Multi‐Omics Analysis and Experimental Verification Reveal Multiple Roles of tRNA‐Derived Fragments in Nasopharyngeal Carcinoma

ABSTRACT tRNA‐derived small RNAs (tsRNAs) are small single‐stranded RNAs cleaved from precursor and mature tRNAs. Aberrant expression of tsRNAs has been reported in multiple cancers, suggesting their potential as novel biomarkers and therapeutic targets. However, the role of tsRNAs in nasopharyngeal carcinoma (NPC) remains unclear. This study aimed to investigate tsRNA expression profiles in serum exosomes of NPC and explore the function of dysregulated tsRNAs. Serum samples from 30 NPC patients and 30 healthy controls, as well as 10 pairs of NPC tumor and adjacent normal tissues were collected. Serum exosomes were isolated by ultracentrifugation and identified using transmission electron microscopy. Exosomal tsRNA expression was analyzed by high‐throughput sequencing in 3 paired samples. Dysregulated tsRNAs were validated by RT‐qPCR in NPC cell lines (5‐8F, CNE1, CNE2), NP69 cells, clinical serum exosomes and tissues. ROC curves were used to evaluate diagnostic values. CCK‐8, EdU, colony formation and Transwell assays were performed to assess cell functions. GO and KEGG enrichment analyses were conducted to functionally annotate target genes and identify significantly enriched biological pathways. The expression of 247 tsRNAs varied significantly in serum exosomes, including 142 upregulated and 105 down‐regulated, in NPC patients compared with healthy controls. RT‐qPCR validation demonstrated that the expression levels of tRNA‐Lys‐CTT‐1‐M5 and tRNA‐Thr‐TGT‐2 were significantly upregulated in NPC cells, tumor tissues, and serum exosomes compared with their normal nasopharyngeal cell, tissue, and serum exosome counterparts from healthy individuals. And in the diagnosis of NPC, the AUC values of exosomal tRNA‐Lys‐CTT‐1‐M5 and tRNA‐Thr‐TGT‐2 based on ROC curve analysis were 0.88 and 0.81, respectively. Functional experiments showed that tRNA‐Lys‐CTT‐1‐M5 and tRNA‐Thr‐TGT‐2 promoted NPC cell proliferation, while tRNA‐Thr‐TGT‐2 enhanced cell migration. These results indicate that tRNA‐Thr‐TGT‐2 plays an important role in the pathogenesis of nasopharyngeal carcinoma (NPC) and has the potential to serve as a novel diagnostic biomarker. Furthermore, tRNA‐Thr‐TGT‐2 is expected to become a promising therapeutic target for the treatment of NPC.