Mir 665 inhibitor

LIMT siRNAs (LIMT siRNA1:LIMT siRNA2:LIMT siRNA3=1:1:1), miR-665 siRNA, miR-665 mimics, miR-665 inhibitor and their corresponding negative controls were designed and manufactured by GenePharma (Shanghai, China). Transfections were performed using lipofectamine 2000 (Invitrogen, Carlsbad, USA) according to the manufacturer’s protocol. The sequences are shown in
Table 1.

Table1The siRNA sequences used in this study

Name	Sequence
LIMT-homo-177	Sense: 5′-CCAUUCAUGUCAGCAGUUATT-3′; Antisense: 5′-UAACUGCUGACAUGAAUGGTT-3′
LIMT-homo-295	Sense: 5′-GCAGAACGUGAGGGUGUAATT-3′ Antisense: 5′-UUACACCCUCACGUUCUGCTT-3′
LIMT-homo-888	Sense: 5′-GCUUCCAACCUCCAUUGCATT-3′; Antisense: 5′-UGCAAUGGAGGUUGGAAGCTT-3′
miR-665 mimic	Sense: 5′-ACCAGGAGGCUGAGGCCCCU-3′; Antisense: 5′-GGGCCUCAGCCUCCUGGUUU-3′
miR-665 inhibitor	5′-AGGGGCCUCAGCCUCCUGGU-3′;

Normal gingival epithelial cells (ATCC^® PCS-200-014™) were acquired from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in minimum essential medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific). Dulbecco’s modified Eagle’s medium (DMEM; Gibco; Thermo Fisher Scientific) with 10% FBS was employed in the culture of the TSCC cell line CAL-27 (ATCC). The other two TSCC cell lines SCC-9 and SCC-15 were maintained in a 1:1 mixture of DMEM and Ham’s F12 medium (Gibco; Thermo Fisher Scientific) containing 10% FBS and 400 ng/mL hydrocortisone. All cells were incubated at 37°C in a humidified incubator supplied with 5% CO₂.
Small interfering RNAs (siRNAs) for NOP14-AS1 (si-NOP14-AS1) and negative control (NC) siRNA (si-NC) were designed and offered by Shanghai GenePharma Co., Ltd. (Shanghai, China). The overexpressed high mobility group box 3 (HMGB3) plasmid pcDNA3.1-HMGB3 was constructed by RiboBio (Guangzhou, China). miRNA-665 (miR-665) mimic, NC mimic, miR-665 inhibitor, and NC inhibitor were also obtained from Shanghai GenePharma. TSCC cells were seeded into six-well plates, and transfection was performed by employing Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific).