Mmp 9 sirna

Full-length human RelA cDNA was amplified by PCR from pCMV4-RelA plasmid (Addgene,
China) using forward primer 5′-GGTCGGTACCATGGACGAACTGTTCCCCCT-3′ and reverse primer
5′-CCATCTCGAGTTAGGAGCTGATCTGACTCA-3′, inserted into pcDNA3.1
vector (Invitrogen, China) tagged with FLAG. P65 siRNA, MMP-9 siRNA and its control
siRNA was purchased from Santa Cruz Biotechnology (China). Transient transfection of
ES-2 cells with pcDNA3.1/p65 cDNA or control pcDNA3.1, P65 siRNA, MMP-9 siRNA and its
control siRNA was carried out using the LipofectAmine reagent (Life Technologies,
China) according to the manufacturer's instructions.

The Human VSMCs were obtained from American Tissue Culture Collection (ATCC, USA). The hVSMCs were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, SiJi-Qing, Hangzhou, China) and 1% penicillin-streptomycin. Then, the cells were kept in a humidified incubator of 5% CO₂ at 37°C. The miR-204-5p inhibitor, miR-204-5p mimic, inhibitor control, and mimic control were obtained from GenePharma (Shanghai, China). MMP-9-plasmid, control-plasmid, MMP9-siRNA and control-siRNA were obtained form Santa Cruz. Then, the miRNAs and plasmids were transfected into VSMCs using Lipofectamine^TM 2000 transfection reagent (Invitrogen) in line with the manufacturer’s protocol.

VSMCs isolated from both AS and S plaques were plated in six‐well plates and grown to 60–80% confluency in smooth muscle cell medium without antibiotics. The cells were transfected with either 40 nmol/L MMP‐9 siRNA or scrambled control siRNA (Santa Cruz Biotechnology, Santa Cruz, CA) using Lipofectamine 2000 (Invitrogen) adhering to the manufacturer's instructions for 6 h. The cells were allowed to recover for 24 h in the medium supplemented with 20% bovine serum albumin. The cells were then stimulated with or without EGF (10 ng/mL) for 24 h in serum‐free medium. After harvesting the cells, qPCR was done to quantify mRNA expression of Col I(α1), Col III(α1), MMP‐1, MMP‐9, EGFR and GAPDH genes using the primers listed in Table 1.