【作者】 王星果；

【导师】 龚道清； 顾志良；

【作者基本信息】 扬州大学 ， 动物遗传育种与繁殖， 2013， 博士

【摘要】 肝脏代谢和骨骼肌发育是鸡十分重要的两个生物过程,microRNA(miRNA)是一类小的非编码的约22个核苷酸长度的单链RNA分子,是基因表达的重要调控因子,其对肝脏代谢和骨骼肌发育发挥了重要的作用。本研究以鸡肝脏和骨骼肌miRNA为研究对象,对其进行了一系列的表达和功能研究,主要研究结果如下：(1)运用实时荧光定量PCR检测miR-122的组织表达,发现其在肝脏中表达量最高。运用靶基因预测软件预测出364个miR-122的靶基因,挑选其中与肝脏代谢相关的预测靶基因P4HA1、PKM2、TGF-β3、FABP5和ARCN1。实时荧光定量PCR显示miR-122在肝脏生长发育过程中上调,在鸡肝细胞离体培养过程中下调,5个基因在肝脏生长发育过程中表达不受影响,P4HA1、TGF-β3和ARCN1在肝细胞离体过程中下调,而FABP5在离体和培养过程中分别下调和上调,PKM2不受影响。体外双荧光素酶报告系统和靶位点突变实验证明5个基因及预测靶位点分别是miR-122的靶基因和靶位点,通过鸡细胞系过表达miR-122证明其对TGF-β3的调控在mRNA水平,而对P4HA1、PKM2和ARCN1的调控不在mRNA水平,表明miR-122可能通过调控这几个基因在鸡肝脏代谢中发挥调控作用。(2)原代鸡肝细胞抑制niR-122后通过转录组测序发现共有123个基因差异表达,其中64个基因表达显著上调,59个基因表达显著下调。GO分析显示21个基因参与鸡肝脏代谢过程,表明miR-122可能通过影响这些基因而调控鸡肝脏代谢。通过比较测序及实时荧光定量PCR结果发现miR-122抑制后靶基因P4HA1、PKM2、TGF-β3上调,FABP5下调,而ARCN1不受影响,Western Blotting分析表明靶基因P4HA1蛋白上调,进一步说明miR-122可调控这些靶基因。(3)使用RNA干扰技术干扰原代鸡肝细胞中miR-122靶基因TGF-β3后发现TGF-β通路下游基因ACTA2表达上调,LEF1和CCNB2表达下调,表明miR-122通过调控TGF-β3可能影响这些基因进而调控肝脏代谢。(4)运用实时荧光定量PCR检测miR-126的组织表达,发现其在肝脏中表达量很少。运用靶基因预测软件预测出54个miR-126的靶基因,挑选其中与肝脏代谢相关的预测靶基因Spredl。实时荧光定量PCR显示miR-126在肝脏生长发育过程中上调,在鸡肝细胞离体培养过程中下调,Spred1表达不受影响。体外双荧光素酶报告系统和靶位点突变实验证明Spred1及预测靶位点分别是miR-126的靶基因和靶位点,通过鸡细胞系过表达miR-126证明其对Spredl的调控不是在mRNA水平,表明miR-126可能通过调控Spredl在鸡肝脏代谢中发挥调控作用。(5)使用鸡生长激素cGH处理原代鸡肝细胞,运用高通量测序技术对其miRNA进行测序,共鉴定出216个鸡miRNA,其中受cGH影响的miRNA有56个,29个表达显著上调,27个表达显著下调,这些miRNA可能参与鸡肝脏代谢过程。(6)运用实时荧光定量PCR检测miR-1a的组织表达,发现其在骨骼肌中表达量很高。运用靶基因预测软件预测出247个miR-1a的靶基因,挑选其中与肌肉发育相关的预测靶基因ACVR1和Spred1,还有一个之前预测是miR-133a的靶基因后来验证其是miR-1a的靶基因CNBP。实时荧光定量PCR显示miR-1a在腿肌生长发育过程中表达有波动,4周时表达量高于其他时期,ACVR1和Spred1表达不受影响,CNBP先下调后上调。体外双荧光素酶报告系统证明3个基因是miR-1a的靶基因,靶位点突变实验证明ACVR1和Spred1的预测靶位点是miR-122的靶位点。通过鸡细胞系过表达miR-1a证明其对Spred1的调控在mRNA水平,而对ACVR1和CNBP的调控则不是在mRNA水平,表明miR-1a可能通过调控这几个基因在鸡骨骼肌发育中发挥调控作用。(7)运用实时荧光定量PCR检测miR-133a的组织表达,发现其在骨骼肌中表达量很高。运用靶基因预测软件预测出287个miR-133a的靶基因,挑选其中与发育相关的预测靶基因BIRC5。实时荧光定量PCR显示miR-133a在腿肌生长发育过程中先下调后上调,BIRC5表达不受影响。体外双荧光素酶报告系统和靶位点突变实验证明BIRC5及预测靶位点分别是miR-133a的靶基因和靶位点,表明miR-133a可能通过调控BIRC5在鸡骨骼肌发育中发挥调控作用。更多还原

