Mir 375 3p inhibitor

LncRNA GAS5 and Klf4 gene knock-down were achieved using RNA interference. shRNA-GAS5, shRNA- Klf4 and its corresponding scramble negative control siRNA (siNC) were purchased from TsingKe Biological Technology (Beijing, China). miR-375-3p mimics, miR-375-3p inhibitor and the corresponding negative control were synthesized by RIBOBIO (Guangzhou, China). The Klf4 over-expression plasmid (pcDNA-Klf4) and empty pcDNA3.1 plasmid (control) were purchased from RIBOBIO (Guangzhou, China).
Plasmids were transfected into the cultured CMs using Lipofectamine 3000 (Cat: L3000-015 Invitrogen) according to the manufacturer’s protocol. The transfection efficiency was detected by qRT-PCR and Western blotting. After 24-h transfection, the cells were incubated in DMEM medium supplemented with 10% FBS and 10 µM ISO for 24 h followed by 30 min OT pretreatment.

Transfection of AMC-HN-8 and Tu-212 cells was performed using Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). miR-375-3p mimic (5′-UUUGUUCGUUCGGCUCGCGUGA-3′), miR-375-3p inhibitor (5′-UCACGCGAGCCGAACGAACAAA-3′) and control miRNA (5′-GGUUCGUACGUACACUGUUCA-3′) were obtained from the Guangzhou RiboBio Co., Ltd. 50 nm of miRNA was diluted with 100 µl OPTI-MEM (Invitrogen; Thermo Fisher Scientific, Inc.) and subsequently incubated with 5 µl Lipofectamine 2000 for 15 min at room temperature. Subsequent experimentation was performed 48 h post-transfection.