C jun sirna

MiR-139 mimic (miR10000250), mimic-NC, inhibitor (miR20000250), inhibitor-NC, antagomiR-139 (miR30000250), and antagomiR-NC (NC: negative control) were synthesized by RiboBio (RiboBio, Guangzhou, China). C-jun siRNA were purchased from GenePharma (Shanghai, China), and c-jun plasmids were purchased from Vigene (Jinan, China). The final concentration of miR-139 mimics, miR-139 inhibitor and C-jun siRNA in the transfection system was 100nM, respectively. The concentration of c-jun plasmid in the transfection system was 1ug/ml. When the confluence of ECFCs was approximately 70% to 80%, the miR-139 mimic, miR-139 inhibitor, C-jun siRNA, and c-jun plasmids were transfected with Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). After 72 hours, the cells were subjected to the process of induction of differentiation.

c-jun siRNA and Scr control siRNA were purchased from Genepharma Company (Suzhou, China). The lentivirus which contain c-jun shRNA and control shRNA respectively were purchased from Genechem Company (Shanghai, China). Cells were transfected using Lipofectamine 2000 for 4 h following which the lipid and siRNA complex was removed and fresh growth medium was added. Cells were lysed 48 h after transfection and specific protein levels were determined by western blot analysis with specific antibodies.

Negative control siRNA (si-NC) and four individual MK5-AS1 siRNAs (si-MK5-AS1 #1, #2, #3, #4), three individual MK5 siRNAs (si-MK5 #1, #2, #3), c-Jun siRNA, let-7f-1-3p mimics, and let-7f-1-3p inhibitor were purchased from Gene Pharma (Shanghai, China). The siRNA sequences are listed in Table S1. Cells were transfected with siRNA when at 30–50% confluence using siLentFect Lipid Reagent (Bio-Rad, CA, USA). MK5-AS1, MK5, and c-Jun were amplified from human cDNA as a template and were cloned into the pcDNA3.1(+) vector (Invitrogen, USA). The c-Jun-S63A sequence was thereafter generated using overlap extension PCR and cloned into pcDNA3.1(+). HCT116 and SW620 cells were grown to 90% confluence before being transiently transfected with plasmids using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s protocol. At 24–48 h posttransfection, cells were harvested for qPCR or western blot analysis.