Patients and clinical specimens
70 primary OSCC tissue specimens were collected from patients with informed consent at West China Hospital of Stomatology, Sichuan University. All of these patients have undergone the curative resection and were devoid of prior treatment or autoimmune diseases. Specimens were identified as squamous cell carcinoma and classified into different grades based on the current Union for International Cancer Control (UICC) criteria. Besides, 20 normal oral mucosa were obtained and all tissues samples were half frozen for RNA and DNA extraction, and half fixed in 10% formalin and embedded in paraffin. The current study was authorized by the Institutional Ethics Committee of the West China Medical Center, Sichuan University, China.
Hpv Detection
Total DNA was extracted using DNA Extraction Kit (Tiangen) according to manufacturer’s instructions. HPV status was determined using a highly sensitive PCR protocol (Invitrogen) and HPV-16/18 primers, which was performed on a C1000 Touch PCR machine (Bio-Rad). Specific primers for HPV16: forward, 5’-CACAGTTATGCACAGAGCTGC-3’, reverse, 5’-CATATATTCATGCAATGTAGGTGTA-3’; HPV-18: forward, 5’-CACTTCACTGCAAGACATAGA-3’, reverse, 5’-GTTGTGAAATCGTCGTTTTTCA-3’. Then, eletrophoresis with 1.5% agarose gels was used to separate PCR products, which were visualized by GCLSTAIN staining.
Mirna Microarray
An Agilent miRNA microarray (8*60K, Design ID: 070156) using clinical specimens from HPV-positive and HPV-negative OSCC patients was carried out to profile miRNA by oeBiotech Limited Company. Total RNA was extracted using TRIzol reagent (Invitrogen) following manufacturer’s protocols and 100 ng RNA per sample was used in microarray.
Qrt-pcr
Total RNA was extracted as previously defined. For mature miRNAs, All-in-One™ miRNA First-Strand cDNA Synthesis Kit (Genecopoeia) was used to generate 10 µL cDNA. Then using miR-specific primers and universal adaptor PCR primers (Genecopoeia), qRT-PCR was performed with All-in-One™ miRNA qRT-PCR Detection Kit (Genecopoeia) on CFX Connect Real-Time System (Bio-Rad). For mRNA, the reverse transcription was carried out using HiScript II Q RT SuperMix (Vazyme), and qRT-PCR was performed with ChamQ™ SYBR Color qPCR Master Mix (Vazyme) on CFX Connect Real-Time System (Bio-Rad) (21). Specific primers for qRT-PCR of mRNA were listed in Supplementary Data. The U6 small nuclear RNA or GAPDH was used as an endogenous control. Each experiment was repeated at least three times and the results were analyzed using 2−△△CT method.
Western Blot
The whole proteins were extracted using cell lysis buffer. Then, SDS-PAGE was used to separate proteins with different sizes. After that, gel was transferred to a PVDF membrane (Millipore) and target protein was immunoblotted with specific primary antibody. Primary antibodies were listed as follows: anti-E-cadherin (1:5000, mouse anti-human, Cell Signaling Technology Inc.), anti-Vimentin (1:2000, mouse anti-human, Abcam), anti-HPV16 E6 + HPV18 E6 (1:1000, mouse anti-human papillomavirus, Abcam), anti-HPV16 E7 (1:1000, mouse anti-human papillomavirus, Abcam), anti-YAP (1:2000, mouse anti-human, Proteintech), anti-TAZ (1:2000, mouse anti-human, Proteintech), anti-CCL2 (1:2000, mouse anti-human, Proteintech), anti-flag-YAP (1:1000, mouse, Abbkine), anti-GAPDH (1:2000, rabbit anti-human, Sigma-Aldrich). After incubating goat anti-rabbit or goat anti-mouse secondary antibody (MultiSciences), immunoblots were visualized using the chemiluminescence (ECL) reagent (Beyotime Biotechnology) and ChemiDoc XRS + System (Bio-Rad).
Mirna Fluorescence In Situ Hybridization (fish)
FISH assays were performed using miRNA Fluorescence In Situ Hybridization Kit (GenePharma) according to the instructions. Oligonucleotide modified probe labeled with cy3 for hsa-miR-550a-3-5p was provided by GenePharma. First, 5 µm formalin-fixed paraffin-embedded sections were preheated for 30 min at 60 ℃ and fresh xylene was used to remove paraffin from the tissue. Then, sections were rehydrated by 5 min incubations in decreasing concentrations of ethanol, and incubated with Proteinase K solution for 15 min at 37 ℃ and denaturing solution for 8 min at 78 ℃. After dehydration, probes were added in the hybridization solution and incubated for 4 h at 50 ℃. Sections were washed and counterstained with DAPI (GenePharma). Images were acquired using a fluorescence microscope (Olympus BX51).
