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。
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 I.SP1 gene was cloned downstream of the firefly luciferase gene in pMIR-REPORT^TM 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 significandy lower levels of luciferase expression compared with pCDNA3.1-miR196a2-T construct group（P 〈 0.05）,when KSP1 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.
出处
《南京医科大学学报（自然科学版）》
CAS
CSCD
北大核心
2009年第6期762-766,共5页
Journal of Nanjing Medical University(Natural Sciences)
基金
国家自然科学基金资助项目(30730080)
