Basic Research
Construction of Dual-luciferase reporter gene vector of 3’UTR of the COL4A3 gene and validation of its targeting relationship with miR-299
Xitao Linghu, Shuai Huang, Yongxiang Luo, Yun Zhang, Jiayu Chen, Xue Wan, Yi Liu, Qingde Wa
Published 2019-06-25
Cite as Chin J Microsurg, 2019, 42(3): 258-263. DOI: 10.3760/cma.j.issn.1001-2036.2019.03.012
Abstract
ObjectiveTo 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.
MethodsThis 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.
ResultsEnzyme 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). In wild COL4A3 gene treated with inhibitor, luciferase activity increased in the miR-299-inhibitor group(The average R/F value was 153.98%) compared with the NC-inhibitor group(The average R/F value was 100.00%), with a statistical significant difference (P<0.05). In mutant COL4A3 gene treated with inhibitor, no obvious statistical differences in luciferase activity were found between miR-299 transfection group (The average R/F value was 102.09%), miR-299-inhibitor group (The average R/F value was 108.51%) and NC group(The average R/F value was 104.70%), NC-inhibitor group (The average R/F value was 105.13%) and P>0.05.
ConclusionThe 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
Contributor Information
Xitao Linghu
Department of Orthopaedic Surgery, The Affiliated Hospital of Zunyi Medical University, Zunyi, Guizhou Province 563003, China
Shuai Huang
Department of Orthopedic Surgery, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, China
Yongxiang Luo
Department of Biomedical Engineering, The Medicine of Shenzhen University, Shenzhen, Guangdong Province 518060, China
Yun Zhang
Department of Emergency, The Sun Yat-sen Memorial Hospital of Sun Yat-Sen University, Guangzhou 510275, China
Jiayu Chen
Department of Biochemistry and Molecular Biology, The Basic Medicine of Zunyi Medical University, Zunyi, Guizhou Province 563006, China
Xue Wan
Department of Key Laboratory of Cell Engineering, The Affiliated Hospital of Zunyi Medical University, Zunyi, Guizhou Province 563003, China
Yi Liu
Department of Orthopaedic Surgery, The Affiliated Hospital of Zunyi Medical University, Zunyi, Guizhou Province 563003, China
Qingde Wa
Department of Orthopaedic Surgery, The Affiliated Hospital of Zunyi Medical University, Zunyi, Guizhou Province 563003, China
