Forty paired samples of OSCC tissues and adjacent healthy tissues were attained from patients with OSCC hospitalized at XXXX. Written informed consents had been acquired from patients and none of the patients underwent chemotherapy or radiotherapy prior to resection. After surgery, tissues were frozen in liquid nitrogen and stored at − 80 °C. This research protocol was licensed by the Ethics Committee of Affiliated Hospital of Chifeng University.
Normal oral keratinocyte (NOK) and OSCC cells (HSC-4, UM1, HSC-3 and SCC-15) were acquired from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were kept in DMEM (Gibco, Grand Island, NY, USA) adding 10% FBS (Gibco) plus antibiotics (Gibco) in a humidified atmosphere of 5% CO2 at 37 °C.
Specific shRNAs against NORAD (sh-NORAD#1 and sh-NORAD#2) and their corresponding NC (sh-NC), along with the pcDNA3.1 vector targeting TPM4 and the empty vector, were gained from Genechem (Shanghai, China). Simultaneously, miR-577 mimics and NC mimics were gained from GenePharma (Shanghai, China). HSC-4 or UM1 cells were selected for the transfection with each of these plasmids through Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA).
Quantitative real-time RT-PCR (RT-qPCR)
With the application of Trizol (Invitrogen), total RNA was isolated and converted to cDNA by using the Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). The cDNA samples were assayed by qRT-PCR with the SYBR Premix Ex Taq Kit (Takara, Tokyo, China) and Stratagene Mx3000P (Agilent Technology, Austin, TX, USA). The expression levels of genes were normalized to the expression level of GAPDH or U6 as per 2−ΔΔCt method.
Cell counting kit-8 (CCK-8) assay
Cell viability was evaluated through Cell Counting Kit-8 (CCK-8) analysis according to the manufacturer’s guidelines. Transfected HSC-4 or UM1 cells were cultured in 96-well culture dishes. After that, 10 µl of CCK-8 solution (Solarbio, Beijing, China) was added to each well upon adherence, and further incubated at 37 °C with 5% CO2 for 2 h. Absorbance of samples was explored at 450 nm via the MRX II microplate reader (Dynex, Chantilly, VA, USA).
Colony formation assay
As for colony formation assay, transfected HSC-4 or UM1 cells were placed into 6-well plates for 14 days of thermostatic culture. Upon this, colonies were gained and washed by using PBS (Sigma-Aldrich, St. Louis, MO, USA) before being immobilized for 15 min using paraformaldehyde (PFA; Sigma-Aldrich) and dyed for 10–30 min in crystal violet (Sigma-Aldrich) at room temperature. The optical microscope (Nikon, Tokyo, Japan) was applied for observing and counting the colonies.
5-Ethynyl-20-deoxyuridine (EdU) incorporation assay
The EdU assay kit (RiboBio, Guangdong, China) was applied for the exploration of cell proliferation. Transfected HSC-4 or UM1 cells were inoculated into 96-well plates overnight, followed by being incubated for 2 h with EdU solution. Cells were sequentially fixed for 30 min utilizing 4% PFA and incubated for 5 min with glycine (Sigma-Aldrich). For the next step, cells were permeabilized for 10 min using PBS of 0.5% Triton X-100 (Sigma-Aldrich) and stained for 30 min using Apollo reaction solution (RiboBio). Upon being washed several times by PBS of 0.5% Triton X-100, cells were stained for 30 min in DAPI (Sigma-Aldrich) in the dark room. Finally, cells were imaged via a microscope (Nikon).
Flow cytometry for apoptotic cells
Transfected HSC-4 and UM1 cells were firstly collected and washed with PBS. Then, they were stained by using the Annexin V-FITC/PI apoptosis detection kit (BD Biosciences, Franklin Lakes, NJ, USA). Apoptotic rate of the cells was assessed by a flow cytometer and the data collected were analyzed via FACScan (BD Biosciences).
