tRNA Ini CAT inhibits proliferation and promotes apoptosis of laryngeal squamous cell carcinoma cell

Background: Transfer RNA (tRNA) is a ubiquitous RNA, present in all life forms and considered a housekeeping gene with limited regulatory function. Recent studies have shown that tRNA is dysregulated and involved in the pathogenic process of cancer. Methods: Quantitative reverse transcription-polymerase chain reaction detected the relative expression levels of tRNA IniCAT in paired LSCC tissues and paracancerous tissues and preoperative, postoperative, and healthy human plasma. We analyzed the relationship between its expression level and the clinicopathological features of patients. We also established the receiver operating characteristic curve and predicted its diagnostic value. High expression of tRNA IniCAT was detected in the LSCC cell line. The proliferation, apoptosis, and cycle of cells were analyzed. High expression of tRNA IniCAT was detected in mice with transplanted tumors. The tumor weight and volume in mice were measured. The transplanted tumor was used for pathological analysis and transmission electron microscope observation. Results: tRNA IniCAT was down-regulated in LSCC tissues and plasma. The area under the plasma receiver operating characteristic curve was 0.808. High expression of tRNA IniCAT in the laryngeal carcinoma cell line inhibited cell proliferation and promoted apoptosis. High expression of tRNA IniCAT in transplanted tumor in nude mice inhibited the growth of the transplanted tumor. Conclusion: tRNA IniCAT acts as a tumor Inhibitor in LSCC, inhibits cell proliferation, and promotes apoptosis in vitro and in vivo. tRNA IniCAT may be used as a new biomarker for the early diagnosis of LSCC.

of the body. More than half of RNA modi cations occur in tRNA. 6 These modi cations affect the structure, stability, and functionality of tRNA, leading to widespread cellular effects; of those, methylation modi cation is the most common effect . 7 Early studies reported that tRNA plays only the role of a "porter", and does not possess regulatory function. Nevertheless, as research progresses, tRNA have been found to be involved in various physiological and pathological processes, including cancer, diabetes, and neuronal disease . 8- 11 The role of tRNA in LSCC remains unknown and there is no complete tRNA expression map on this disease. This study established tRNA expression pro les from eight pairs of LSCC and paracancerous tissues via a microarray platform. In addition, the relative expression levels of tRNA Ini CAT in tumor and paracancerous tissues, preoperative and postoperative plasma of patients with LSCC and plasma of healthy human were examined. The tRNA Ini CAT was analyzed to determine the relationship between its expression level and clinicopathological features. The work characteristic curves of the subjects were established to predict their diagnostic value. The tRNA Ini CAT was upregulated in laryngeal cancer cell lines. The proliferation, apoptosis, and cycle of cells were analyzed. High expression of tRNA Ini CAT was observed in mice transplanted with tumors. The tumor weight and volume in mice were measured, and the tumors were analyzed by pathology and transmission electron microscopy (TEM). Finally, the usefulness of tRNA Ini CAT as biomarker and potential therapeutic target for LSCC was examined. 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.

Materials And Methods
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 xed 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 ow 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 ow 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 sacri ced, 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 xed in 10% formalin, embedded in para n, 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 xed with 2.5% glutaraldehyde for 1 h and washed with phosphate-buffered saline for 1 h. The tumor tissue was subsequently xed 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 tRNA Ini CAT 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 tRNA Ini CAT 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 signi cant differences.

