Mir 218 5p mimics

The conserved binding sites between miR-218-5p and HO-1 were obtained using the online predict software TargetScan (http://www.targetscan.org) and miRDB (http://www.mirdb.org/miRDB/). miR-Con, miR-218-5p mimics and inhibitors were synthesized by RiboBio (Guangzhou, China). GMCs were seeded in 6-well plates and transfected with miR-Con, miR-218-5p mimics and inhibitors using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 48 h at 37 °C according to the manufacturer’s protocol. The wild-type (WT) and mutant-type (MT) 3′-UTR of HO-1 were inserted into the multiple cloning sites of the luciferase expressing pMIR-REPORT vector (Ambion; Thermo Fisher Scientific, Inc.). For the luciferase assay, GMCs (1 × 10⁵) were seeded into 24-well plates and co-transfected with luciferase reporter vectors containing the WT or MT 3′-UTR of HO-1 (0.5 μg) and mimic sequences of miR-218-5p (100 nM) using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). The luciferase activity was measured using a luciferase reporter assay kit (Promega, Madison, WI, USA) according to the manufacturer’s protocol.

Four OSCC cell lines (TSCCA, CAL-27, SCC-9, and Tca8113) and NHOK cells were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, PRC). All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco, Waltham, MA, USA), 100 IU/mL of penicillin, and 100 μg/mL of streptomycin at 37°C incubator with 5% CO₂.
shRNA containing the HOXA-AS3 interference sequence (sh-HOXA-AS3) and negative control (sh-NC) were purchased from GeneChem (Shanghai, PRC) and miR-218-5p mimics, anti-miR-218-5p and negative control (miR-NC) were purchased from RiboBio (Guangzhou, PRC). Transfection was performed using Lipofectamine® 2000 Reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer’s protocol in OSCC cells. The culture medium was replaced 6 h after transfection, and transfection efficiency was examined with the expression vector of red fluorescent protein (RFP) at 48 h after the transfection.