Clinical specimens of OSCC
OSCC specimens were randomly selected from 88 patients who had undergone surgery at Affiliated Haikou Hospital and Xiangya Hospital and surgically proven primary OSCC. None of the OSCC patients had received chemoradiotherapy prior to surgery. We collected tumor tissues and their adjacent non-cancerous tissues from each case. All specimens were immediately frozen in liquid nitrogen until used for the subsequent RNA extraction. This study was approved by the Ethics Committee of Affiliated Haikou Hospital and Xiangya Hospital. All patients have been informed and written the consents.
Cell culture and transfection
The CAL27(ATCC® CRL-2095), SCC9(ATCC® CRL-1629) and SCC25(ATCC® CRL-1628) cell lines were purchased from American Type Culture Collection (Manassas, VA, USA), The HSC3(HTX2055), NOK(HTX2992) and CAL33(CBP60579) cell lines were kindly gifted from Guanghua School of Stomatology of Sun yet-san University. CAL27, CAL33 and HSC3 cells were cultured in DMEM (Gibco) supplemented with 10% FBS (Invitrogen, Carlsbad, CA, USA). SCC25 and SCC9 cells were cultured in DMEM/F-12 (Gibco) supplemented with 10% FBS. Normal oral keratinocytes (NOKs) were cultured in KSFM medium (Gibco) supplemented with EGF. Linc01234 siRNAs and control siRNAs were purchased from RiboBio (Guangzhou, China). CAL27 and SCC25 cells were transfected with Linc01234 siRNAs or negative controls (50nM) using Lipofectamine 2000 (Invitrogen, Carlsbad, USA) according to the manufacturers’ indications.
Real‐time quantitative PCR(RT-qPCR)
Total RNA was extracted from OSCC tissues and cells by TRIzol Reagent (Invitrogen Life Technologies). 1000ng total RNAs were reverse transcribed using the PrimeSxript RT reagent Kit (Takara, Tokyo, Japan). RT-qPCR was performed using SYBR Green qPCR Mix (Takara, Tokyo, Japan) with a Roche LightCycler 480 system. The ΔCt values of Linc01234 were normalized to GAPDH or U6.
Cell proliferation assays
For EDU assays, CAL27 and SCC25 cells (2x104/well) were cultured in 6-well plates prior to the transfection. 48h later, the ability of cell proliferation was detected by 5-ethynyl-2′-deoxyuridine (EDU) assay (Life Technologies Corporation, USA) as previously reported [16]. Then, CAL27 and SCC25 cells were stained with DAPI and visualized by a fluorescence microscope (Olympus, Tokyo, Japan). The EDU+ rate was measured with the ratio of the number of EDU-positive cells (green cells) to the total number of DAPI-positive cells (blue cells). For the CCK8 assays, CAL27 and SCC25 cells were cultured in the 96-well plate for 4 consecutive days. Then, the new medium containing 10μl CCK8 solution was replaced in each well, followed by a 1h incubation at 37 °C, and the absorbance was measured at 450nm.
Transwell assays
1×105 OSCC cells were suspended in serum-free DMEM medium and seeded in the upper chambers (BD Biosciences, San Jose, CA, USA) pre-coated with Matrigel (Corning, New York, NY, USA) (for invasion assays) or boyden chamber without Matrigel (for migration assays) and incubated for 24h. The migrated and invaded OSCC cells on the lower compartment were stained and counted with a microscope (Leica, Wetzlar, Germany).
Wound-healing assays
Two OSCC cells were cultured in 6-well plates until 90% confluent. After scratch on the bottom, CAL27 and SCC25 cells were washed with PBS and taken photographs using a microscope (Leica, Wetzlar, Germany) at 0 h and 48 h, respectively.
Subcellular fraction
The subcellular fractionation was performed with a PARIS Kit purchased from Invitrogen (Carlsbad, CA, USA). 1 × 107 CAL27 and SCC25 cells were collected and isolated using a previously established protocol [15].
Dual-luciferase reporter assays
The Linc01234 fragments or 3′-UTR sequence of PAK4 with potential miR-433-3p binding site or mutants were cloned into the pMIR-REPORT plasmids. The wild type or mutant plasmids were co-transfected into OSCC cells as well as miR-433-3p mimics or miR-NC. 48h later, luciferase activity was examined with a dual luciferase assay kit (Promega, Madison, WI, USA) and these results were normalized to Renilla activity.
Western blot assays
CAL27 and SCC25 cells were collected and lysed by RIPA buffer (Beyotime, China). The protein extracts were separated with SDS-PAGE gel and transferred into PVDF membranes (Millipore Corporation, USA). Then, the PVDF membranes were blocked in 5% nonfat milk solution at RT and cultured with were incubated with anti-PAK4 (ab62509, Abcam, Cambridge, UK) and anti-GAPDH (AC003, ABclonal, China) overnight at 4℃, followed by incubating with the suitable secondary antibodies. The reaction was detected by an enhanced chemiluminescence (ECL) detection system (Millipore, MA, USA)
Statistical analysis
All data were calculated with SPSS 22.0 (IBM Corp., Armonk, NY, USA) and expressed as the mean ± standard deviation of at least three independent experiments. For CCK8 experiments, colony formation assay, Transwell, wound-healing assays, dual-luciferase reporter detection, RT-qPCR and western blotting, each of them was independently repeated 3 times. Comparisons were performed using two-tailed Student’s t-test or one-way ANOVA. The correlation between Linc01234 expression and clinicopathological parameters was analyzed using the χ2 test. P<0.05 was considered to indicate a statistically significant difference.