文章摘要
陈佳萍,胡志斌,田 甜,周小毅,苗瑞芬,靳光付,马红霞,沈洪兵.miR-196a2成熟区单核苷酸多态性对靶基因LSP1表达的影响[J].南京医科大学学报,2009,29(6):762~766
miR-196a2成熟区单核苷酸多态性对靶基因LSP1表达的影响
Single nucleotide polymorphism associated with mature miR-196a2 influences the expression of Lymphocyte-specific protein 1(LSP1) gene
投稿时间:2008-10-13  
DOI:10.7655
中文关键词: miR-196a2  单核苷酸多态性  LSP1
英文关键词: miR-196a2  single nucleotide polymorphism  LSP1
基金项目:国家自然科学基金资助项目(30730080)
作者单位
陈佳萍 南京医科大学流行病与卫生统计学系,江苏 南京 210029 
胡志斌  
田 甜  
周小毅  
苗瑞芬  
靳光付  
马红霞  
沈洪兵  
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中文摘要:
      目的:研究miR-196a2成熟区单核苷酸多态性rs11614913(T/C)对预测靶基因LSP1表达的影响?方法:构建含有不同基因型miR-196a2的真核表达载体pCDNA3.1-miR196a2-C和pCDNA3.1-miR196a2-T;同时利用化学合成含有不同基因型的成熟miR-196a2探针miR-196a2-C和miR-196a2-U?将靶基因LSP1的3’UTR序列克隆至pMIR-REPORTTM Luciferase 载体中而获得LSP1报告基因载体pMIR-LSP1?采用所构建的miR-196a2真核表达载体以及化学合成的成熟miR-196a2 探针分别与靶基因LSP1报告基因载体组建两套转染体系,分别共转染于HEK293?A549和CHO三种细胞后进行荧光素酶活性分析?结果:miR-196a2真核表达载体与靶基因LSP1报告基因表达载体共转染于HEK293?A549和CHO三种细胞后进行荧光素酶活性分析,pCDNA3.1-miR196a2-C等位基因荧光素酶相对活性与pCDNA3.1-miR196a2-T等位基因相比均有显著降低(P < 0.05);含不同基因型的化学合成的成熟miR-196a2 探针与靶基因LSP1报告质粒共转染HEK293? A549和CHO三种细胞,miR-196a2-C等位基因荧光素酶活性与miR196a2-T等位基因相比也有显著降低(P < 0.05)?结论:荧光素酶活性强弱间接反映了miR-196a2与靶基因LSP1结合能力的大小,当miR-196a2 成熟区rs11614913位点为C时,miR-196a2可能更为有效地与预测靶基因LSP1结合,从而在转录后水平调控靶基因表达?
英文摘要:
      Objective:To investigate the effect of a common polymorphism T/C(rs11614913) in mature miR-196a2 on the expression of LSP1 gene. Method:The pCDNA3.1-miR196a2-C and pCDNA3.1-miR196a2-T expression plasmids were created containing T or C allele of pre-miR196a2. Meanwhile,mature miR-196a2-C and miR-196a2-U probes were chemically synthesized for the investigation. The 3’UTR from LSP1 gene was cloned downstream of the firefly luciferase gene in pMIR-REPORTTM Luciferase Vector. Two sets of cotransfection were performed with CHO,HEK293 and A549 cells,and the luciferase activity with the Dual-Luciferase Reporter Assay System were analyzed. Results:pCDNA3.1-miR196a2-C construct group showed significantly lower levels of luciferase expression compared with pCDNA3.1-miR196a2-T construct group(P < 0.05),when LSP1 3’UTR luciferase reporter plasmids were cotransfected with miR-196a2 expression plasmids in CHO,HEK293 and A549 cells. Mature miR-196a2-C probe group showed significantly lower luciferase activity compared with miR-196a2-T probe group(P < 0.05),while LSP1 3’UTR luciferase reporter plasmids were cotransfected with mature miR-196a2 probes in CHO,A549 and HEK293 cells. Conclusion: The miR-196a2 could effectively bind the predicted target gene of LSP1 and influence the expression on the level of transcription,while miR-196a2 carrying the rs11614913 C allele.
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