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

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