Differences in tRNA expression in LSCC and adjacent tissues
The cDNA microarray results indicated that the expression levels of 88 tRNA (including intracellular tRNA and mitochondrial tRNA) in LSCC and paracancerous tissues were detected in eight pairs. The results showed that the expression pro les of tRNA in LSCC tissues were different from those observed in adjacent tissues (Figs. 1 and 2). We found that two tRNA doubled their expression levels in LSCC and paracancerous tissues: tRNA Ini CAT (2.24-fold change) and mt-tRNA Glu TTC (− 2.21-fold change). Meanwhile, the rst 20 tRNA had higher expression levels in LSCC than in adjacent tissues and were all intracytoplasmic tRNA. In contrast, 18 of the rst 20 tRNA with lower expression in LSCC tissues than in adjacent tissues were mitochondrial tRNA (Figs. 3 and 4).
Low expression of tRNA Ini CAT in LSCC tissues To validate the results of the PCR array, tumor and paracancerous tissue samples were collected from a total of 100 patients with LSCC through quantitative reverse-transcription PCR to determine the relative expression levels of tRNA Ini CAT . However, as shown in Fig. 5, the expression levels of tRNA Ini CAT in tumor tissues were signi cantly lower than those measured in adjacent paracancerous tissues (p = 0.0077).
Among those, the expression levels of tRNA Ini CAT in 63 LSCC tissues (63%) were lower than those recorded in the corresponding paracancerous tissues (Fig. 6).
tRNA Ini CAT in plasma of patients with LSCC was downregulated By exploring the relative expression levels of tRNA Ini CAT in plasma, we obtained consistent results with those reported for tumor tissues. The expression levels of tRNA Ini CAT in the preoperative plasma of patients with LSCC were signi cantly lower than those noted in postoperative plasma (p = 0.0032) and healthy human plasma (p < 0.0001). Meanwhile, the expression levels in postoperative plasma were signi cantly lower than those observed in healthy human plasma (p = 0.0458) (Fig. 7).
tRNA Ini CAT expression levels and clinicopathological characteristics of patients with LSCC and potential diagnostic value Tissues were divided into low and high expression groups according to the expression levels of tRNA Ini CAT . Subsequently, we analyzed the relationship between the expression levels of tRNA Ini CAT and clinicopathological factors. The results showed that the expression levels of tRNA Ini CAT were not associated with clinicopathological features ( Table 1). The area under the ROC curve for tRNA Ini CAT in tissues and plasma was 0.575 and 0.808, respectively (Fig. 8). High expression of tRNA Ini CAT inhibits LSCC cell growth AMC-HN-8 cells were transfected using LV3-tRNA Ini CAT to upregulate tRNA Ini CAT and negative control (LV3-NC) to assess the role of tRNA Ini CAT in tumor progression. Following transfection of LV3-tRNA Ini CAT and LV3-NC in AMC-HN-8 cells, green uorescent protein uorescence expression was observed (Fig. 9). The levels of tRNA Ini CAT in the cells showed a 2.1-fold increase (Fig. 10). A cell inhibition rate analysis was performed to determine the effect of tRNA Ini CAT on LSCC cell inhibition. The results showed that overexpression of tRNA Ini CAT signi cantly inhibited cell growth compared with the NC group (Fig. 11).
Effects of tRNA Ini CAT on cell cycle and apoptosis For further exploration of the potential regulatory mechanisms of tRNA Ini CAT in cell proliferation, cell cycle and apoptosis were assessed by ow cytometry. The upregulated tRNA Ini CAT did not have a signi cant effect on the cell cycle (Fig. 12). Furthermore, the results of the apoptosis analysis showed that transfection of LV3-tRNA Ini CAT signi cantly promoted apoptosis in AMC-HN-8 cells (Fig. 13). Collectively, these results indicated that tRNA Ini CAT promoted apoptosis.
tRNA Ini CAT inhibits growth of LSCC xenograft We used an in vivo xenograft mouse model to investigate the role of tRNA Ini CAT in inhibiting cancer. The mice were divided into three groups: LV3-tRNA Ini CAT (high and low doses) and LV3-NC (Fig. 14). The results indicated that upregulation of tRNA Ini CAT signi cantly inhibited tumor growth (Fig. 15). Among them, the weight of the transplanted tumor in the high dose group was signi cantly smaller than that in the NC group. During the experiment, there was no signi cant difference in tumor volume and mouse weight in these groups (Figs. 16 and 17). Pathological ndings indicated that the administration of LV3-tRNA Ini CAT signi cantly increased tissue necrosis in nude mice transplanted with tumors, showing a certain role of tRNA Ini CAT in promoting apoptosis (Fig. 18). Through TEM, apoptotic cells were shown to have special structural characteristics. The autophagy and apoptosis of the tumor in the LV3-tRNA Ini CAT treatment group were signi cantly increased, the phagocytic structure in the bubble was upgraded, and the nucleus had slightly shrunk (Fig. 19). Therefore, we concluded that tRNA Ini CAT promotes LSCC cell apoptosis in vivo.

