at 0 h after PH,that is helpful for the expression of the G_(0) phase-related genes promoted by CEBPαand for the hepatocytes to be in G_(0) phase.On the contrary,the interaction of miR-144-3 p and rno-LOC100365958_0001,rno-Usp3_0010 and rno-AC241873_0010 leads to CEBPαmRNA to bind with miR-144-3 p,to CEBPαbeing formed difficultly,and to the expression of the G_(1) phase-related genes inbibited by CEBPαprobablely,and to the hepatocytes being in G_(1) phase at 6 hours after PH.