Construction and Functional Analysis of Luciferase Reporter Plasmid Containing Vimentin Gene Promoter

Abstract

Breast cancer is the main cause of cancer deaths in women around the world, and is closely related to metastasis. We know that MRTF-A and STAT3 play an important role in the migration of breast cancer. Vimentin is an important marker gene for epithelial-mesenchymal transition (EMT), which is closely related to the process of breast cancer metastasis. The construction of the mutant luciferase reporter gene plasmid will demonstrate that MRTF-A and STAT3 can modulate the transcription of vimentin. In this study, the Vimentin promoter luciferase reporter gene was derived from PCR amplification of the Vimentin promoter. The PCR amplified fragment was cloned into the PGL-3 vector by restriction enzyme digestion. The promoter sequence was confirmed by sequencing. The sequencing results showed that the Vimentin promoter luciferase reporter plasmid was constructed successfully. MDA-MB-231 cells were respectively transfected with the Vimentin luciferase reporter plasmids and pcDNA3.1 and pcDNA3.1-MRTF-A and pcDNA3.1-STAT3, and were analyzed by luciferase reporter assay. The results showed that MRTF-A could significantly enhance the transcriptional activity of vimentin gene promoter compared with transfected pcDNA3.1 group, while STAT3 group could slightly up-regulate the transcriptional activity of vimentin gene promoter.