LSCC tissue specimens and patient follow up
In this study, a total of 99 LSCC tissues were obtained from patients who underwent surgical resection at the Sun Yat-sen University Cancer Center between October 2007 and December 2009. All patients with LSCC were confirmed by pathology. The clinico-pathological features were collected and stored in our database. Patients were followed up every 3 months to assess survival status. Overall survival (OS) was recorded from the date of surgery to the date of death or last follow-up (31st May 2015), as previously reported . This retrospective study on tumor material was approved by the Research Ethics Committee of Guangdong General Hospital & Guangdong Academy of Medical Sciences and Sun Yat-sen University Cancer Center (No.GDREC2018029H).
Two human head and neck squamous carcinoma cell lines, Hep-2 and TU212, were purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences (Chinese Academy of Science, Shanghai, China). Both cell lines were routinely tested for mycoplasma contamination every two months in our lab. Tests for mycoplasma were negative for all duration of the experiment. Cells were cultured in RMPI-1640 medium (Gibco, Waltham, MA,USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Waltham, MA,USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Hyclone, Logan, UT, USA). Cells were cultured at 37°C in 5%CO2/95% air in a humidified atmosphere.
Plasmid construction and cell transfection
Two lentivector-mediated short-hairpin OTX1 (sh-OTX1-1 and sh-OTX1-2) and non-targeting plasmids (sh-control) were designed and synthesized by GenePharma (Shanghai, China). A scramble, empty lentiviral vector was used as the control. Lipofectamine 2000 system was used for transfection. The lentivirial vectors carried puromycin resistance. The transfected cells were screened with 0.5 mg/ml of puromycin. Stably transfected cell lines were tested using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and western blot analysis. The transfection procedure was performed according to the manufacturer’s instruction. The following short-hairpin RNA (shRNA) sequences were used: sh-OTX1-1: GCAACACCTCGTGTATGCA；sh-OTX1-2: GCCGACTGCTTGGATTACA.
Immunohistochemistry (IHC) analysis
LSCC tissues were paraffin-embedded and sliced. Slides were then subjected to de-paraffinization/ rehydration, followed by the antigen retrieval in a microwave. Slides were blocked with 5% milk for 1 hour at 25°C, and then incubated with the corresponding anti-human OTX1 antibodies (1:400, MAD5602, Millipore, USA) at 4°C overnight. After PBS washes, the slides were incubated with secondary antibodies at 25°C for 1 hour. Signals for each slide were developed in pre-made 0.05% diaminobenzidine (DAB) containing 0.01% hydrogen peroxidise (H2O2). The results of immunohistochemistry (IHC) were evaluated by two independent investigators who were blinded to the clinical and prognostic data based on the Shimizu criteria . Based on the score criteria, the expression of OTX1 in LSCC tissues was graded as negative (-), weakly positive (+), moderately positive (++), and strongly positive (+++). The expression levels of OTX1 protein in LSCC tissues were dichotomized into the low (-/+) and high (++/+++) expression groups, respectively.
Quantitative real-time PCR
Total RNA was extracted from LSCC tissue samples and cells using Trizol reagent (Invitrogen, USA) according to the manufacturer’s instructions from LSCC tissue samples and cells. Complementary DNA (cDNA) was synthesized from total RNA using the cDNA Takara Kit (NHK, Japan). The transcriptional levels were detected in amplification by RT-PCR using the Power SYBR Green PCR Master Mix (NHK, Japan). Relative OTX1 mRNA expression was calculated using the 2-ΔΔCT method and normalized to the internal control β-actin. The primers used in the study were as follows: OTX1 forward, 5’-GCGTCGTCGCTGAGTACAC-3’ and reverse, 5’-ACATGGGATAAGAGGCTGCTG-3’; β-actin forward, 5’-TCACCAACTGGGA CGACAT-3’ and reverse, 5’-GCACAGCCTGGATAGCAAC-3’.
Cells were lysed in ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer containing 0.1% protease inhibitor. The concentration of total protein was measured using a bicinchoninic acid (BCA) assay kit (Boster, Wuhan, China). Equal amounts of protein were loaded onto 10% sodium dodecyl sulfate ployacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). After blocking with 5% skimmed milk, the membranes were incubated with anti-OTX1 (1:1000 dilutions, MAD5602, Millipore, MA, USA) and β-actin (1:3000 dilutions, CST4967, Cell Signaling Technology, Boston, USA) at 4 °C overnight. Subsequently, the membranes were washed with TBST buffer three times and incubated with the corresponding secondary antibodies for 1 hour at room temperature. Finally, the immunoreactive bands on the membrane were visualized using enhanced chemiluminescence (ECL) reagents (Millipore, Plano, TX, USA).
