Non specific control sirna

The ORF of human Twist1 and Snail genes were amplified by PCR in 293FT cell line, the amplified fragments were subcloned into the pcDNA3.1/myc-hisA vector. The pE-CAD-wt and pE-CAD-mu were a gift from Kumiko UiTei. The Twist1 3′-UTR containing miR-129-5p binding site or miR-129-5p binding site mutated fragments were cloned into pGL3-Control vector (Promega, Madison, WI, USA; Tw-3′UTR-wt and Tw-3′UTR-mu). The miR-129 promoter region (−300 to +1) and the E-box mutated fragments were cloned into pGL3-Basic vector (Promega; miR-129-Ebox-wt and miR-129-Ebox-mu). The miR-129-5p mimic and inhibitor were purchased from RiboBio (Shanghai, China). The Twist1 gene-specific short interfering (siRNA), Snail siRNA, and non-specific control siRNA were also purchased from RiboBio.

The ORF of human SNAI2 and ERα generated from MDA-MB-231 and MCF7 cells, the resultant PCR product of which was connected together with pcDNA3.1 tagged HA. The SNAI2 3′-UTR containing miR-1271 binding sites were amplified and cloned into psiCHECK2 vector (Promega, Madison, WI, USA) to generate Luc-SNAI2. Site-directed mutagenesis was performed using the Site-Directed Mutagenesis Kit (TransGene, Beijing, China) to generate the SNAI2 3′-UTRmut reporter vector (SNAI2M). The miR-1271 promoter region (P1: − 1100 to + 1; P2: -705 to + 1), Estrogen Receptor 1 (ESR1) promoter region (− 1000 to + 1) or the E-box mutated fragments were cloned into pGL3-Basic vector (Promega; pGL3-P1/P2 and pGL3-ESR1-wt/mut). All constructs were confirmed by sequencing. The miR-1271 mimic, miR-1271 inhibitor, or the appropriate scrambled controls were purchased from RiboBio (Shanghai, China). The ERα and SNAI2 gene-specific siRNAs, and non-specific control siRNA were also purchased from RiboBio.
miRNA or siRNAs were transfected into different cell lines using FuGENE HD Transfection Reagent (Promega, Madison, WI, USA) and plasmids were transfected using TransFast Transfection Reagent (Promega) according to the manufacturer’s recommendations.