Immunohistochemistry (ihc)
Formalin-fixed paraffin-embedded sections were firstly deparaffinized using xylene and rehydrated in alcohol with decreasing concentrations. Then boiling for 3 min in 0.01 M citrate buffer (pH = 6.0) was used for antigen retrieval, and 3% hydrogen peroxide was added for blocking endogenous peroxidase activity. After that, we used 5% goat serum for antigen blocking and incubated sections with the primary antibody at 4 ℃ overnight in a moist chamber. Sections were washed with PBS, and incubated first with biotinylated anti-mouse/rabbit IgG and sequent with streptavidin-biotin peroxidase both for 15 min. DAB was added to detect the primary antibody, and hematoxylin was used to stain the nucleus. As negative controls, sections were analyzed in parallel except incubating with isotype-specific immunoglobulin (IgG) instead of the primary antibody. And the following primary antibodies were listed: anti-p16 (1:500, rabbit anti-human, Bioss), anti-Ki-67 (1:200, rabbit anti-human, HUABIO), anti-E-cadherin (1:500, mouse anti-human, Cell Signaling Technology Inc.), anti-Vimentin (1:200, mouse anti-human, Abcam), anti-CD31 (1:300, rabbit anti-human, Biorbyt), anti-CD34 (1:300, mouse anti-human, HUABIO), anti-YAP (1:600, mouse anti-human, Proteintech), anti-CCL2 (1:200, mouse anti-human, Proteintech), anti-CD163 (1:500, rabbit anti-human, Biorbyt). HE staining was referred to as hematoxylin and eosin staining. Staining of IHC was evaluated in ten randomly selected fields at 400X magnification. Quantification was evaluated by three independent investigators who were blinded to patient characteristics. IHC staining scores were calculated as staining intensity × percentage of positive cells. Staining intensity was assigned as: 0 (negative), 1 (weak), 2 (moderate), 3 (strong). Percentage of positive cells was defined as: 0 (no positive cells), 1 (༜10% positive cells), 2 (10–50% positive cells), 3 (༞50% positive cells). For CD163 staining, positive cells in each field were counted and expressed as the mean value (22).
Cell Culture And Reagents
HPV-positive human OSCC cell lines UPCI:SCC090 and UM-SCC-47, HPV-negative human OSCC cell lines Cal-27, SCC25, and HSC3, normal human oral keratinocytes (NOK), dysplasia human oral keratinocytes (DOK), and human monocyte cell line THP-1 were obtained from State Key Laboratory of Oral Disease, Sichuan University. UPCI:SCC090 and UM-SCC-47 were cultured in MEM (Gibco) containing 1X non-essential amino acids (Sigma-Aldrich), 1 mM sodium pyruvate (Sigma) and 10% fetal bovine serum (FBS, Gibco), Cal-27 and HSC3 were cultured in DMEM (Gibco) containing 10% FBS, SCC25 was cultured in DMEM/F12 (Gibco) containing 10% FBS, NOK and DOK were cultured in EpiLife (Gibco), and THP-1 was cultured in RPMI 1640 medium (Gibco) with 10% FBS. All of them were maintained at 37 ℃ in a humidified atmosphere with 5% CO2. For macrophage generation, THP-1 cells were induced by 200 nM PMA (Sigma-Aldrich) for 24 h. Then OSCC cells whether transfected or not, were cultured with serum-free medium for 24 h and the supernatant was collected and centrifuged at 1000 x g for 20 min, known as the conditioned media (CM). We obtained tumor-associated macrophages (TAMs) by culturing PMA-induced THP-1 cells in CM of OSCC cells for 24 h. Morphology and polarization of TAMs were observed and detected. Co-cultivation of TAMs and OSCC cells was performed with the non-contact transwell system (pore size 0.4 µm, corning). TAMs seeded in the upper chamber communicated with corresponding OSCC cells in the lower chamber for 48 h, and OSCC cells were harvested for further analysis.
Verteporfin (VP, Selleck) was dissolved in DMSO and used in the culture medium at a final concentration of 10 µM for 24 h. Recombinant human CCL2 (Sino Biological) was added to CM at 500 ng/mL before inducing PMA-THP-1 cells.
Transfections
miR-550a-3-5p mimic or inhibitor were obtained from GenePharma Co. Ltd., China. Small-interfering RNAs (siRNAs) targeting E6, E7, and YAP were designed and synthesized also by GenePharma Co. Ltd., China. The plasmid pCDH-EF1-YAP-5SA (FLAG-tagged) and the empty Vector were kindly provided by Dr. Fanyuan Yu in West China School of Stomatology, Sichuan University and used for overexpression of activated YAP. These transfections were performed using EndoFectin™ (GeneCopoeia) according to the manufacturer’s instructions. 48 h after transfection, further experiments could be carried out. Stably-transfected OSCC cells with miR-550a-3-5p overexpression were derived from the parental cells by a fluorescence microscope (Olympus BX51) detection and puromycin (Sigma-Aldrich) selection.
Cell Proliferation Assay And Flow Cytometry-based Apoptosis Analysis
CCK-8 assay was used to study cell proliferation. Cells were seeded in 96-well plates at the density of 1 × 103 (cells/well), and the absorbance at 450 nm was measured on 24, 48, 72, 96, 120 hours with 10 µL of CCK-8 solution. Proliferative comparison of mimic-transfected cells or inhibitor-transfected cells was calculated by normalization with respect to NC: % cells = (mimic or inhibitor – corresponding NC)/ corresponding NC × 100. Difference of OD Value on 120 h and OD Value on 24 h was used for calculation. Each assay was carried out in triplicate.
