Ethics statement and tissue samples
The OSCC tumor tissues and matched adjacent normal tissues (ANTs) in this study were collected from 82 patients with OSCC under surgery at the Department of Oral and Maxillofacial Surgery, Nanfang Hospital, Guangzhou, Guangdong Province after obtaining informed consent. The diagnosis was pathologically confirmed by the Department of Pathology, Nanfang Hospital. ANTs located at least 1.5 cm from the edge of the tumor were defined as a normal control. This study was approved by Research Scientific Ethics Committee of NanFang Hospital (NFEC-2018-027).
Cell culture
The human tongue squamous cell lines (SCC9, SCC15, SCC25), normal human oral keratinocyte cell line (HOK) and human embryonic kidney cell line (HEK 293T) were obtained from the Institute of Antibody Engineering, Southern Medical University (Guangzhou, China). All cells were cultured in DMEM culture medium (Gibco, NY, USA) with 10% Fetal bovine serum (ExCell Bio, Shanghai, China) and 100U/ml Penicillin-Streptomycin (Invitrogen, CA, USA) at 37°C in a humidified 5% CO2.
Expression plasmid, RNA oligonucleotides and transfection
The AC007271.3 overexpression vector pcDNA3.1(+)-AC007271.3 and its corresponding control vector pcDNA3.1(+), specific siRNAs targeting AC007271.3 (si-AC007271.3), Slug(si-Slug 1, si-Slug 2, si-Slug 3) and the scramble negative control siRNA (si-AC007271.3 NC, si-Slug NC) were obtained from Sangon Biotech Corp., Ltd. (Shanghai, China). The miR-125b-2-3p inhibitor, miR-125b-2-3p micmic and the negative control (miR-125b-2-3p micmic NC, miR-125b-2-3p inhibitor NC) were purchased from RiboBio Corp, Ltd. (Guangzhou, China). Transfections were performed with Lipofectamine 3000 reagent (Invitrogen, CA, USA) following the manufacturer's protocols.
RNA extraction and Quantitative real-time polymerase chain reaction (qRT-PCR)
Total cellular RNA from the cells was extracted by RNA isolater Total RNA Extraction Reagent (Vazyme, Nanjing, China) following the manufacturer’s instructions. HiScript Q RT SuperMix for qPCR(+g DNA wiper)(Vazyme, Nanjing, China) and miRNA 1st Strand cDNA Synthesis Kit (by stem-loop) (Vazyme, Nanjing, China) were used to generate cDNA. With ChamQ® SYBR® qPCR Master Mix (Vazyme, Nanjing, China), qRT-PCR was performed on the Real-Time PCR detection system (Applied Biosystems, CA, USA). All expression levels were normalized against the GAPDH or U6 (for miRNA) mRNA level. The primer sequences are listed in Supplemental Data 3.
RNA pull‐down assay
Purified RNAs were biotin-labeled using Pierce RNA 3′End Desthiobiotinylation Kit (Thermo Fisher Scientific, CA, USA). Biotin-labeled wild-type miR-125b-2-3p (miR-125b-2-3p-Bio), Biotin-labeled mutant-type miR-125b-2-3p (miR-125b-2-3p-Bio-MUT) and negative control (NC-Bio) were incubated with SCC9 cell lysates over night at 4°. Magnetic beads were added to each mixture for 1 hour at room temperature. The eluted RNAs were detected by qRT-PCR.
Western blot and Anti-body
After 72-hours transfection with RNA oligonucleotides or plasmid, OSCC cells were lysed with RIPA buffer. Cell lysates were separated on 10% SDS–polyacrylamide gel (16mA,1.5h) and then transferred to polyvinylidene fluoride membrane (PVDF) (100V,1.5h) (Bio-Rad Laboratories). After blocking (5% non-fat dry milk in PBS Tween) for 2.5h at room temperature, the primary antibodies were used for incubation at 4°C overnight and the secondary antibodies were incubated one hour at room temperature. ECL reagent (Millipore, Bedford, MA) was used for chemiluminescence. Primary antibodies were listed as follow: Rabbit anti-Slug (1:1000, CST, MA, USA, #9585), Rabbit anti-α-tubulin (1:1000, Abclonal, Wuhan, China, #AC007), Rabbit anti-N-cadherin (1:1000, Abclonal, Wuhan, China, #A3045), Rabbit anti-E-cadherin (1:1000, Abclonal, Wuhan, China, #A11509), Rabbit anti-Vimentin (1:2000, Proteintech, Chicago, USA, #10366-1-AP), Rabbit anti-NFKB1 (1:1000, CST, MA, USA, #13586), Rabbit anti-phospho-NFKB1-S337 (1:1000, Abclonal, Wuhan, China, #AP0125), Rabbit anti-p65 (1:1000, CST, MA, USA, #8242), Rabbit anti- Phospho-p65-Ser536 (1:1000, CST, MA, USA, #3033), Rabbit anti-Histone H3 (1:2000, CST, MA, USA, #4499).
