tRNAIni CAT inhibits proliferation and promotes apoptosis of laryngeal squamous cell carcinoma cells

Abstract Background Laryngeal squamous cell carcinoma (LSCC) brings a heavy blow to the patient's voice. Transfer RNA (tRNA) is a common RNA, the roles of tRNAs in LSCC are largely unknown. Methods The tRNA expression profile in LSCC tissues and adjacent normal tissues was measured by a tRNA qRT‐PCR array. The expression level of tRNAIni CAT in LSCC tissues and plasmas was detected by qRT‐PCR. The receiver operating characteristic (ROC) curve was established. tRNAIni CAT was upregulated by a lentivirus vector in the LSCC cell line. Moreover, tRNAIni CAT was upregulated in LSCC xenograft nude mouse model and the xenografts were used for pathological analysis and transmission electron microscope (TEM) observation. Results The top 10 upregulated tRNAs were tRNALys CTT‐1, tRNALeu TAA, tRNAPhe GAA, tRNALeu CAG, tRNATyr ATA, tRNAMet CAT, tRNATyr GTA‐1, tRNAThr CGT, tRNATyr GTA‐2, tRNAAla AGC; and the top 10 downregulated tRNAs were tRNAIni CAT, mt‐tRNAGlu TTC, tRNAVal CAC‐3, mt‐tRNATrp TCA, mt‐tRNATyr GTA, mt‐tRNALys TTT, mt‐tRNAThr TGT, mt‐tRNAAsp GTC, mt‐tRNAAsn GTT, mt‐tRNAPro TGG. tRNAIni CAT was downregulated in LSCC tissues and plasma. The area under the ROC curve (AUC) in LSCC tissues and the plasma of patients with LSCC was 0.717 and 0.808, respectively. tRNAIni CAT inhibited LSCC cell proliferation and promoted apoptosis. The in vivo results showed that tRNAIni CAT inhibited the growth of the xenografts and promoted apoptosis. Conclusions This is the first study to provide tRNA expression profiles for LSCC tissues. tRNAIni CAT may be used as a new biomarker for the early diagnosis of LSCC. tRNAIni CAT inhibits cell proliferation and promotes apoptosis in vitro and in vivo.


| INTRODUC TI ON
Laryngeal cancer is a common malignant tumor of the head and neck. It is common among men aged 50-70 years. Its incidence is 1.5-3/100,000, accounting for approximately 1% of all malignant tumors. Laryngeal squamous cell carcinoma (LSCC) is the most common type, accounting for approximately 90% of cases. 1 At present, the incidence of laryngeal cancer is increasing. Treatment is mainly through surgical resection, combined with preoperative or postoperative chemotherapy, radiotherapy, and other comprehensive therapies. However, vocal disorders occur following surgery, affecting the quality of life of patients. Globally, more than 80,000 patients with laryngeal cancer expire each year. 2 Because the symptoms of early laryngeal cancer are not obvious, early detection is very important to reduce the hazard of laryngeal cancer.
On the one hand, it can improve the survival rate of patients after surgery, on the other hand, it preserves the voice function of the larynx as much as possible to reduce postoperative complications.
Transfer RNA (tRNA) is a universal RNA present in all forms of life. A mature tRNA is characterized by its secondary structure which consists of three loops and four stems: D-loop, anticodon loop, Tloop, and D-stem, anticodon stem, T-stem, acceptor stem; as well as an L tertiary structure maintained by hydrogen bonds. 3 As a basic component of the translation process, tRNA transport amino acids to the ribosome; the genetic information of the nucleotide sequence is transformed into the corresponding polypeptide chain in the manner of codon (mRNA)-anticodon (tRNA) interaction 4 ; this process is essential to maintain the normal life activities of the body. More than half of RNA modifications occur in tRNA. 5 These modifications affect the structure, stability, and functionality of tRNA, leading to widespread cellular effects; of those, methylation modification is the most common effect. 6 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. [7][8][9] The regulatory role of tRNA in LSCC remains unknown and there is no complete tRNA expression spectrum on this disease. This is the very first study to provide tRNA expression profiles for LSCC tissues. We analyzed the diagnostic value of tRNA Ini CAT through detecting the expression level in tissues and plasma. In addition, we proved that tRNA Ini CAT acts as a tumor inhibitor in LSCC through cell culture and nude mouse xenograft experiments.

| 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/

| 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. The primers of 88 tRNAs were bought from Arraystar (Rockville, MD, USA). U6 was used as a control, and the relative expression level of tRNA was determined by the ΔΔC q 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

| Cell cycle and apoptosis analysis
The cells were serum-starved to synchronize the cell cycle.
Subsequently, the tissues were dewaxed with xylene for 30 min,

| Low expression of tRNA Ini CAT was detected in LSCC tissues and plasma and potential diagnostic values
To validate the results of the expression profile, tumor and adjacent normal tissue samples were collected from a total of 37 patients with CAT was 0.808 in plasmas, with 74.2% sensitivity and 79.3% specificity, the cutoff was −2.9. The AUC in tissues was 0.717, with 89.2% sensitivity and 46% specificity, the cutoff was 0.8 ( Figure 2C).  Figure 4B). Furthermore, the results of the apoptosis analysis showed that transfection of LV3-tRNA Ini CAT significantly promoted apoptosis in AMC-HN-8 cells ( Figure 4C). Collectively, these results indicated that tRNA Ini CAT inhibited proliferation and promoted apoptosis of LSCC cell.

