microRNA125对无义突变的人凝血因子Ⅸ基因调控的分子机制  

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

作　　者：王刚[1] 杨林花[1] 柴宝峰[2] 张翠明[1] 王梅芳[1] 王琨[1] 陈玙[1] 李玲[1] 

机构地区：[1]山西医科大学第二临床医学院、山西医科大学第二医院血液科,太原030001 [2]山西大学生物技术研究所、化学生物学与分子工程教育部重点实验室

出　　处：《中华血液学杂志》2016年第5期388-392,共5页Chinese Journal of Hematology

基　　金：国家自然科学基金（81270587）;山西省自然科学基金（2015011119）;山西医科大学博士后基金;山西医科大学第二医院博士基金

摘　　要：目的构建人凝血因子Ⅸ小基因（Mini．hF9）及无义突变体，检测其mRNA表达水平，分析microRNA125对无义突变的F9基因表达调控的分子机制。方法利用PCR定点突变技术构建若干无义突变的人Mini—hF9并转染哺乳动物细胞HEK293T等，同时转染miR－125模拟物，通过实时荧光定量PCR（q—PCR）检测Mini—hF9基因mRNA表达水平。结果成功构建了Mini—hF9并在细胞中正确剪接。成功构建了无义突变体M1（nt34G〉TinExon7）、M2（nt52G〉TinExon7）和M3（nt85G〉TinExon7）。M1和M2突变体中Mini．hF9基因mRNA表达水平分别为野生型的14．1％（t=-15．464，P=0．004）和22．4％（t=-15．755，P=0．004），通过放线菌酮处理实验证实这是由于无义介导的mRNA降解（nonsense—mediatedmRNAdecay,NMD）所致。分别转染miR-125a模拟物和miR-125b模拟物后，M1突变体中Mini-hF9基因mRNA水平分别升高到1．70倍（t=-4．883，P=-0．039）和2．40倍（t=-17．537，P=0．003），M2突变体Mini—hF9mRNA水平分别升高到2．02倍（t=-19．264，P=0．003）和2．07倍（t=-9．158，P=0．012）。结论无义突变发生的位置是触发NMD途径的一个关键因素；microRNA125能够通过抑制NMD途径提高无义突变的凝血因子Ⅸ的mRNA水平。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.

关 键 词：因子IX 微RNAS 基因表达调控 无义介导的mRNA降解 Mini—hF9基因 

分 类 号：R3416[医药卫生—基础医学]