摘要
目的:利用microRNA靶位点预测网站预测了miR-29靶基因及结合位点,构建含野生型及其突变型整合素β_1(integrinβ_1,ITGβ_1)-3'端非翻译区(UTR)双荧光素酶报告基因(DLR)表达系统(p MIR-ITGβ_1-3'UTR),通过转染细胞、双荧光素酶活性分析初步确定miR-29的靶位点,以此研究miR-29对ITGβ_1基因靶向调控的作用。方法:提取人全血RNA,逆转录mRNA成c DNA,以之为模板,逆转录PCR(RT-PCR)扩增ITGβ_1-3'UTR片段,经酶切后连接至荧光素酶报告载体p MIR-Report上,构建出p MIR-ITGβ_1-3'UTR的荧光素酶报告基因载体及突变载体并进行鉴定。将p MIR-Report Luciferase载体,所构建的p MIR-ITGβ_1-3'UTR及突变载体分别同miR-29 mimics共转染至大鼠心肌成纤维细胞,采用双荧光素酶实验分析miR-29与ITGβ_1的作用机制。结果:通过酶切及基因测序的方法证实所构建质粒序列正确,克隆获得的DNA片段大小及序列与Genbank报道的一致;成功构建p MIR-ITGβ_1-3'UTR双荧光素酶报告基因,双荧光素酶实验证实miR-29可以结合在ITGβ_1-3'UTR相应的碱基位点,并显著下调荧光素酶的表达。结论:该荧光素酶报告基因载体构建成功,转染miR-29 mimics后能显著下调萤火虫荧光素酶的表达。
Objective： To build with wild type and mutant integrins beta 1（ integrin beta 1,ITGβ1）-3＇ end of translation region（ UTR） dual luciferase reporter gene expression system（ DLR）（ p MIR-ITGβ1-3＇ UTR）,through the dual luciferase report gene detects the kits ITGβ1,this study miR-29 ITGβ1gene targeting regulatory role. Which based on regulating ITGβ1transcription level after the screening of tanshinone. Method： Extraction of whole blood RNA,reverse transcriptase mRNA into c DNA,for the template,RT-PCR amplification ITGβ1-3 ＇UTR fragments,after enzyme digestion to connect to the luciferase report carrier p MIR-Report,construct p MIR ITGβ1-3 ＇ UTR luciferase reporter gene vector and mutation carriers and identified. Will p MIR-Report Luciferase control,constructed p MIR ITGβ1-3 ＇ UTR and mutation carriers respectively with miR-mimics a total of 29 transfection to ovarian granulosa cells,with double Luciferase experimental analysis of the mechanism of action of miR-29 and ITGβ1. Result： By enzyme cutting and gene sequencing method confirmed by construction of plasmid sequence is correct; dual luciferase experiments have established that miR-29 can combine in ITGβ1-3 ＇ UTR.Successful build p MIR ITGβ1-3＇UTR dual luciferase report gene,confirmed by the method of enzyme cutting and gene sequencing constructed plasmid sequence is correct. Cloning of DNA sequence fragment size and consistent with the reported in Genbank. Conclusion： The luciferase report gene build successful,miR-29 can combine after transcription ITGβ1-3＇UTR.
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2016年第17期102-107,共6页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金青年项目(81503435)