Cell proliferation was evaluated using the MTS (Qiagen, Hilden, German) assay. Briefly, Logarithmically growing cells were seeded in 96-well plates at a density of 3,000 cells/well in triplicate. After incubation for 24, 48, 72, 96,120, or 144 h, 20 μL of working solution containing MTS and serum-free RPMI-1640 medium was added into each well, followed by another incubation in humidified air with 5% CO2 at 37°C for 2 h. The absorbance was measured at 490 nm using the Synergy™ Multi-Mode Microplate Reader (Biotek, Vermont, USA).
Colony formation assay
For the colony formation assay, 500 cells were seeded in a 6-well plate and cultured in complete medium for 10 days. Cell colonies were fixed with methanol and stained with 0.2% crystal violet for 30 min at room temperature. The number of colonies was counted using Quantity One software (Bio-Rad, Hercules, CA, USA).
Migration, and invasion assays
Cell migration was evaluated using the Transwell migration assay (8-μm pore; BD Biosciences). Briefly, cells were starved in serum-free medium for 2 h. Next, cells were resuspended in serum-free RPMI-1640 medium, and 200 μL of the cell suspension (3 × 106 cells) was added to the upper chamber. A volume of 600 μL of RPMI-1640 medium containing 10% FBS was added to the bottom chamber. After 24 h, cells that attached to the lower surface of the membrane were fixed with methanol and stained with 0.2% crystal violet for 30 min. Finally, migratory cells were counted in five random fields. Th cell invasion assay was performed following the same procedures as described above, except that diluted Matrigel (Corning, New York, USA) was precoated on the upper well of the transwell chambers and incubated for 48h.
Xenografted tumors in nude mice
All animal experiments were performed in accordance with the guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health. This study was approved by the Ethics Committee of Guangdong Provincial People’s Hospital (No.GDREC2018029A). Four-to-five-week old BALB/c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and were raised under the specific pathogen-free environment at 20-22°C and 40-60% humidity. After 1 week of adaptive feeding, the nude mice were randomly divided into six groups (n = 5 for each group) for Hep-2 and TU212 cells: Control, sh-OTX1-1, and sh-OTX1-2 respectively. To stably knock down OTX1, the shRNA-OTX1-1 and shRNA-OTX1-2 were constructed, packaged into lentiviruses, and used to infect Hep-2 and TU212 cells. After this, Hep-2 and TU212 cells infected with shRNA-OTX1-1 and shRNA-OTX1-2 or their corresponding controls were suspended in normal saline (1 × 106 cells in 0.1 mL) and then subcutaneously injected into the right armpit of each mouse to establish a xenograft tumor mouse model. The tumor volume was calculated every 4 days. Twenty-eight days later, the mice were sacrificed under anesthesia. All animals were anesthetized by intravenous injection of barbiturate at a final concentration of 100 mg/kg. The tumors were isolated and fixed in 4% paraformaldehyde solutions and embedded in paraffin before being cut into 4μm thick sections. The prepared sections were stained using haematoxylin and eosin (H&E) and Ki-67 following the routine staining procedure and examined using a microscope.
MiR-129-5p or miR-129-5p-mut mimics were purchased from RiboBio Co., Ltd (Guangzhou, China). DNA fragments from the 3′-untranslated region (UTR) of OTX1 containing the predicted complementary sites of miR-129-5p were constructed into a pGL3-basic plasmid (Addgene, Cambridge, USA). LSCC cells (1× 104) were seeded in 48-well plates in triplicate and settled for 24h. Next, the pGL3-OTX1 -3’-UTR reporter plasmids (100ng) plus 5 ng of pRL-TK Renilla plasmid (Promega, Madison, USA), and increasing amounts (10 and 50nM) of negative control (NC) or miR-129-5p or miR-129-5p-mut mimic were co-transfected into LSCC cells using Lipofectamine LTX reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’s recommendation. Luciferase and Renilla signals were measured 24 h after transfection using the Dual Luciferase Reporter Assay Kit (Promega, Madison, USA) according to a protocol provided by the manufacturer. The sequences of miR-129-5p and miR-129-5p-mut were 5'-cuuuuugcggucugggcuugc-3' and 5'-cuuuuugcggucugguugugc-3', respectively.
All experimental assays were conducted at least three times. Data were represented as means ± standard deviation (SD). The two-tailed Student’s t-test was used to determine the statistical significance of differences between groups. Chi-square or Fisher’s exact test was used to analyse the categorical data. Survival analysis was performed using the Kaplan-Meier method and compared using the log-rank test. Statistical analysis was performed using the Statistical Program for Social Sciences 19.0 software (SPSS, CA, USA) and presented with GraphPad Prism 6.0 (GraphPad Software, CA, USA). Values of p < 0.05 were considered as statistically significant.