Figure 3

FSCN1 is the target gene of miR-133a in breast cancer cells. A. Putative conserved target site in the FSCN1 3'UTR was identified with the TargetScan database: The conserved 7-bp seed sequence of miR-133a is aligned to FSCN1 mRNA. B. QRT-PCR analysis of FSCN1 mRNA expression. Breast cancer MCF-7 cells were grown and transiently transfected with miR-133a mimic and then subjected to RNA extraction and qRT-PCR analysis. C. Western blot analysis of FSCN1 protein expression. MCF-7 cells were grown and transfected with miR-133a mimic or negative control. Total cellular protein was isolated and subjected to Western blot analysis of FSCN1 expression. ß-actin was used as an internal control. D. Luciferase reporter assay. MCF-7 cells were transfected with a reporter vector psiCHECK2-FSCN1 3'UTR or psiCHECK2 control vector and miR-133a mimic or negative control. Each transfection was carried out in triplicate. Luciferase assay was performed 48 h after gene transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. E. Transwell migration and matrigel invasion assays. MCF-7 cells were grown and transiently transfected with miR-133a ASO, miR-133a ASO plus FSCN1 siRNA, or scrambled sequence oligonucleotides as negative control for 2 days and subjected to migration and invasion assays. Magnification: ×200.