Clinical specimens of OSCC
OSCC specimens were randomly selected from 88 patients who had undergone surgery at Affiliated Haikou Hospital and Xiangya Hospital and had surgically proven primary OSCC. None of the OSCC patients had received chemoradiotherapy prior to surgery. We collected tumor tissues and their adjacent noncancerous tissues from each case. All specimens were immediately frozen in liquid nitrogen until use for subsequent RNA extraction. This study was approved by the Ethics Committee of Affiliated Haikou Hospital and Xiangya Hospital. All patients were informed about the study and provided written consent.
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, NY, USA) supplemented with 10% FBS (Invitrogen, Carlsbad, CA, USA). SCC25 and SCC9 cells were cultured in DMEM/F-12 (Gibco, NY, USA) supplemented with 10% FBS. Normal oral keratinocytes (NOKs) were cultured in KSFM (Gibco, NY, USA) 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 (50 nM) using Lipofectamine 2000 (Invitrogen, Carlsbad, USA) according to the manufacturers’ instructions.
Real‐time quantitative PCR(RT-qPCR)
Total RNA was extracted from OSCC tissues and cells by TRIzol Reagent (Invitrogen Life Technologies). Total RNA (1000g) was reverse transcribed using the PrimeScript 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 those of GAPDH or U6.
Cell proliferation assays
For Edu assays, CAL27 and SCC25 cells (2x104/well) were cultured in 6-well plates prior to transfection. Four-eight hours later, cell proliferation was detected by the 5-ethynyl-2′-deoxyuridine (Edu) assay (Life Technologies Corporation, USA) as previously reported . Then, CAL27 and SCC25 cells were stained with DAPI and visualized by a fluorescence microscope (Olympus, Tokyo, Japan). The Edu+ rate was measured as the ratio of the number of Edu-positive cells (green cells) to the total number of DAPI-positive cells (blue cells). For the CCK8 assay, CAL27 and SCC25 cells were cultured in 96-well plates for 4 consecutive days. Then, new medium containing 10 μl CCK8 solution was replaced in each well, followed by a 1 h incubation at 37°C, and the absorbance was measured at 450 nm.
OSCC cells (1×105) were suspended in serum-free DMEM and seeded in the upper chamber (BD Biosciences, San Jose, CA, USA) precoated with Matrigel (Corning, New York, NY, USA) (for invasion assays) or a Boyden chamber without Matrigel (for migration assays) and incubated for 24 h. The migrated and invaded OSCC cells on the lower compartment were stained and counted under a microscope (Leica, Wetzlar, Germany).
Two OSCC cell lines were cultured in 6-well plates until 90% confluence. After generating a scratch on the bottom of the well, CAL27 and SCC25 cells were washed with PBS and images were acquired using a microscope (Leica, Wetzlar, Germany) at 0 h and 48 h.
Subcellular fractionation was performed with a PARIS Kit purchased from Invitrogen (Carlsbad, CA, USA). CAL27 and SCC25 (1 × 107) cells were collected and isolated using a previously established protocol .
Dual-luciferase reporter assays
The Linc01234 sequence and 3′-UTR sequence of PAK4 with potential miR-433-3p binding site or mutated binding site were cloned into the pMIR-REPORT plasmid. The wild-type or mutant plasmid were cotransfected into OSCC cells as long with miR-433-3p mimics or miR-NC. Four-eight hours 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 in RIPA buffer (Beyotime, China). The protein extracts were separated with SDS-PAGE gel and transferred onto PVDF membranes (Millipore Corporation, USA). Then, the PVDF membranes were blocked in 5% nonfat milk solution at RT and cultured with anti-PAK4 (ab62509, Abcam, Cambridge, UK) and anti-GAPDH (AC003, ABclonal, China) antibodies overnight at 4 ℃, followed by incubation with the corresponding secondary antibodies. The reaction was detected by an enhanced chemiluminescence (ECL) detection system (Millipore, MA, USA)
All data calculation was performed with SPSS 22.0 (IBM Corp., Armonk, NY, USA) and are expressed as the mean ± standard deviation of at least three independent experiments. CCK8 experiments, colony formation assay, Transwell, wound-healing and dual-luciferase reporter assays, RT-qPCR and Western blotting, were each 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.