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目的 :检测脊索瘤组织中micro RNA(mi RNA)的差异性表达情况,探讨其RNA编辑。方法 :使用RTq PCR(Real-time quantitative polymerase chain reaction)技术检测骶骨脊索瘤组织11种候选mi RNA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mi RNA前体(pri-mi RNAs)的DNA和c DNA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mi RNA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q RT-PCR检测mi RNA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mi RNA的靶基因ERBB2和HOXA1进行活性分析,检测ADAR与测得异常表达的mi RNA的靶基因的关系。结果:与髓核组织相比,脊索瘤组织中mi R-10a和mi R-125a的相对表达程度明显下调(P<0.05)。脊索瘤组织中mi R-10a和mi R-125a的前体c DNA序列中出现了腺苷酸向鸟苷酸(A-G)突变,而在髓核组织中mi R-10a和mi R-125a前体的c DNA序列中无此改变,mi R-10a和mi R-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mi RNA抑制物的HEK293T细胞中ADAR1表达出现上调,而mi R-10a和mi R-125a的表达出现下调,mi R-10a的靶基因ERBB2和mi R-125a的靶基因HOXA1的荧光素酶活性显著增高;相反,转染了mi RNA模拟物的HEK293T细胞中ADAR1表达出现下调,而mi R-10a和mi R-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mi R-10a和mi R-125a的成熟和表达,参与脊索瘤发生中的细胞异常增殖调控。
Objective: To detect the differential expression of micro RNA (miRNA) in chordoma tissue and to explore its RNA editing. Methods: Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of 11 candidate miRNAs in sacral chordoma and to compare them with the expression level in nucleus pulposus. (pri-mi RNAs) DNA and c DNA sequences were compared to detect the presence of RNA editing. Western blot was used to detect the expression of adenosine deaminase acting on RNA (ADARs), a key RNA-editing enzyme in chordoma tissues. The expression vectors of ADAR were constructed and the 3’-untranslated region reporter vectors of ERBB2 and HOXA1 were constructed and transfected into miRNA mimics and inhibitors, and co-transfected with ADAR expression vector into human embryonic kidney cell 293T (HEK293T cells ). The expression of mi RNA was detected by q RT-PCR. The activity of ERBB2 and HOXA1, the target genes of miRNAs that had been abnormally expressed, was detected by luciferase reporter assay. The mi RNA targets the gene’s relationship. Results: Compared with nucleus pulposus, the relative expression of mi R-10a and mi R-125a in chordoma was significantly decreased (P <0.05). Adenine to guanylic acid (AG) mutations were found in the precursor c DNA sequences of mi R-10a and mi R-125a in chordoma, whereas in the nucleus pulposus prior to mi R-10a and mi R-125a There was no such change in the c DNA sequence of the body, and AI RNA editing occurred during maturation of mi R-10a and mi R-125a. There were 3 ADAM1 overexpression in 4 chordoma tissues and 2 ADAR2 overexpression. Upregulation of ADAR1 expression was observed in HEK293T cells transfected with miRNA inhibitors, whereas expression of mi R-10a and mi R-125a was down-regulated, and fluorescence of HOXA1, a target gene of mi R-10a and a target gene of mi R-125a In contrast, expression of ADAR1 was down-regulated in miRNA-mock-transfected HEK293T cells, whereas expression of mi R-10a and mi R-125a was up-regulated and luciferase activities of ERBB2 and HOXA1 were significantly reduced. CONCLUSION: Overexpression of ADAR1 may be involved in the regulation of abnormal proliferation of chordoma through the regulation of the maturation and expression of mi R-10a and mi R-125a mediated by A-I RNA editing.