【Abstract】 Liver metabolism and skeletal muscle development are two important biological processes in chicken. microRNAs (miRNAs) are a class of small non-coding RNAs about22nuleotides in length, which regulate gene expression by interacting with the3’untranslated region (UTR) of target mRNA. miRNAs play important roles in liver metabolism and skeletal muscle development. In the current study, a series of expression and function studies about the miRNAs in chicken liver and skeletal muscle were conducted. The main results are as follows:(1) miR-122expression in various tissues was detected using real-time quantitative RT-PCR (qRT-PCR). The result showed miR-122was highly expressed in liver. Using miRNA target prediction software,364potential target genes of miR-122were predicted, of which the genes P4HA1, PKM2, TGF-β3, FABP5and ARCN1related with liver metabolism were selected for further investigation. By qRT-PCR it was found that miR-122was up-regulated during the liver development and down-regulated during the isolation and cultivation of hepatocytes, the expression of the5genes were not affected during the liver development, P4HA1, TGF-β3and ARCN1were down-regulated during the isolation of hepatocytes, and FABP5was down-regulated and up-regulated during the isolation and cultivation of hepatocytes, respectively. By dual luciferase reporter assays and target site mutation assays it was validated that the5genes and their predicted target sites were bona fide target genes and target sites of miR-122, respectively. Through the overexpression of miR-122in chicken cell line, it was shown that miR-122regulates TGF-(33in mRNA level, whereas it does not regulate P4HA1, PKM2and ARCN1in mRNA level. These results indicated that miR-122plays roles in liver metabolism by regulating the5genes in chicken.(2) After the inhibition of miR-122in primary chicken hepatocytes, the genes were analyzed by trancriptome sequencing. The result showed123genes were differentially expressed, of which64were up-regulated and59were down-regulated. Gene ontology (GO) analysis showed21genes were involved in liver metabolism, indicating that miR-122may regulate liver metabolism by affecting these genes. Through comparison of sequencing and qRT-PCR, it was found that after the inhibition of miR-122P4HA1, PKM2and TGF-β3were up-regulated, FABP5was down-regulated, and ARCN1was not affected. The Western Blotting analysis showed the P4HA1protein was up-regulated by the inhibition of miR-122. These results further validated that miR-122regulate these target genes.(3) After the RNA interference of TGF-β3in primary chicken hepatocytes, it was found that3genes downstream the TGF-β3signaling pathway were differentially expressed, of which ACTA2was up-regulated, and LEF1and CCNB2were down-regulated, indicating that miR-122may affect these genes to regulate liver metabolism by targeting TGF-P3.(4) miR-126expression in various tissues was detected using qRT-PCR. The result showed miR-126was expressed in liver very low. Using miRNA target prediction software,54potential target genes of miR-126were predicted, of which the gene Spredl related with liver metabolism was selected for further investigation. By qRT-PCR it was found that miR-126was up-regulated during the liver development and down-regulated during the isolation and cultivation of hepatocytes, the expression of Spredl was not affected. By dual luciferase reporter assays and target site mutation assays it was validated that Spredl and its predicted target site were bona fide target gene and target site of miR-126, respectively. Through the overexpression of miR-126in chicken cell line, it was shown that miR-126does not regulate Spred1in mRNA level. These results indicated that miR-126plays roles in liver metabolism by regulating Spredl in chicken.(5) After the cGH treatment of primary chicken hepatocytes, the miRNAs were analyzed using deep sequencing. The result showed216chicken miRNAs were identified. Among the identified chicken miRNAs56were affected by cGH, of which29were up-regulated and27were down-regulated, indicating these miRNAs may take part in liver metabolism.(6) miR-1a expression in various tissues was detected using qRT-PCR. The result showed miR-la was highly expressed in skeletal muscle. Using miRNA target prediction software,247potential target genes of miR-la were predicted, of which the genes ACVR1and Spredl related with muscle development were selected for further investigation. Another further investigated gene was CNBP, which was previously predicted as miR-133a target gene and validated as miR-la target gene later. By qRT-PCR it was found that miR-la was wavily expressed during the thigh muscle development and it was highly expressed at4week-old stage, the expression of ACVR1and Spredl was not affected, and CNBP was at first down-regulated and then up-regulated. By dual luciferase reporter assays it was validated that the3genes were bona fide target genes of miR-la, and by target site mutation assays it was validated that the predicted target sites in ACVR1and Spredl were bona fide target sites of miR-la. Through the overexpression of miR-la in chicken cell line, it was shown that miR-la regulates Spredl in mRNA level, whereas it does not regulate ACVR1and CNBP in mRNA level. These results indicated that miR-la plays roles in skeletal muscle development by regulating the3genes in chicken.(7) miR-133a expression in various tissues was detected using qRT-PCR. The result showed miR-133a was highly expressed in skeletal muscle. Using miRNA target prediction software,287potential target genes of miR-133a were predicted, of which the gene BIRC5related with development was selected for further investigation. By qRT-PCR it was found that miR-133a was down-regulated firstly and up-regulated later during the thigh muscle development, the expression of BIRC5was not affected. By dual luciferase reporter assays and target site mutation assays it was validated that BIRC5and its predicted target site were bona fide target gene and target site of miR-133a, respectively. These results indicated that miR-133a plays roles in skeletal muscle development by regulating BIRC5in chicken.更多还原