Construction of Dual-luciferase reporter gene vector of 3'UTR of the COL4A3 gene and validation of its targeting relationship with miR-299
10.3760/cma.j.issn.1001-2036.2019.03.012
- VernacularTitle:COL4A3基因3'UTR双荧光素酶报告基因载体构建及其与miR-299靶向关系验证
- Author:
Xitao LINGHU
1
;
Shuai HUANG
;
Yongxiang LUO
;
Yun ZHANG
;
Jiayu CHEN
;
Xue WAN
;
Yi LIU
;
Qingde WA
Author Information
1. 遵义医科大学附属医院骨科
- Keywords:
Collagen type Ⅳ alpha3 chain;
MicroRNA;
3'Untranslated region;
Dual-luciferase reporter genevector;
Chondrogenic differentiation
- From:
Chinese Journal of Microsurgery
2019;42(3):258-263
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a dual-luciferase reporter gene vector and validate the targeting relation ship 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 pre dicted 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).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/9＞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.