Cell migration and matrigel invasion assay
OSCC cells transfected with RNA oligonucleotides or plasmid for 48h were digested and resuspended in DMEM culture. 5×104 cells were seeded into the upper chambers of 8-μm transwell inserts (Corning, NY, USA), and 500μl DMEM containing 20% FBS was added to the lower chamber. After 36 hours incubation at 37 °C, the membranes were fixed with methanol and stained with 0.1% crystal violet. Removed the cells on the top of the membranes and then counted the number of cells attached to the membranes under an inverted microscope. For invasion assay, matrigel (Corning, NY, USA) was diluted with DMEM (at 1:10) and covered the top membranes before cells seeding. Other steps were the same as migration assay.
Dual luciferase reporter assay
PmiRGLO Dual Luciferase miRNA Target Expression Vector (Promega, WI, USA) was used to construct the wild type plasmids (AC007271.3-WT, Slug-WT) which containing the potential binding sites. The mutant type plasmids (AC007271.3-MUT, Slug-MUT) were obtained from the wild type plasmid by replacing the targeted sequence (Replaced ACTTGTG with TCATCTC for AC007271.3-MUT, replaced ACTTGTGA with ACTGTCGA for Slug-MUT). All vectors were constructed by TsingKe Biological Technology Corp, Ltd. (Beijing, China). AC007271.3 WT (MUT) or Slug WT(MUT) and miR-125b-2-3p mimics (miR-125b-2-3p micmic NC) were co-transfected into HEK 293T cells. The Luc-Pair Dual-Luciferase Assay Kit 2.0 (GeneCopoeia, MD, USA) was used to detect the firefly and renilla luciferase activity. Firefly/renilla luciferase activity ratio represents the relative luciferase activities.
Hematoxylin & Eosin (H&E) and Immunohistochemical (IHC) staining
For histological examination, tissues were fixed with 4% paraformaldehyde at room temperature for 24 hours and washed with 70% alcohol. All tissues were embedded in paraffin and cut into sections (4 µm thick). Hematoxylin and eosin were used for H&E staining. For IHC staining, after dewaxing, washing, rehydration, antigen retrieval and endogenous peroxidase blocking following the manufacturer's protocols. Antibody Rabbit anti-Slug (1:50, Abclonal, Wuhan, China,#A1057) was used to incubate at 4°C overnight. Secondary biotinylated conjugated goat anti-rabbit antibody was incubated at room temperature for 30mins. DAB (3,3'-diaminobenzidine) were used as chromogens and hematoxylin used as counterstains. Images were acquired under the light microscope (Leica, DM 2500, Wetzlar, Germany). The comparison of staining results between tumor tissues and ATNs was performed by two independent pathologists who were blinded to the clinical information of patients. The scoring criteria of staining were as follow, first was staining intensity: 0-none staining, 1-light yellow, 2-deep yellow, or 3-brown; and the second criterion was staining cells proportion: 0 (<5%), 1(5–25%), 2 (25–50%), 3 (51–75%) or 4 (>75%). The product of the two scores was considered as the final score. The final scores were divided into two levels: 0–5 (Slug low expression) and more than 5 (Slug high expression).
Animal experiment
Lentivirus-NC and lentivirus-AC007271.3 were purchased from Obio Technology Corp., Ltd. (Shanghai, China) and transfected into SCC9 according to the operating instructions. Puromycin was used to select the AC007271.3 stably expressed cells (SCC9-AC007271.3) and its control cells (SCC9-NC). Five-weeks-old female BALB/c nude mice were randomly divided into three groups (blank, SCC9-AC007271.3 and SCC9-NC). Approximately 1 × 106 SCC9-AC007271.3 cells or SCC9-NC cells were resuspended in 500 μL of PBS and then injected via tail vein. Simultaneously, injection of 500 μL of PBS without cells suspension was regarded as blank group. After 8 weeks, all mice were sacrificed, and the visible lung metastases were counted. Lung metastases were excised for HE or IHC staining. The expression of AC007271.3 and miR-125b-2-3p were detected by qRT-PCR and the expression of Slug and EMT markers were inspected by western blot. This animal experiment was approved by Southern Medical University Experimental Ethics Committee (L2018199), and all BALB/c nude mice were purchased from the Animal Care Unit of Southern Medical University.
