Mir 205 mimic

Myomaker sequences that contain miRNA binding sites were cleaved using SacI/XhoI and cloned into the pmirGLO plasmid (Promega, Madison, WI, USA). We named the recombinant pmirGLO vectors with the Myomaker sequence as pmirGLO-Myomaker, which were also synthesized by TSINGKE (TSINGKE, Beijing, China). Meanwhile, the corresponding information of miRNA sequences was found from miRbase. miR-205 mimics (50 nM) and NC mimics (50 nM) were purchased from RIBOBIO (RIBOBIO, Guangzhou, Guangdong, China). When Hela cell density in a 48-well plate reached 70%, pmirGLO-Myomaker was co-transfected with miRNA mimic into Hela cells using Lipofectamine 3000, according to the manufacturer’s instructions. Cells were collected after 48 h; dual-luciferase activity was measured using the Dual-Luciferase Reporter Assay System kit (Promega, Madison, WI, USA), according to the manufacturer’s instructions.

Ovarian cancer cell line SKOV3 in the logarithmic growth phase was adopted and the cells were separated into three groups: the blank group: cells without transfection; the mimics negative control (NC) group: cells transfected with miR-205 mimics NC or Cy3-mimics NC; the miR-205 mimics group: cells transfected with miR-205 mimics or Cy3-miR-205 mimics. Cy3-miR-205 mimics, Cy3-mimics NC, miR-205 mimics and mimics NC were all obtained from Guangzhou RiboBio Co., Ltd. (Guangdong, China). Cy3-miR-205 mimics, Cy3-mimics NC or miR-205 mimics and mimics NC were transfected by Lipofectamine™ RNAiMAX (Invitrogen, Carlsbad, CA, USA) on the basis of the kit instruction.

MiR-205 mimics and matched NC were from RiboBio Co., Ltd (Guangzhou, China). The control and SMAD4 siRNAs were prepared as previously described [36 (link)]. The target sequences of the siRNA was: 5′-GTACTTCATACCATGCCGA-3′; Cell transfections were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instruction. After 72 h of transfection, the cells were collected for further experiments.