TGFβR3基因3’UTR双荧光素酶报告载体的构建及其与miR-let-7a靶向关系的验证

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Abstract Objective: To construct a luciferase reporter vector containing the 3’untranslated region (3’UTR) of TGFβR3 gene and evaluate the targeting correlation between TGFβR3 and miR-let-7a. Methods: The potential complementary binding sites of miR-let-7a and TGFβR3 were predicted by targetscan. The 3’UTR fragment of TGFβR3 amplified by PCR was firstly cloned into pGEM-T vector and the potential complementary binding sites was mutated. Then, the amplified fragment and the mutated fragment of TGFβR3 3’UTR were subcloned into pmirGLO dual-luciferase miRNA target expression vector within the PmeI/SalI restriction sites. The luciferase reporter vector and miR-let-7a mimics/miR-let-7a NC were co-transfected into HEK293T cells respectively. The relative luciferase activity was detected by luciferase reporter assay. Results: Results of double enzyme digestion and DNA sequencing confirmed that the luciferase reporter vector was successfully constructed. The relative luciferase activity of TGFβR3 3’UTR reporter vector and miR-let-7a mimics co-transfected group were decreased significantly comparing with miR-let-7a NC group (P<0.05), but the relative luciferase activity was no significance in TGFβR3 3’UTR mutated reporter vector. Conclusion: The luciferase reporter vector containing the 3’UTR-WT or 3’UTR-MUT of TGFβR3 is constructed successfully. And TGFβR3 is a direct novel target gene of miR-let-7a.

Key words： microRNAs miR-let-7a transforming growth factor beta receptor 3 luciferase reporter vector

Received: 21 November 2016

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	ZHOU Huandong
	GU Rui
	WU Dazhou
	CHEN Xiaoming.

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https://xb.wmu.edu.cn/EN/ OR https://xb.wmu.edu.cn/EN/Y2017/V47/I5/313

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