AC007271.3 Promoter Region Cloning
The sequences of the 2000bp before the 5’UTR of AC007271.3 were regarded as promoter region and obtained from Ensemble database (http://asia.ensembl.org/index.html). According to the sequences, we designed several primers with specific primers containing restriction enzyme protection sites of KpnI and Bgl II, respectively, for amplification the fragments (-1998/-2, -1508/-2, -1000/-2, -519/-2). Genomic DNA was used as a template to amplify by KOD-Plus-Neo (TOYOBO, Osaka, Japan) amplification enzyme. The reaction conditions were set following the instruction. 1% agarose gel electrophoresis was performed to select the bands in correct size and confirmed through sequencing (Sangon, Shanghai, China). The pGL3-Basic vector and correct fragments were digested with KpnI (TakaraBio, Dalian, China) and Bgl II (TakaraBio, Dalian, China) restriction enzymes. DNA ligation kit ver.2.1 (TakaraBio, Dalian, China) was used to connect the fragments and pGL3-Basic vector. DH5α competent cells were used for transformation. After the monoclonal colony sequence confirmation, the plasmids were extracted by using HiPure Plasmid EF Mini Kit (Magen, Guangzhou, China) for identification of core promoter of AC007271.3 by Dual luciferase reporter assay.
Chromatin Immunoprecipitation (ChIP) assay
ChIP assay were carried out by Magna ChIPTM A / G Kits (Merck, Darmstadt, German) according to the manufacturer's protocols. Briefly, SCC9 cells were crosslinked with 1% formaldehyde, glycine quenching and lysed. The nuclear DNA was fragmented by sonication. NFKB1 (p50) specific antibody (1:50, CST, MA, USA, #13586) was used for immunoprecipitation of cross-linked protein / DNA. Protein / DNA complexes were eluted and reverse cross-linked into free DNA. Purified DNA was used for qRT-PCR analysis to detect the enrich fragments.
Bioinformatics analysis
Coding Potential Caculator (http://cpc.cbi.pku.edu.cn/) were performed to predict the protein coding ability of AC007271.3. LncBase Predicted v.2 database (http://carolina.imis.athena-innovation.gr/diana_tools/web/index.php?r=lncbasev2%2Findex-predicted) were used to predict the potential miRNAs regulated by AC007271.3. TargetScan (http://www.targetscan.org/) were carried out to predict the target gene’s mRNA of miRNAs. The transcriptome profiling datum of OSCC were download from the The Cancer Genome Atlas (TCGA) database(https://portal.gdc.cancer.gov/) through GDC Data Transfer Tool. The Slug mRNA sequence data and relevant clinical information of 319 cases of OSCC and 32 cases of cancer adjacent normal tissues were extracted from transcriptome profiling by R software (Version 3.6.1). The miR-125b-2-3p expression analysis results of OSCC and the GEO accession number (GSE45238) were obtained from dbDEMC 2.0 (https://www.picb.ac.cn/dbDEMC/). The miRNA sequence data of GSE45238 were downloaded from Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE45238) and extracted the expression data of miR-125b-2-3p. Gene-regulation online website (http://gene-regulation.com/pub/programs.html) and JASPAR database (http://jaspar.genereg.net/) were used for transcription factor prediction.
Statistical analysis
All experimental assays were performed in triplicate. All data were presented as mean ± standard deviation (SD) of triplicate replicates and analyzed by SPSS statistics 20.0 (Chicago, USA). All statistical charts were manufactured in Graphpad Prism 7.0 (CA, USA). Student’s t-test was performed to compare the differences of two groups. The correlation between miR-125b-2-3p (or Slug) and clinicopathological features was analyzed by using Fisher’s exact tests. Pearson’s correlation coefficient analysis was used to analyze the correlation between AC007271.3 and miR-125b-2-3p. Kaplan–Meier (K–M) curve was applied for detecting the comparison of overall survival in two groups.