Patient tissue samples
Human OSCC tissues and para-cancerous specimens (more than 2 cm away from tumor tissues) were obtained from Tianjin Stomatological Hospital, and the category of all OSCC tissues was confirmed by pathological analysis. The patients referred to the 8th edition of UICC/AJCC oral squamous cell carcinoma tumor node metastasis (TNM) staging criteria. Moreover, none of the patients received the radiotherapy or chemotherapy before the operation. Clinical characteristics and demographics of the patients in this study were summarized in Table 1. All patients signed informed consent prior to using the tissues for this study according to the principles of the Declaration of Helsinki. Te tumor tissues were immediately frozen in liquid nitrogen and then stored at –80 °C for further research. This study was approved by the Ethics Committee of Tianjin Stomatological Hospital (Tianjin, People’s Republic of China [PRC]).
Cell culture and transfection
Four OSCC cell lines (TSCCA, CAL–27, SCC–9, and Tca8113) and the normal human oral keratinocyte (NHOK) were purchased from the Cell Bank of Type Culture Collection of 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.
RNA extraction and quantitative real time-polymerase chain reaction (qRT-PCR)
Total RNA was extracted from OSCC tissues or cells using the TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. qRT-PCR was performed using the All-in-One™ miRNA qRT-PCR detection kit (GeneCopoeia, Rockville, MD, USA) for miR–218–5p and U6 as the internal control. The relative expression level of mRNA was detected using SYBR Green qRT-PCR assay (Bio-Rad Laboratories Inc, Hercules, CA, USA), and GAPDH was used as the internal control. All qRT-PCR procedure was performed on the ABI 7500 thermocycler (Thermo Fisher Scientific, Waltham, MA, USA). The sequences of the primers were: HOXA-AS3, F: 5’-GCTGAATTAACGGTGGCTCC–3’, R:5’-ATGGCGAGCGAAGGGAAG–3’; GAPDH, F: 5’-GGAATCCACTGGCGTCTTCA–3’, R: 5’-GGTTCACGCCCATCACAAAC–3’. The specific primers for miR–218–5p and U6 were purchased from RiboBio (Guangzhou, PRC). The relative expression levels of detective genes were calculated using the 2−ΔΔCt method.
Cell proliferation analysis
Cell Counting Kit–8 reagent (Dojindo, Kumamoto, Japan) was used to measure the proliferation of OSCC cells. The OSCC cells were seeded into 96-well plates after transfection for 48 h. After culturing for 12 h, 24 h, 48 h and 72 h respectively, and then CCK–8 reagent was added to each well with 10 μl and further incubated for 4 h. The optical density (OD) value of each well was detected using an enzyme labeling instrument at 450 nm.
Colony formation assay
The OSCC cells were inoculated into 6-well plates with 200 cells in each well after transfection for 48 h. Subsequently, the cells were cultured in the complete medium for 14 d. At the first time, the medium was replaced after 5 d, and then replaced every 3 d. When the cell colonies formed, the medium was sucked dry. Then, the cells were washed twice with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde at 4 °C for 1 h. Next, the cells were stained with 0.1% crystal violet staining solution for 20 min. Finally, the number of cell colonies containing >50 cells in each well was calculated and photographed.
Luciferase reporter assay
Online Software Starbase v2.0 (http://starbase.sysu.edu.cn) was used to predict the target miRNAs of lncRNA HOXA-AS3. The wild-type HOXA-AS3 3’-UTR containing the miR–218–5p seed sequence fragment (HOXA-AS3 Wt) and mutant-type (HOXA-AS3 Mut) luciferase vectors were conducted. The OSCC cells were seeded into 48-well plates and were co-transfected with the luciferase vectors and miR–218–5p mimics or negative control using Lipofectamine® 2000 Reagent for 48 h. This assay was normalized with 0.05 μg of the RFP expression vector pDsRed2-N1 (Clontech, USA). Subsequently, cells were lysed with RIPA lysis buffer, and the luciferase activity and RFP intensity were detected with the F–4500 Fluorescence Spectrophotometer (Hitachi, Japan) according to the manufacturer’s instructions.
RNA immunoprecipitation (RIP) assay
RIP assay was used to detect the sponge function of HOXA-AS3 on miR–218–5p by using Magna RIPTM RNA Immunoprecipitation Kit (Millipore, Bedford, MA,USA). Briefly, the OSCC cells were transfected with miR–218–5p mimics, Vector-HOXA-AS3 or corresponding controls for 48 h, and then were lysed using the lysis buffer. Next, cell lysates were incubated with anti-Ago2 (Abcam, UK) or anti-IgG (Abcam, UK) and protein A/G magnetic beads. Finally, co-precipitated RNAs were detected by qRT-PCR.
Statistical analysis
Statistical analyse was performed using SPSS version 20.0 (IBM, Armonk, NY, USA) Data are presented as the mean ± standard deviation. Differences among multiple groups were analyzed by ANOVA (one-way) followed by Tukey t-test, and differences between the two groups were analyzed using the student’s t-test. Correlation analysis between HOXA-AS3 and miR–218–5p expression was assessed using Pearson’s correlation coefficient. The prognosis survival time of patients was evaluated using Kaplan-Meier analysis, and Log-rank test was used to examine the difference between different curves. P<0.05 was considered to indicate a statistically significant difference.