Serum transfer RNA‐derived fragment tRF‐31‐79MP9P9NH57SD acts as a novel diagnostic biomarker for non‐small cell lung cancer

Abstract Background tRNA‐derived fragments (tRFs) have been found to have a crucial function in the pathophysiology of cancers. However, the function of tRFs in non‐small cell lung cancer (NSCLC) is yet unknown. The goal of this study was to assess the tRF‐31‐79MP9P9NH57SD serum expression from NSCLC patients and to determine its diagnostic usefulness. Methods By using stem‐loop quantitative real‐time PCR, we were able to detect various tRF‐31‐79MP9P9NH57SD expressions in 96 NSCLC serum samples, 96 healthy controls, and 20 pairs of NSCLC serum samples pre‐ and post‐surgery (qRT‐PCR). After that, we analyzed its diagnostic effectiveness using the receiver operating characteristic (ROC) curve. Results Serum tRF‐31‐79MP9P9NH57SD expression was higher in NSCLC patients, and levels of tRF‐31‐79MP9P9NH57SD were linked to the clinical stage (p = 0.002) and the malignancy of lymph node (p = 0.012). In addition, after the procedure, the serum tRF‐31‐79MP9P9NH57SD expression in NSCLC patients dropped. With 48.96 percent sensitivity and 90.62 percent specificity, the area under ROC curve (AUC) was 0.733. Conclusion serum tRF‐31‐79MP9P9NH57SD possibly is a new and groundbreaking biomarker for the NSCLC.

enolase (NSE) and cytokeratin fragment 21-1 (CYFRA21-1), were restricted in clinical utilization due to inferior sensitivity and specificity. 4,5 Therefore, it is critical to explore novel and effective biomarkers in NSCLC.
Small non-coding RNAs (sncRNAs) formed from matured tRNAs or pre-tRNAs are known as transfer RNA (tRNA)-derived small RNAs (tsRNAs). 6 Based on the cleavage site and length, they are divided into two categories: tRNA-derived fragments (tRFs) and tRNA halves (tiRNAs). tRFs are further split into tRF-1, tRF-3, and tRF-5 based on the tRNA splicing site, whereas tiRNAs are separated into two subtypes based on the anticodon cleavage site: 5'tiRNAs and 3'tiRNAs. 7,8 Growing evidence suggests that tsRNAs play important roles in multifarious human illnesses due to their part in cancer cells growth, translation regulation, and genetic silencing. [9][10][11] For example, tRF-3027 (tRNA Gly-GCC ) participates in the development of RNA-induced silencing complex and silences the replication protein RPA1 to inhibit tumor cell proliferation. 12 tRF-5026a (tRF-18-79MP9P04) expression was dramatically decreased in the tissues of gastric cancer, according to Zhu et al., and tRF-5026a could modulate the cancerous cell growth and pathological assault via the signaling pathway of PTEN/ PI3K/AKT. 13 More crucially, some research has looked into the idea of using tRFs as new biological markers for different diseases. [14][15][16] According to tRFdb 17 and related databases, 18 tRF-31-79MP9P9NH57SD may be associated with lung cancer. Its diagnostic efficacy and biological involvement in lung cancer, however, are unknown. Here, we investigated the tRF-31-79MP9P9NH57SD serum expression in patients suffering from lung cancer. Then, the multifactorial connections of clinical and pathological characteristics were assessed. Our data indicate that tRF-31-79MP9P9NH57SD may be used as a noninvasive indicator for diagnosing lung cancer.

| Patients and samples
Serum from 96 NSCLC patients before surgery, 20 patients with NSCLC pre-and post-surgery, and 96 healthy human subjects were acquired from 2020 to 2022 at the Ningbo University's affiliated People's Hospital. Before usage, the samples were kept at −80°C.
NSCLC diagnosis was confirmed via pathological examination in appropriate patients. Chemotherapy and radiography were not used on the participants who took part in this trial. The present research was performed with the approval of the Ningbo University's affiliated People's Hospital and their Ethical Committee. Informed consent was obtained from all the recruited human subjects.

| RNA preparation
TRIzol™ LS Reagent (Invitrogen) was utilized to extract the entire RNA from serum as per the manufacturer's directions. For isopropanol precipitation, glycogen 100 μg/ml (Thermo Fisher) was added, to boost the precipitation of RNA.

