脊索瘤中microRNA的差异性表达及其RNA编辑

The differential expression and RNA editing of microRNAs in chordoma

中文关键词:  脊索瘤  microRNA  RNA编辑  RNA腺苷脱氨酶

英文关键词:Chordoma  MicroRNA  RNA editing  Adenosine deaminase acting on RNA

中文摘要:

  【摘要】 目的：检测脊索瘤组织中microRNA（miRNA）的差异性表达情况，探讨其RNA编辑。方法：使用RT-qPCR（Real-time quantitative polymerase chain reaction）技术检测骶骨脊索瘤组织11种候选miRNA的表达水平，将其与髓核组织中的表达水平进行比较，对表达异常的miRNA前体（pri-miRNAs）的DNA和cDNA序列进行对比，检测是否存在RNA编辑。使用蛋白印迹法（Western blot）检测脊索瘤组织中RNA编辑的关键酶RNA腺苷脱氨酶（adenosine deaminase acting on RNA，ADARs）的表达。构建ADAR的表达载体，同时构建ERBB2和HOXA1的3′-非翻译区报告载体并转染miRNA模拟物和抑制物，将其和ADAR的表达载体共转染至人胚肾细胞293T（HEK293T细胞），用qRT-PCR检测miRNA的表达水平，并通过荧光素酶报告基因活性分析法对已测得异常表达的miRNA的靶基因ERBB2和HOXA1进行活性分析，检测ADAR与测得异常表达的miRNA的靶基因的关系。结果：与髓核组织相比，脊索瘤组织中miR-10a和miR-125a的相对表达程度明显下调（P<0.05）。脊索瘤组织中miR-10a和miR-125a的前体cDNA序列中出现了腺苷酸向鸟苷酸（A-G）突变，而在髓核组织中miR-10a和miR-125a前体的cDNA序列中无此改变，miR-10a和miR-125a在成熟过程中出现了A-I RNA编辑。4组脊索瘤组织中有3组存在ADAR1过度表达，2组存在ADAR2过度表达。转染了miRNA抑制物的HEK293T细胞中ADAR1表达出现上调，而miR-10a和miR-125a的表达出现下调，miR-10a的靶基因ERBB2和miR-125a的靶基因HOXA1的荧光素酶活性显著增高；相反，转染了miRNA模拟物的HEK293T细胞中ADAR1表达出现下调，而miR-10a和miR-125a的表达出现上调，ERBB2 和HOXA1的荧光素酶活性显著降低。结论：ADAR1的过度表达可能通过介导A-I RNA编辑影响miR-10a和miR-125a的成熟和表达，参与脊索瘤发生中的细胞异常增殖调控。

英文摘要:

  【Abstract】 Objectives: To investigate the differential expression of miRNAs and the RNA editing in chordoma. Methods: The expressions of 11 candidate miRNAs were detected by using real-time quantitative polymerase chain reaction(RT-qPCR) in both the chordoma and the nucleus pulposus tissues. In order to explain the changed expressions of the miRNAs, the DNA and cDNA sequences of the miRNAs′ precursors(pri-miRNAs) were compared to see if there existed RNA editing. The western blot was used to detecte the expression of adenosine deaminase acting on RNA(ADARs), which were key enzymes in RNA editing, in chordoma and control tissues. An ADAR expression vector was constructed. The ERBB2 and HOXA1 3′-UTR luciferase reporter vectors were also constructed and transfected with miRNA mimics and miRNA inhibitors. After that, the ADAR expression vector and the 3′-UTR luciferase reporter vectors were co-transfected into HEK293T cells which had been cultivated in vitro. After the cells were harvested, the total extracted RNAs were detected by RT-qPCR and the relative luciferase activities were assayed by the Dual-Luciferase Assay to investigate the relationship between ADARs and target genes of the miRNAs. Results: The expressions of miR-10a and miR-125a were down-regulated significantly in chordoma tissues compared with controls(P<0.05), while the expressions of their precursors remained unchanged. An A to G variation was found in miR-10a and miR-125a cDNA sequence obtained from chordoma tissues, but it was not found in genome DNA or cDNA from nucleus pulposus tissues. There was an A-I RNA editing in the maturation process of miR-10a and miR-125a in chordoma. The expression of ADAR1 was over-expressed in 3 of 4 chordoma samples with up-regulated ADAR2 in 2 samples. In HEK293T cells transfected with miRNA inhibitors, ADAR1 over expressed when miR-10a and miR-125a were down-regulated, while the relative luciferase activities of ERBB2 and HOXA1, which were target genes of miR-10a and miR-125a, were significantly raised. On the contrary, in HEK293T cells transfected with miRNA mimics, ADAR1 underexpressed when miR-10a and miR-125a were up-regulated, while the relative luciferase activities of ERBB2 and HOXA1 significantly decreased. Conclusions: The over-expression of ADAR1 enhances A-I RNA editing in pri-miR-10a and pri-miR-125a, the maturation and expression of miR-10a and miR-125a are hence affected, which contribute to the pathogenesis of chordoma.