Discussion
The expression spectrum of early LSCC mainly focused on mRNA and miRNA, such as DJ-1, 12 HuR, 13 and miR-34a, 14 which were found to be associated with this disease. In the current study using gene chip technology, the expression pro les of tRNA in LSCC and normal tissues were found to differ. This study provides a complete expression pro le of tRNA in LSCC.
tRNA was widely regarded as a housekeeping gene with limited regulatory function. There are only few tRNA with known additional functions, 15 other than their usefulness as adapters for protein synthesis. In recent years, a growing body of evidence suggests that tRNA and its derivatives are dysregulated and participate in the pathogenic process of cancer. 16 Mutations in tRNA and the involvement of co-oproteins produced by tRNA biogenesis and modi cation were found to be associated with cancer. [17][18][19] Studies have found that tRNA synthesis was controlled by various oncogenic and tumor suppressor genes. Ras 20 and c-Myc 21 facilitate RNA Pol III transcription; however, Rb 22 and p53 23 inhibit RNA Pol III transcription, leading to severe dysregulation of tRNA levels in multiple types of cancer. In addition, in several types of cancer, tRNA modi ed enzymes can add some tRNA modi cation, which changes their codon preferences and leads to an increase in protein expression levels of mRNA with "preference" codons. 24,25 Meanwhile, mutations in mitochondrial tRNA cause mitochondrial dysfunction which is also associated with tumorigenesis. 26 Contrary to the pro ling result, the present study con rmed the relative levels of tRNA Ini CAT expression in 100 pairs of paired tissue samples, and revealed that tRNA Ini CAT expression in LSCC tissues was downregulated (Fig. 5). tRNA Ini CAT was downregulated in LSCC preoperative plasma compared with postoperative plasma; this nding was consistent with the results obtained in tissues (Fig. 7). Moreover, this study demonstrated that tRNA Ini CAT may act as tumor inhibitor in LSCC in vitro and in vivo. Firstly, we used a LV3-tRNA Ini CAT lentivirus to upregulate tRNA Ini CAT in LSCC cells (Fig. 10). Cell inhibition rate analysis was conducted to determine the effect of tRNA Ini CAT overexpression on cell proliferation. The data showed that overexpression of tRNA Ini CAT inhibited cell growth (Fig. 11). Moreover, cell cycle and apoptosis were evaluated to further investigate the biological functions of tRNA Ini CAT in LSCC cells. The results showed that the upregulation of tRNA Ini CAT did not signi cantly affect the cell cycle (Fig. 12). However, overexpression of tRNA Ini CAT improved the rate of apoptosis in treated AMC-HN-8 cells (Fig. 13). Lastly, we reached the same conclusion based on experiments in BALB/c mouse xenografts; tRNA Ini CAT inhibited tumor growth and promoted tumor cell apoptosis in vivo. Overexpression of tRNA Ini CAT signi cantly decreased the tumor weight of the xenografts (Fig. 15). By pathological examination and TEM, we found that overexpression of tRNA Ini CAT signi cantly increased tumor cell apoptosis and autophagy (Figs. 18 and 19).
The ndings of this study are consistent with those of previous studies. Numerous studies have con rmed the important role of tRNA in regulating gene expression. Change in bacterial activity tRNA content plays an adaptive role with environmental signal changes. 27 The binding of tRNA to cytochrome C inhibits the effects of cytochrome C and apoptotic proteases, thereby inhibiting apoptosis and enzyme activity . 28 Overexpression of the initial tRNA (tRNA i Met ) was observed in normal mammary epithelial cells. Changes in the whole cell tRNA expression levels accelerate the speed of cell proliferation. 29 In carcinoma-associated broblasts, tRNA i Met promotes tumor proliferation and angiogenesis. 30 Studies revealed that tRNA Ile expression was higher in breast cancer-associated broblasts than in normal broblasts . 30,31 These abnormally expressed tRNA molecules are expected to be new prognostic markers for such diseases. 32 tRNA Glu UUC and tRNA Arg CCG are highly expressed in breast cancer and enhance tumor invasion. EXOSC2 and GRIPAP1 are downstream target proteins of tRNA Glu UUC to promote the progression of cancer invasion and metastasis. 33 In human epidermal growth factor receptor 2-positive breast cancer cell lines, free tRNA Leu may interact with human epidermal growth factor receptor 3 (ErbB3) and binding protein (EBP1), which in turn activates the ErbB2/ErbB3 signaling pathways, and ultimately promotes cancer cell proliferation. 34 tRNA plays a key role in translation, and its dysregulation may affect cell protein expression. Cancer cells regulate the expression of multiple promoters of cancer progression by regulating the levels of tRNA. This leads to the notion that cancer cells exhibit tRNA regulatory effects in addition to the numerous known regulatory mechanisms that alter the expression of cancer progression promoters. Disorders of tRNA Ini CAT we found in LSCC may cause dysregulation of downstream target protein expression, which in turn changes the ability of tumor proliferation and apoptosis.

Conclusion
The present study provides a comprehensive tRNA expression spectrum for LSCC. Availability of data and material The datasets used and analyzed during the current study are available from the corresponding author upon request.  Relative expression levels of tRNAIniCAT in 100 pairs of LSCC and paracancerous tissues. LSCC, laryngeal squamous cell carcinoma tRNAIniCAT ROC curves in tissues and plasma ROC, receiver operating characteristic    Tumor weight in the high-dose treatment group was signi cantly higher than that measured in the NC group. *p<0.05. NC, negative control There was no signi cant difference in tumor volume between each group and the NC group. NC, negative control Page 24/25

Figure 17
There was no signi cant difference in the weight of mice between each group and the NC group. NC, negative control Figure 18 Pathological examination showed that tumor necrosis in the high-dose treatment group was markedly decreased compared with that observed in the NC group (magni cation ×100). (a) NC. (b) Low dose. (c)