The differential expression and RNA editing of microRNAs in chordoma

English Keywords:Chordoma  MicroRNA  RNA editing  Adenosine deaminase acting on RNA

English Abstract:

  【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.