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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 predict the miR-29 target gene and its binding site using the microRNA target site prediction website and to construct a recombinant plasmid containing wild-type and its mutated integrinβ_1 (ITGβ_1) -3’untranslated region (UTR) The target gene of miR-29 was initially determined by transfection of cells and luciferase activity assay to study the effect of miR-29 on the expression of ITGβ_1 gene target (pRIR-ITGβ_1-3’UTR) The role of regulation. Methods: Human whole blood RNA was extracted and the mRNA was reverse transcribed into c DNA. The ITGβ_1-3’UTR fragment was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and ligated into luciferase reporter vector p MIR -Report, a luciferase reporter gene vector and a mutant vector of p MIR-ITGβ_1-3’UTR was constructed and identified. The p MIR-Report Luciferase vector, the constructed p MIR-ITGβ_1-3’UTR and the mutant vector were cotransfected into rat cardiac fibroblasts respectively with miR-29 mimics. Dual luciferase assay was used to analyze miR-29 and Mechanism of action of ITGβ_1. Results: The plasmid sequence was confirmed by restriction enzyme digestion and gene sequencing. The size and sequence of the cloned DNA fragment was consistent with that reported in Genbank. The p MIR-ITGβ_1-3’UTR dual luciferase reporter gene was successfully constructed, The enzyme experiment confirmed that miR-29 could bind to the corresponding base site of ITGβ_1-3’UTR and significantly down-regulate luciferase expression. CONCLUSION: The luciferase reporter gene vector was successfully constructed and the expression of firefly luciferase was significantly down-regulated after miR-29 mimics transfection.