Si scramble

The miR-106a-5p mimics (5′-AAAAGUGCUUACAGUGCAGGUAG-3′), miR-106a-5p inhibitor (5′-UAUGGCUUUUUAUUCCUAUGUGA-3′) and scrambled mimic or inhibitor were designed and synthesized by Shanghai GenePharma Co., Ltd. si-STAT3 (5′-GCAGCAGCTGAACAACATG-3′) and si-Scramble (5′-UUCUCCGAACGUGUCACGUTT-3′) were also designed and purchased from Shanghai GenePharma Co., Ltd. miR-106a-5p mimics (50 nM), miR-106a-5p inhibitor (50 nM) or si-STAT3 (100 nM) were transfected into cells (2×10⁴ cells) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. After transfection for 48 h, cells were then exposed to ox-LDL for 48 h and collected for further experiments. Inhibitor experiments were performed using 30 µm STA-21 (Enzo Life Sciences, Inc.) as previously described (28 (link)). Briefly, HUVEC cells were co-treated with miR-106a-5p inhibitor and either 30 µm STA-21 or DMSO for 24 h and then exposed to ox-LDL for 48 h and collected for further experiments.

siRNA targeting SPP1 (siSPP1) and a scrambled siRNA (siScramble) were synthesized by GenePharma (GenePharma, Shanghai, P.R. China) to decrease the expression of SPP1. The siScramble and siSPP1 were transfected to the HCC827AR and PC9AR cells by Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific Inc.) according to the manufacturer’s instructions. After 48 h, the cells were collected for further experiments. To validate the interference efficiency, SPP1 expression was examined by real-time quantitative (qRT)-PCR.

The miR-144-3p mimics and mimics negative control (mimics NC) were obtained from GenePharma. The EZH2 overexpression vector, pcDNA-EZH2, and pcDNA vector were constructed by GenePharma. In addition, EZH2 siRNA (si-EZH2) and si-Scramble were also purchased from GenePharma.
CAL-27 and SCC-4 cells (8.0×10⁵/well) in a 6-well plate grown to approximately 80% confluence, and then respectively transfected with miR-144-3p mimics (20 nM), mimics NC (20 nM), si-EZH2 (50 nM), or 2 µg pcDNA-EZH2 using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). In addition, inhibition experiments were performed using the EZH2 inhibitor, GSK126 (a compound competes with S-adenosyl-methionine for binding to EZH2, thereby inhibiting histone methyltransferase activity without affecting EZH2 protein expression), as previously described (16 (link)). Briefly, the CAL-27 and SCC-4 cells were treated with 5 µM GSK126 (Shanghai HanXiang Life Technology Limited Corporation) or DMSO for 48 h and then collected for use in further experiments. The concentration of DMSO used was ≤1% to ensure the lack of cytotoxicity.