| qRT-PCR
To reverse transcribe total RNA, the ImProm-II Reverse Transcription System (Promega,) was utilized according to the directions provided by the manufacturer. Based on the reported literatures, 14,19 stemloop qRT-PCR method was used for quantification of mature tRFs, and miR-16 was selected as an internal control for the quantifica-

For data comparison, statistical techniques such as Student's t-test
and chi-square test were utilized when needed. The ROC curve studies were performed using MedCalc 11.0 (MedCalc, Ostend, Belgium).
In these studies, the significance was considered to be at p < 0.05.
Then, to amplify the tRF-31-79MP9P9NH57SD, we created one set of primers, and the amplified products were analyzed through melting curve and Sanger sequencing. Our findings revealed that the amplified product only produced a single peak. (Figure 1C), and the sequences were coincident with that in MINTbase v2.0 ( Figure 1D).
The data showed that tRF-31-79MP9P9NH57SD could be amplified by qRT-PCR availably.

| Highly expressed tRF-31-79MP9P9NH57SD in serum of NSCLC patients
qRT-PCR detected the tRF-31-79MP9P9NH57SD expression in serum samples of the 96 NSCLC patients and healthy controls. The expression of tRF-31-79MP9P9NH57SD was substantially increased in NSCLC patients as to that of the healthy controls, according to our findings ( Figure 2A). Next, tRF-31-79MP9P9NH57SD expression in another group including 20 NSCLC pre-and post-surgical patients and 20 healthy human subjects (termed as controls) was detected.
The resulting outcome reveals that tRF-31-79MP9P9NH57SD was enriched in NSCLC patient's serum before the surgical procedure ( Figure 2B).

| Clinical implications of serum tRF-31-79MP9P9NH57SD in NSCLC
We further explored the connection between the serum tRF-31-   Figure 3. Moreover, we also found that the diagnostic accuracy of the Panel was higher than that of either single biomarker (Table S1). Collectively, these results suggested that serum tRF-31-79MP9P9NH57SD might have potential values for NSCLC.

| Exploration of the downstream and function prediction of tRF-31-79MP9P9NH57SD
In the TargetScan, miRanda, and TargetRank databases, we predicted downstream targets of tRF-31-79MP9P9NH57SD in a Venny diagram ( Figure 4A). Among the linked genes that demonstrated the MAP3K2, GALNT10, RMND5A, ZNF33B and OAS3). We then sought to look into the molecular mechanism of tRF-31-79MP9P9NH57SD.

KEGG Enrichment analysis indicated that tRF-31-79MP9P9NH57SD
was enriched in virus infection, a variety of tumors and mTOR signaling pathway ( Figure 4B). tRF-31-79MP9P9NH57SD may have a role in RNA biosynthesis and transcription control, according to GO functional enrichment analysis of the target genes ( Figure 4C).
Therefore, the mechanisms of tRF-31-79MP9P9NH57SD in the regulation of NSCLC need to be further investigated.

| DISCUSS ION
The primary function of tRNAs is to transport amino acids to the ribosome, allowing the synthesis of the appropriate protein to proceed more quickly under the supervision of the mRNA. tRFs and tiRNAs have lately been discovered in a range of malignancies, 20  Recently, several tRFs have been reported to be diagnostic biomarkers in lung cancer and other malignancies. 15,26,27 In this study,

| CON CLUS ION
To conclude, this research discovered that serum tRF-31-79MP9P9NH57SD expression is elevated in NSCLC, and has strong diagnostic importance, insinuating that tRF-31-79MP9P9NH57SD serum may be a novel diagnostic marker for NSCLC.

AUTH O R CO NTR I B UTI O N S
The study was conceived and carried out by LJ and YW. CC and FL were involved in obtaining ethical approval, collecting samples, performing qRT-PCR, and analyzing data. The manuscript's first version was written by LJ. The final manuscript was reviewed and approved by all authors.

ACK N OWLED G M ENT
The study was carried out with the support of National Natural Science Foundation of China (NO.81872433).

CO N FLI C T O F I NTE R E S T S
None.

DATA AVA I L A B I L I T Y S TAT E M E N T
On reasonable request, the corresponding author will provide the datasets used and/or analyzed during the current work.

CO N S E NT FO R PU B LI C ATI O N
Not applicable.