Mir 187 inhibitor

The coding sequences of CD276 were cloned into pcDNA3.1 (+) to generate CD276 expression vectors. The wild-type CD276 3′UTR was cloned into the pMIR-REPORT luciferase vector (Ambion, Austin, TX, USA). Mutant CD276 3′UTR was generated based on the pMIR-CD276-3′UTR by mutating 3 nt that are recognized by miR-187. The primers for CD276 were: 5′-TGTGGATCCCTGTCATCTGGGAAGTAACAACGCA-3′ (forward) and 5′-AAGTCTAGAGAGCCACTACTGCCTGTTGTCTTTG-3′ (reverse). The primers for CD276 3′UTR were: 5′-TCTGAGCTCGCTAAACAGCCATAAACGGAAACGC-3′ (forward) and 5′-ACCACGCGTGCGTAGATTCTCCTTTATGGGGCTG-3′ (reverse). MiR-187 mimics, miR mimic control, miR-187 inhibitor, miR inhibitor control, and small interfering RNA targeting CD276 were purchased from GenePharma (Shanghai, China). Transfection was performed according to the manufacturer's instructions.

HeLa and CaSki cell lines purchased from American Type Culture Collection (Manassas, VA, USA) and the cell bank of the Chinese Academy of Science (Shanghai, China), respectively, were cultured at 37°C in 5% CO₂ in a humidified incubator in Dulbecco's modified Eagle's medium and RPMI 1640 medium, respectively. All cells were authenticated by short tandem repeat profiling before receipt and were propagated for less than 6 months after resuscitation.
miRNAs were transfected at a working concentration of 100 nmol/L using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). miR-187 mimics, a nonspecific mimic control, miR-187 inhibitor, and a nonspecific inhibitor control were all purchased from GenePharma (Shanghai, China). Protein and RNA samples were extracted from subconfluent cells during the exponential growth phase.