Collection of specimens and clinical data
Patients with LSCC who underwent otorhinolaryngology and head and neck surgery at Li Huili Hospital affiliated to Ningbo University (Ningbo, China) from January 2013 to April 2019 without receiving radiotherapy, chemotherapy, or other antitumor treatments were selected. Tumor tissue and paracancerous tissue samples were obtained from surgery. All LSCC samples were confirmed by at least two senior pathology experts. Healthy human plasma samples were used to obtain plasma samples before and after surgery. Tumor staging was determined according to the American Joint Committee on Cancer tumor-lymph node-metastasis (TNM) staging (2017, 8th edition). This study was approved by the Ethics Committee of Human Research of Ningbo University (No.KY2015PJ012). Written informed consent was provided by all subjects.
Total RNA preparation and synthesis of cDNA
The TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and TRIzol LS reagents (Invitrogen) was used to extract the total RNA of tissues/cells and plasma through the SmartSpec Plus spectrophotometer (BioRad, Hercules, CA, USA). The purity and concentration of total RNA were determined. The RNA quality control standards were as follows: A260:A230 ratio > 1.7; A260:A280 ratio 1.8–2.1. According to the rtStar™ tRNA-optimized First-Strand cDNA Synthesis Kit (Arraystar, Rockville,MD, USA) instructions, 2 µg of total RNA was used for demethylation reactions and cDNA synthesis. For assessing the quality of synthetic cDNA, 1 µl of RNA Spike-in quantitative polymerase chain reaction (qPCR) Primer Mix, use µl cDNA, 2 5 µl GoTaq qPCR Mixture (Promega, Madison, WI, USA) and 2 µl of enzyme-free water were added; only samples with cycle threshold (Ct) values < 30 were used for qPCR detection.
qPCR
The cDNA was diluted with enzyme-free water (1:20). The Applied Biosystems 7900 real-time PCR system (Thermo-Fisher, USA) was used to qPCR. 88 kinds of tRNA primers were bought from Arraystar,Inc,Rockville,MD,USA. U6 was used as a control, and the relative expression level of tRNA was determined by the ΔΔCt method. U6 primers were purchased from BGI (Shanghai, China). The U6 sequences were as follows: justice, 5'- GCTTCGGCAGCACATATACTAAAAT-3'; Antimony 5'- CGCTTCACGAATTTGCGTCAT-3'.
Cell culture and transfection
The LSCC cell line AMC-HN-8 was purchased from BeNa Culture Collection, Shanghai, China. The cells were cultured in RPMI 1640 (HyClone, Logan, UT, USA) 10% fetal bovine serum (PAN Biotech, Aidenbach, Germany) at 37 °C and 5% CO2. Cells were counted using a TC10 automatic cell counter (BioRad). The lentivirus was purchased from GenePharma (Shanghai, China). Stably transfected cells were selected through purine mycin. The tRNAIniCAT of the overexpression lentivirus is termed LV3-tRNAIniCAT. Sequences: 5'- AGCAGAGTGGCG CAGCGGAAGCGTGCTGGGCCCATAACCCAGAGGTCGATGGATCGAAACCATCCTCTGCTA-3', LV3-NC used as a control, sequence: 5'- TTCTCCGAACGTGTCACGT-3'.
Cell inhibition rate test
AMC-HN-8 cells were digested with trypsin to form a single-cell suspension and inoculated on a six-well plate. A culture medium containing 3 H-TdR was added for 16 h, and the cells were digested and collected. Finally, a Micro Beta 2450 liquid scintillation counter (Perkin Elmer,Waltham, Mass, USA) was used to determine the minutes (cpm), and the inhibition rate was calculated by the average cpm of three wells.
Cell cycle and apoptosis analysis
The cells were starved (serum) to synchronize the cell cycle. Subsequently, the cells were collected, washed with phosphate-buffered saline, and fixed in 70% ethanol at − 20 °C for 24 h. Next, staining with DNA dye solution was performed using the Cell Cycle Detection Kit (KeyGEN, Jiangsu, China), and the cells were incubated for 30 min. Lastly, BD FACSCanto II flow cytometry (BD Biosciences, San Jose, CA, USA) Testing was performed. Cells were digested with trypsin (no ethylenediaminetetraacetic acid) into a single-cell suspension, and resuspended in binding buffer. The cells were then stored at room temperature. Using an Annexin V-FITC/PI apoptosis kit (CoWin Biosciences, Beijing, China), the cells were stained for 15 min without light, and the rate of apoptosis was detected using BD FACSCanto II flow cytometry (BD Biosciences).
Mouse models of xenotransplantation
All animal studies were conducted in accordance with the animal protocol approved by the animal protection and use committee of Zhejiang University of Traditional Chinese Medicine (No.2019-007),Hangzhou, Zhejiang, China. BALB/c female nude mice were purchased from Charles River (Beijing, China) and raised in the experimental animal center of Zhejiang University of Traditional Chinese Medicine. The mice were anesthetized with pentobarbital sodium (45 mg/kg); the laryngeal skin was removed and the larynx was inoculated with AMC-HN-8-Luc cell suspension. The mice were randomly divided into three groups (9 or 10 mice per group). Tumor growth was observed using an IVIS Lumina XRMS small animal in vivo imager (Perkin Elmer) after 1 week. The mice in each group were injected with 50 µl of corresponding medicine solution within the tumor (twice per week for 21 consecutive days). The weight and tumor volume in mice were measured twice per week. The following day, the mice were sacrificed, and the solid tumor was resected and weighed. Conventional hematoxylin-eosin pathological examination and electron microscopy were performed on the tumor tissue.
Staining and TEM of hematoxylin-eosin
The tumor tissue was fixed in 10% formalin, embedded in paraffin, cut into sections (thickness: 8 µm) and baked at 45 °C for 5 h. Subsequently, the tissues were dewaxed with xylene for 30 min, treated with different concentrations of ethanol (100%, 90%, 70%); hydrated with distilled water, stained with hematoxylin for 15 min, differentiated in ethanol and ammonia, dehydrated with ethanol, stained with eosin, dehydrated again with ethanol, and washed with xylene. Tissue morphology was observed by optical microscopy. The tumor tissue was fixed with 2.5% glutaraldehyde for 1 h and washed with phosphate-buffered saline for 1 h. The tumor tissue was subsequently fixed in 1% osmium acid for 30 min–1 h, and dehydrated in ethanol and acetone. Finally, the tissue was embedded in epoxy resin and stained with uranyl acetate and lead citrate. The Hitachi H-7650 TEM (Tokyo, Japan) was used to observe the changes in autophagy and apoptosis morphology.
Statistical analysis
The SPSS version 20.0 software (IBM Corp., Armonk, NY, USA) and GraphPad 8.0 (San Diego, CA, USA) were used for data analysis. The relative expression level of tRNAIniCAT between LSCC tissues and adjacent non-tumor tissues was analyzed by paired sample t test. One-way analysis of variance was used to analyze the level of tRNAIniCAT expression in the preoperative plasma, postoperative plasma, and plasma of patients with LSCC. The correlation between the expression level and clinicopathological factors was analyzed using the chi-squared test. The diagnostic value was assessed using the Receiver operating characteristic curve (ROC). P-values < 0.05 denoted statistically significant differences.