TsRNA-49-73-Glu-CTC: A promising serum biomarker in non-small cell lung cancer

Abstract

Objective: Lung cancer has the highest incidence and mortality rates globally, with the majority of cases classified as non-small cell lung cancer (NSCLC). Due to the absence of specific tumor biomarkers, most lung cancer cases are diagnosed at an advanced stage. Therefore, the identification of novel molecular biomarkers with high sensitivity and specificity for early diagnosis is deemed crucial for enhancing the treatment of NSCLC. Transfer RNA-derived small RNA (tsRNA) is closely associated with malignant tumors and holds promise as a potential biomarker for tumor diagnosis. This study aimed to investigate whether serum tsRNA could serve as a biomarker for NSCLC.

Methods: Differentially expressed tsRNAs were identified through high-throughput sequencing of serum samples obtained from patients with NSCLC and healthy individuals. Additional serum samples were collected for validation using Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR). The diagnostic performance of these tsRNAs was assessed through Receiver Operating Characteristic (ROC) Curve Analysis. Furthermore, preliminary functional exploration was undertaken through cell experiments.

Results: tsRNA-49-73-Glu-CTC is highly expressed in the serum of patients with NSCLC and demonstrates superior diagnostic value compared to commonly used tumor markers in clinical practice, such as Carcinoembryonic Antigen (CEA), Neuron-Specific Enolase (NSE), and Cytokeratin 19 Fragment (CYFRA). A combined diagnostic approach enhances the accuracy of NSCLC detection. Additionally, tsRNA-49-73-Glu-CTC is highly expressed in A549 cells, and transfection with a tsRNA-49-73-Glu-CTC inhibitor significantly reduces both proliferation and migration capabilities.

Conclusions: tsRNA-49-73-Glu-CTC has the potential to serve as a novel molecular diagnostic biomarker for NSCLC and plays a significant role in the biological processes associated with NSCLC proliferation and migration.

Fig 1. Serum tsRNA expression profiles in NSCLC patients.

(A) Venn diagram showing the number of known and detected tsRNAs. (B) Number of tsRNAs expressed in the serum of NSCLC patients and healthy controls. (C, D) Distribution of tsRNA subtypes in NSCLC and healthy serum samples. (E, F) The number of subtype tsRNA against tRNA isodecoders. The X axes represents tRNA decoders and the Y axes shows the number of all subtype tsRNA against tRNA isodecoders. The color represents the subtype tsRNA.

Fig 2. Differentially expressed tsRNAs.

(A) Scatter plot of differentially expressed tsRNAs between NSCLC and healthy controls. The values on the X and Y axes represent the average CPM values for each group (log2 scaled). Red dots above the top line (upregulated) or green dots below the bottom line (downregulated) indicate tsRNAs with a fold change greater than 1.5 between the two comparison groups. Gray dots represent non-differentially expressed tsRNAs. (B) Volcano plot of significantly differentially expressed tsRNAs between NSCLC and healthy controls. (C) Heatmap of significantly differentially expressed tsRNAs between NSCLC and healthy controls. (D, E, F) Expression levels of tsRNA-49:73-Glu-CTC, tsRNA-19:32-Lys-CTT, and tsRNA-18:32-Lys-CTT in the serum of NSCLC patients and healthy controls, ****P <  0.0001.

Fig 3. Secondary structures of tsRNA-49:73-Glu-CTC, tsRNA-19:32-Lys-CTT, and tsRNA-18:32-Lys-CTT.

Fig 4. Diagnostic value analysis of tsRNA-49:73-Glu-CTC in serum.

(A) Expression levels of tsRNA-49:73-Glu-CTC in the validation cohort. (B) ROC curve analysis of tsRNA-49:73-Glu-CTC. (C) ROC curve analysis of tsRNA-49:73-Glu-CTC combined with CEA, NSE, and CYFRA individually. (D) ROC curve analysis of tsRNA-49:73-Glu-CTC combined with CEA, NSE, and CYFRA. AUC: Area Under the Curve; NSE: Neuron-Specific Enolase; CYFRA: Cytokeratin 19 Fragment; tsRNA: tsRNA-49:73-Glu-CTC. *  P < 0.05.

Fig 5. Preliminary functional exploration of tsRNA-49:73-Glu-CTC.

(A) Expression levels of tsRNA-49:73-Glu-CTC in BEAS-2B and A549 cells. (B) Expression of tsRNA-49:73-Glu-CTC in A549 cells after transfection with tsRNA-49:73-Glu-CTC inhibitor. (C) CCK-8 cell proliferation assay. (D) Cell scratch assay. NC-IN: inhibitor NC; tsRNA-IN: tsRNA-49:73-Glu-CTC inhibitor. *  P < 0.05, **** P < 0.0001.