Molecular mechanism of microRNA125 regulating human coagulation factor IX gene with nonsense mutation

Objective To construct human coagulation factor Ⅸ mini-gene (Mini-hF9) and some nonsense mutants, detect the levels of the Mini-hF9 mRNA, and analyze the molecular mechanism of microRNA125 regulating F9 gene with nonsense mutation. Methods Three nonsense mutants were obtained by using PCR mutagenesis to analyze the mechanism of plasma thromboplastin component recognition. The Mini-hF9 gene mRNA levels were detected by Real-time PCR in mammalian cells co-transfected with nonsense mutant expression vectors and miR-125 mimics. Results Mini-hF9 gene was constructed successfully and cloned into the mammalian expression vector. The only normal transcript was detected in cells transfected with the Mini-hF9 gene expression vectors. Three nonsense mutants, M1 (nt 34 G>T in Exon 7), M2 (nt 52 G>T in Exon 7) and M3 (nt 85 G>T in Exon 7), were obtained by using PCR mutagenesis. The levels of the Mini-hF9 mRNA decreased to 14.1% (t=15.464, P=0.004) in M1 and 22.4% (t=15.755, P=0.004) in M2 mutants after transfection, respectively. It was proved to be caused by nonsense-mediated mRNA decay (NMD) in CHX experiment. The levels of Mini-hF9 mRNA increased to 1.70 times (t=-4.883, P=0.039) and 2.40 times (t=-17.537, P=0.003) in M1 mutant after miR-125a or miR-125b mimics treatment, respectively. The levels of Mini-hF9 mRNA increased to 2.02 times (t=-19.264, P=0.003) and 2.07 times (t=-9.158, P=0.012) in M2 mutant after miR-125a or miR-125b mimics treatment, respectively. Conclusion Nonsense mutant location is a key determinant for triggering NMD. MicroRNA125 could improve the stability of Mini-hF9 mRNA in M1 and M2 mutants by repressing NMD. MicroRNA125, a short non-coding RNA molecule, could be a potential therapeutic target in conditions caused by the NMD pathway. Key words: Factor Ⅸ; MicroRNAs; Gene expression regulation; Nonsense-mediated mRNA decay; Mini-hF9 gene