Flow cytometry using the Annexin V-FITC Apoptosis Detection Kit (Invitrogen) was used to measure cell apoptosis. Cells were seeded in 6-well plates and digested after 48 h. Then, 1X Binding Buffer was used to resuspend cells. We stained cells with 5 µL of Annexin V-FITC Conjugate and 5 µL of Propidium Iodide Solution and incubated them for 30 min. Stained cells were analyzed using a FACScalibur (Becton-Dickinson). For comparison, % apoptotic cells = (mimic or inhibitor – corresponding NC)/ corresponding NC × 100. Experiments were conducted in triplicate.
Wound Healing Assay And Cell Invasion Assay
Cells were cultured in 6-well plates and starved in serum-free medium for 24 h after arriving at 100% confluence. A pipette tip was used to scratch the cells and form the gap space. After washing out the dead cells, photomicrographs were taken immediately and at a timepoint of 24 h. Closed scratch areas were calculated by ImageJ software. Each experiment was conducted in triplicate.
24-well transwell plates (pore size 8 µm, Millipore) were used for conducting Cell invasion assays. The upper chamber was coated with Matrigel (BD Bioscience) before 5 × 104 cells being seeded in it. After 24 h of cell adherence, medium was changed into serum-free in upper chamber and complete medium was added to lower chamber. Then, cells which have invaded through the Matrigel after 24 h incubation were stained with 0.1% crystal violet and counted under a microscope. Five pre-selected fields (× 200) were observed and assays were conducted in triplicate.
Imaging Of Macrophage Morphology
Fluorescent staining was used for observation of macrophage morphology. Macrophages were washed with PBS for three times, and fixed with 4% paraformaldehyde for 20 min. Then, PBS containing 0.5% Triton X-100 was used to permeabilize the cells for 5 min. After incubating cells with FITC-phalloidin (Solarbio) for 30 min, nuclei were stained using DAPI (Sigma-Aldrich). All photographs were taken using a fluorescence microscope (Olympus BX51).
Dual Luciferase Reporter Gene Assays
The wild-type or mutant YAP 3’UTR pEZX-FR02 plasmids designed by GeneCopoeia Co. Ltd., was cotransfected with miR-550a-3-5p mimic or NC in UPCI:SCC090 and UM-SCC-47 cells using EndoFectin™ (GeneCopoeia). 48 h after transfection, cell lysates were collected and used for detection of firefly and Renilla luciferase activities following the protocol of a Luc-Pair™ Duo-Luciferase Assay Kit 2.0 (GeneCopoeia). Renilla luciferase activity was used for normalization in the study.
Animal Experiments
All animal experiments were approved by the Institutional Animal Care and Use Committee of West China Medical Center, Sichuan University. For subcutaneous xenograft model of nude mice, twenty 4-week-old BALB/c male nude mice (Dashuo) were divided into four groups after 1 week acclimation. Stably miR-550a-3-5p-overexpressed UPCI:SCC090 and Cal-27 cells and their relevant empty vector-transfected control cells were injected into the right flank region of nude mice subcutaneously. 0.2 mL containing 5 × 106 cells per aliquot was used in each mouse. Tumor volumes were evaluated every three days and calculated using formula as below: length × (width)2 × ∏/6. Mice were killed using isoflurane after 8 times taking notes of tumor volume, and tumors were collected for weights measuring and further examination.
For 4NQO-indued OSCC model of transgenic mice, a total of sixteen 7-week-old female Rosa26-E6-E7 constitutive knock-in C57BL/6 mice (Cyagen, ID: TOS150814BA1) were used and divided into two groups randomly after one week of acclimation: 4NQO + VP group (n = 8) and 4NQO group (n = 8). A solution of 4NQO (Sigma-Aldrich) was added to the distilled drinking water at a concentration of 100 µg/mL for 8 weeks, and then switched to distilled water for another 8 weeks to generate OSCC as we previously described (23). After that, VP (Selleck) was injected every three days intraperitoneally at 100 mg/kg in 4NQO + VP group, while Vehicle-treated mice were injected with DMSO. Mice were anesthetized using isoflurane after 6 doses of VP, and tongue was collected, carefully observated and longitudinally bisected. We fixed one part of each tongue tissue with 10% formalin and embedded it with paraffin, and froze the other part immediately and stored it at -80 ℃.
Statistical analysis
The correlations between miR-550a-3-5p expression and other clinicopathlogical factors were estimated using Chi-square analysis. Means comparisons were conducted with Student t-test or one-way ANOVA. The associations between miR-550a-3-5p and YAP, YAP and CCL2, and CCL2 and CD163 expressions in HPV-positive OSCC specimens were assessed using 2-tailed Pearson’s statistics. All cellular experiments were conducted independently for at least three times and in triplicate each time. GraphPad Prism 7.0 (GraphPad Software) was used for processing all data and values were presented as means ± SD. P༜0.05 was considered to be statistically significant.