Construction of Dual-luciferase reporter gene vector of 3’UTR of the COL4A3 gene and validation of its targeting relationship with miR-299

中华显微外科杂志 Pub Date : 2019-06-25 DOI:10.3760/CMA.J.ISSN.1001-2036.2019.03.012

Xitao Linghu, Shuai Huang, Yong-Mei Luo, Yun Zhang, Jia-Yu Chen, Xuechao Wan, Yi Liu, Qingde Wa

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引用次数: 0

Abstract

Objective To construct a dual-luciferase reporter gene vector and validate the targeting relationship between miR-299 and the COL4A3 gene, laying a foundation for the study on the effect of miR-299 in the chondrogenic differentiation of stem cells by regulating the COL4A3 gene. Methods This study was made from March, 2018 to December, 2018. Firstly, the potential binding sites between miR-299 and COL4A3-3'UTR were predicted using bioinformatics. Then, the wild and mutant COL4A3-3’UTR sequences were amplified by PCR and cloned into psiCHECK-2 plasmid to construct corresponding recombinant vectors. The vectors were validated by enzyme digestion and gene sequencing. Finally, the cells were resuscitated, amplified, transfected and divided into 4 groups: COL4A3-WT+miR-299/NC group, COL4A3-WT+miR-299-inhibitor/NC-inhibitor group, COL4A3-MUT+miR-299/NC group and COL4A3-MUT+miR-299-inhibitor/NC-inhibitor group. Each group contains 3 holes, respectively. Luciferase activity in each group was determined using a dual-luciferase assay kit. The statistical analysis was conducted and differences between groups were compared by t test. Probabilities lower than 5%(P<0.05) were considered statistically significant. Results Enzyme digestion and DNA sequencing showed that the dual-luciferase reporter gene vector of psiCHECK-2-COL4A3 was constructed successfully. Luciferase assay demonstrated that in wild COL4A3 gene, luciferase activity reduced in the miR-299 transfection group (The average R/F value was 59.38%) compared with the NC group (The average R/F value was 100.00%), with a statistical significant difference (P 0.05. Conclusion The dual-luciferase reporter gene vector of the 3’UTR of the COL4A3 gene is constructed successfully. In addition, dual-luciferase assay further verifies the authenticity of miR-299 directly targeting the 3’UTR of the COL4A3 gene. Key words: Collagen type IV alpha3 chain; MicroRNA; 3’Untranslated region; Dual-luciferase reporter genevector; Chondrogenic differentiation

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COL4A3基因3’UTR双荧光素酶报告基因载体的构建及其与miR-299靶向关系的验证

目的构建双荧光素酶报告基因载体，验证miR-299与COL4A3基因的靶向关系，为研究miR-299调控COL4A3在干细胞软骨分化中的作用奠定基础。方法本研究时间为2018年3月至2018年12月。首先，利用生物信息学预测了miR-299和COL4A3-3'UTR之间的潜在结合位点。然后，通过PCR扩增野生和突变的COL4A3-3'UTR序列，并将其克隆到psiCHECK-2质粒中，构建相应的重组载体。通过酶切和基因测序对载体进行了验证。最后，将细胞复苏、扩增、转染并分为4组：COL4A3-WT+miR-299/NC组、COL4A3-WT+miR-299-inhibitor/NC抑制剂组、COL4A 3-MUT+miR-299/2C组和COL4A3-MUT+miR-299-inhibiton/NC抑制剂组。每组分别包含3个孔。使用双荧光素酶测定试剂盒测定各组中的荧光素酶活性。进行统计学分析，并通过t检验比较各组之间的差异。概率低于5%（P<0.05）被认为具有统计学意义。结果酶切和DNA测序结果表明，成功构建了psiCHECK-2-COL4A3双荧光素酶报告基因载体。萤光素酶测定表明，在野生COL4A3基因中，miR-299转染组的萤光素酶活性（平均R/F值为59.38%）与NC组（平均R/F值为100.00%）相比降低，具有统计学显著性差异（P 0.05）。结论成功构建了COL4A3基因3’UTR双荧光素酶报告基因载体。此外，双荧光素酶测定进一步验证了miR-299直接靶向COL4A3基因3’UTR的真实性。关键词：胶原蛋白IV型alpha3链；微小核糖核酸；3'未翻译区域；双荧光素酶报告基因；软骨分化

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来源期刊

中华显微外科杂志

CiteScore

0.50

自引率

0.00%

发文量

6448

期刊介绍： Chinese Journal of Microsurgery was established in 1978, the predecessor of which is Microsurgery. Chinese Journal of Microsurgery is now indexed by WPRIM, CNKI, Wanfang Data, CSCD, etc. The impact factor of the journal is 1.731 in 2017, ranking the third among all journal of comprehensive surgery. The journal covers clinical and basic studies in field of microsurgery. Articles with clinical interest and implications will be given preference.