Transwell migration assay
Migration ability of transfected HSC-4 and UM1 cells was evaluated by utilizing the Transwell System (Costar, Cambridge, MA, USA). Cells in serum-free medium were added to the upper wells, whereas medium with 10% FBS was put to the lower chambers. Twenty-four hours later, cells in the upper layer were removed with caution by a cotton swab and then fixed in methanol solution for 15 min. Crystal violet was adopted to stain the membranes for 10 min, and the invaded or migrated cells were observed and counted under a microscope (10 × 10).
Western blot analysis
Total protein was extracted from transfected HSC-4 or UM1 cells with RIPA buffer (Beyotime, Shanghai, China). After quantification, total protein was separated by SDS-PAGE (Bio-Rad, Hercules, CA, USA), and electrically transferred onto PVDF membranes (Millipore, Bedford, MA, USA). Membranes were blocked for 1 h employing 5% defatted milk, followed by individually incubated with primary antibodies against Cleaved Caspase-3 (ab2302), Caspase-3 (ab13847), Bax (ab32503), Bcl-2 (ab32124), E-cadherin (ab40772), N-cadherin (ab76057), TPM4 (ab181085) and GAPDH (ab8245) acquired from Abcam (Cambridge, USA). Upon being washed for three times using 0.1 % TBST (Sigma-Aldrich), membranes were incubated for additional 1 h with secondary antibodies. The washing process was repeated and bands were examined by an ECL reagent (Thermo Fisher Scientific).
Fluorescence in situ hybridization (FISH) assay
HSC-4 and UM1 cells were washed with PBS after being inoculated to glass coverslips in 24-well plates, followed by the fixation of 30 min in 4% formaldehyde (Sigma-Aldrich). After the permeabilization in 70% ethanol (Sigma-Aldrich) overnight, cells were rinsed twice by PBS. Hybridization solution (Sigma-Aldrich) as well as fluorescently labeled NORAD probe (GenePharma) was added during the overnight incubation. Three hours after hybridization, DAPI was adopted to stain cell nuclei and the cells were washed with saline-sodium citrate (SSC; Sigma-Aldrich). In the end, fluorescence microscope was utilized to observe and analyze the stained cells (Olympus Corp., Tokyo, Japan).
RNA pull-down assay
The Pierce™ RNA3′ End Desthiobiotinylation Kit was utilized for RNA pull-down assay. Cell lysates of HSC-4 and UM1 cells were incubated with Bio-miR-577-Wt/Mut or Bio-NC. Cell protein extracts were then prepared to be mixed with magnetic beads (Invitrogen) and Bio-NC or Bio-miR-577. NORAD or TPM4 level in the complex which was pulled down by biotinylated RNAs was assayed by RT-qpCR analysis.
Luciferase reporter assay
The wild-type or mutant interacting sequences of miR-577 in NORAD or 3′-UTR of TPM4 were sub-cloned into pmirGLO dual-luciferase vector (Promega, Madison, WI, USA) to establish pmirGLO-NORAD-Wt/Mut or pmirGLO-TPM4-Wt/Mut which was co-transfected into HSC-4 and UM1 cells with miR-577 mimics or NC mimics. To examine luciferase activities, dual luciferase reporter assay system (Promega) was adopted after 48 h.
RNA immunoprecipitation (RIP) assay
With the RNA-Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA), RIP assay in HSC-4 and UM1 cells was achieved with the specific antibodies and normal control anti-IgG (Abcam) and anti-Ago2 (Abcam). Lysates were obtained from OSCC cell lines using RIP lysis buffer. The lysis was incubated with the magnetic beads conjugated with the Ago2 antibody or IgG antibody (negative control). The precipitated RNAs were analyzed by RT-qPCR assay.
Data were presented as mean ± SD. All assays were conducted for three times. To analyze the differences between groups, Student’s t-test or one-way ANOVA was applied. With the application of Prism 7.0 software (GraphPad, San Diego, CA, USA) or SPSS 17.0 software (SPSS, Chicago, IL, USA), data were analyzed statistically, with the probability (P) < 0.05 as threshold.