Clinical specimens of OSCC
Eighty-eight pairs of OSCC samples were randomly collected from OSCC patients who underwent surgery at the Affiliated Haikou Hospital and Xiangya Hospital, Central South University from 2012 to 2014. None of OSCC patients received radio- or chemotherapy before their surgery. All tumor tissues and their adjacent noncancerous tissues were immediately frozen stored in liquid nitrogen for subsequent RNA extraction. This study was approved by the Ethics Committee of Affiliated Haikou Hospital and Xiangya Hospital, and all OSCC patients provided the informed consent.
Cell culture
The CAL27(ATCC® CRL-2095), SCC9(ATCC® CRL-1629) and SCC25(ATCC® CRL-1628) cell lines were obtained from American Type Culture Collection (Manassas, VA, USA), The HSC3, NOK and CAL33 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. All cell lines were cultured in a 37°C, 5% CO2 incubator.
Cell transfection
Linc01234 siRNAs and control siRNAs were purchased from RiboBio (Guangzhou, China). CAL27 and SCC25 cells were seeded in the 6-well plate and added with 5μL of siRNAs or siNCs (50 nM) and 5μL of Lipofectamine 2000 (Invitrogen, Carlsbad, USA) in each well following the manufacturers’ instructions.
Subcellular fraction and Real‐time quantitative PCR(RT-qPCR)
The PARIS Kit purchased from Invitrogen (Carlsbad, CA, USA) was applied to isolate cytoplasmic and nuclear RNAs using a previously established protocol, followed by RT-qPCR detection [15]. Total RNA from OSCC tissues and cells was collected using the TRIzol Reagent (Invitrogen Life Technologies) and was subsequently reverse transcribed to cDNA using the PrimeScript RT reagent Kit (Takara, Tokyo, Japan). RT-qPCR detection was performed on a Roche LightCycler 480 system (Bio-Rad, Hercules, CA, USA) using a SYBR Green qPCR Mix (Takara). The relative RNA expression was calculated using the (2−ΔΔCt) method. The ΔCt values were normalized to these of GAPDH or U6.
EdU assay
After 48h transfection, CAL27 and SCC25 cells (2x104/well) were seeded in 24-well plates. The 5-ethynyl-2′-deoxyuridine (EdU) assay (Life Technologies Corporation, USA) was used to evaluate the proliferation ability of OSCC cells as previously reported [16]. Briefly, CAL27 and SCC25 cells were incubated with 100μL EdU reagent for 2h at 37°C, and stained with DAPI and visualized by a fluorescence microscope (Olympus, Tokyo, Japan).
Cell counting Kit-8 (CCK-8) assay
CAL27 and SCC25 cells were cultured in 96-well plates at 8000cells/well after transfection. Following the 4 consecutive days culture, each well was replaced by the fresh medium containing 10% CCK-8 solution (Yeasen, Shanghai, China). After a 2h incubation at 37°C, the absorbance of 450nm was measured using a microplate reader (Bio-Rad, Hercules, CA, USA)
Transwell assays
The migration and invasion ability were assessed using Transwell chamber with 8 μm pore (Corning, New York, NY, USA). To evaluate the invasion capacity, OSCC cells (1×105) suspended in serum-free DMEM were added into the upper chamber precoated with Matrigel matrix (BD Biosciences, San Jose, CA, USA). For migration assay, OSCC cells (1×105) were cultured in the Boyden chamber without Matrigel. After 24h incubation, the migrated and invaded cells were fixed and stained, and counted under a microscope (Leica, Wetzlar, Germany).
Wound-healing assays
CAL27 and SCC25 cells were seeded in the 6-well plate and transfected with siRNAs or siNCs. Until a 90% confluence, we generated a scratch on the bottom of each well. After washing with PBS, cells were photographed using a microscope (Leica, Wetzlar, Germany) at 0 h and 48 h.
Dual-luciferase reporter assays
The sequence of Linc01234 or PAK4 3′-UTR containing the putative or mutated binding sites for miR-433-3p were cloned into the pMIR-REPORT vector (Promega, Madison, WI, USA). The wild-type or mutant pMIR-REPORT vectors were co-transfected into CAL27 and SCC25 cells as long with miR-433-3p mimics and miR-NC. 48h later, the relative luciferase activity was assessed using a dual luciferase assay kit (Promega) and these values were normalized to Renilla activity.
Western blot assays
Total protein from CAL27 and SCC25 cells was lysed in RIPA buffer (Beyotime, China). Then, the lysates were treated with a 10% SDS-PAGE gel and transferred onto PVDF membranes (Millipore Corporation, USA). After 1h incubation in 5% nonfat milk solution, the PVDF membranes were cultured with anti-PAK4 (ab62509, Abcam, Cambridge, UK) and anti-GAPDH (AC003, ABclonal, China) antibodies overnight at 4 ℃. After TBST washing, the membranes were incubated with the matched secondary antibodies (Proteintech, wuhan, China) at 37°C for 1 h. The reaction was visualized by an enhanced chemiluminescence (ECL) detection system (Millipore, MA, USA)
Statistical analysis
All data in this study were performed with SPSS 22.0 (IBM Corp., Armonk, NY, USA) and were 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.