| tRNA Ini CAT inhibits growth of LSCC xenograft
We used a LSCC xenograft mouse model to investigate the antitumor role of tRNA Ini CAT in vivo. The mice were divided into three groups: LV3-tRNA Ini CAT -high dose, LV3-tRNA Ini CAT -low dose and LV3-NC ( Figure 5A). The results indicated that upregulation of tRNA Ini CAT significantly inhibited xenograft growth ( Figure 5B). Among them, the weight of the xenograft in the high-dose group was significantly smaller than that in the NC group. During the experiment, there was no significant difference in xenograft volume ( Figure 5C) and mouse weight ( Figure 5D) in these groups. Pathological findings indicated that the treatment of LV3-tRNA Ini CAT significantly increased tissue necrosis in xenograft, showing a certain role of tRNA Ini CAT in promoting apoptosis ( Figure 5E). Through TEM, apoptotic cells were shown to have special structural characteristics. The autophagy and apoptosis of xenograft in the LV3-tRNA Ini CAT treatment group were significantly increased, the phagocytic structure in the bubble was degraded, and  Figure 5F). Therefore, we concluded that tRNA Ini CAT promotes LSCC cell apoptosis in vivo.

| DISCUSS ION
In the past, the ncRNA expression spectrum of LSCC mainly focused on miRNA and lncRNA, our previous research revealed that miR-34a, 10 AC026166.2-001 11 and RP11-169D4.1-001 12 were closely related to the occurrence of LSCC. A recent study found that lncRNA MALAT1 was overexpressed in LSCC tissues and highly correlated with the 5-year survival of patients; it triggers the resistance of LSCC to chemotherapy drugs by promoting metastasis and inhibiting tumor cell apoptosis. 13 However, there has not been any report about tRNA in LSCC. tRNA was widely regarded as a housekeeping gene with limited regulatory CAT -treated groups was significantly lower than the NC group. *p < 0.05. C, There was no significant difference in tumor volume between each group. D, There was no significant difference in the weight of mice between each group. E, Pathological examination showed that tumor necrosis in the high-dose treatment group was markedly increased compared with that observed in the NC group (magnification ×100).
function. Compared with other additional functions of tRNA, 14 the researchers understand that tRNA acts an adaptor for protein synthesis better.
In recent years, a growing body of evidence suggests that tRNA and its derivatives are dysregulated and participate in the pathogenic process of cancer. 15 Mutations in tRNA and the involvement of co-proteins produced by tRNA biogenesis and modification were found to be associated with cancer. [16][17][18] Meanwhile, mutations in mitochondrial tRNA cause mitochondrial dysfunction which is also associated with tumorigenesis. 19 Studies have found that tRNA syn- In carcinoma-associated fibroblasts, tRNA i Met promotes tumor proliferation and angiogenesis. 29 Studies revealed that tRNA Ile expression was higher in breast cancer-associated fibroblasts than in normal fibroblasts. 30 These abnormally expressed tRNA molecules are expected to be new prognostic markers for such diseases. 31 tR-NA 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. 32 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. 33 In the current study using a tRNA qRT-PCR array, the expression profiles of tRNA in LSCC and normal tissues were found to differ. Consistent with the profiling result, the present study confirmed the lower expression levels of tRNA Ini CAT in LSCC tissue and plasma samples (Figure 2A,B). More importantly, we first identified its potential diagnostic value. As a biomarker, the AUC of tRNA Ini CAT in plasma is 0.808, the sensitivity and specificity were 74.2% and 79.3%, respectively ( Figure 2C). Then we explored its impact on LSCC cells in vitro and in vivo. Firstly, we used a LV3-tRNA Ini CAT lentivirus to upregulate tRNA Ini CAT in LSCC cells (Figure 3). The 3 H-TdR penetration test results showed that overexpression of tRNA Ini CAT inhibited cell proliferation ( Figure 4A). Moreover, the results of flow cytometry showed that the upregulation of tRNA Ini CAT did not affect the cell cycle ( Figure 4B), but boosted the cell apoptosis ( Figure 4C). In conclusion, our study provided a comprehensive tRNA expression spectrum for LSCC. We found that tRNA Ini CAT may be used as a new biomarker for LSCC. tRNA Ini CAT acted as a tumor suppressor in LSCC.

ACK N OWLED G EM ENTS
The author thanks all participants for their contributions.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data are included in this article.