Expression profiles of tsRNAs
A total of 4,021 tsRNAs mapped to 334 unique tRNAs, including tRNAs in the cytosol or mitochondria, were identified. Generally, tRFs are classified into four subtypes according to their original sites in pre-tRNA or mature tRNA, including tRF-1, tRF-3, tRF-5 and i-tRF. tRF-1 is derived from the 3'end of the precursor tRNA, and tRF-5 and tRF-3 are derived from the 5'and 3'ends of mature tRNA. While i-tRF is produced by internal fragments of mature tRNA. tiRNA could produced tiRNA-3 and tiRNA-5 . Based on this classification, we further analysed the proportion of tRF subtypes in patients with sarcoidosis and healthy controls. The pie charts show the distribution of the number for each subtype of tsRNA with a threshold average CPM ≥ 20. A total of 231 tsRNAs for exact matches were identified in the serum (207 in sarcoidosis, 214 in healthy controls). In the sarcoidosis group, two tRF-2, 33 tRF-3a, 22 tRF-3b, 38 tRF-5a, 24 tRF-5b, 76 tRF-5c, and 12 tiRNA-5 were identified in the sarcoidosis group (Figure 1A). In the control group, four tRF-1, two tRF-2, 34 tRF-3a, 26 tRF-3b, 38 tRF-5a, 23 tRF-5b, 72 tRF- 5c, and 15 tiRNA-5 were identified (Figure 1B). In addition, the numbers of tsRNAs derived from the variable anticodon tRNAs are shown in the stacked plots (Figure 1C-D).
Differentially expressed tsRNAs in patients with sarcoidosis vs. healthy controls were identified through RNA sequencing. All differentially expressed tsRNAs are shown in the cluster heatmap in Figure 2A. The heatmap revealed systematic variations between the sarcoidosis and control groups in terms of tsRNA expression. Additionally, we identified 13 significantly dysregulated tsRNAs: four were upregulated, while nine were downregulated (FC > 1.5, P < 0.05; Figure 2B and Table 4).
RT‑qPCR validation of RNA sequencing results
Six tsRNAs (tRF-iMet-CAT-001, tRF-Gln-TTG-005, tiRNA-Glu-TTC-001, tiRNA-Lys-CTT-003, tiRNA-Gly-GCC-002, and tRF-Ser-TGA-007) were randomly selected to validate the RNA sequencing results using RT-qPCR in sarcoidosis and control samples. The expression profiles of tiRNA-Glu-TTC-001, tiRNA-Lys-CTT-003, and tRF-Ser-TGA-007 agreed with results obtained from RNA sequencing. There were significant differences in tsRNA expression levels between patients with sarcoidosis and healthy controls (P < 0.05; Figure 3). These findings indicated that the RNA sequencing data were reliable enough to warrant further analysis.
Correlation between differentially expressed tsRNAs and the clinical parameters of sarcoidosis
The relative expression levels of tiRNA-Glu-TTC-001 were negatively correlated with age (Figure 4A). The relative expression levels of tiRNA-Lys-CTT-003 were significantly correlated with age and the number of affected systems (Figure 4B-C). There was a significant positive correlation between the relative expression of tRF-Ser-TGA-007 and blood calcium levels (Figure 4D).
Prediction of target genes of differentially expressed tsRNAs
Although the mechanism of tsRNA action is yet to be elucidated, recent evidence suggests a miRNA-like mode of action. miRNAs can recognise their mRNA targets using their seed sequence (positions 2-7nt at their 5ʹ ends) and suppress global mRNA translational activities. Based on this rule, we used two types of algorithms, TargetScan and miRDB, to predict tsRNA targets. In total, miRDB targeted a total of 646 mRNAs, TargetScan targeted a total of 228 mRNAs, and the two target a total of 96 mRNAs (Figure 5B); tiRNA-Glu-TTC-001, tiRNA-Lys-CTT-003, and tRF-Ser-TGA-007 could target 637, 111, and 39 transcripts, respectively (Figure 5A).
Biological function analysis revealing potential therapeutic effects of differentially expressed tsRNAs
The tsRNAs regulated mRNA translational activities, therefore we conducted bioinformatics analyses of the functions of target mRNAs to understand their biological functions. We used GO biological processes and KEGG pathways to explore the functions of target mRNAs. The target mRNA of tiRNA-Glu-TTC-001 was enriched in BP and involved in the regulation of transcription, DNA-templated, transcription, negative regulation of transcription from RNA polymerase II promoter, Ras protein signal transduction, heterophilic cell-cell adhesion via plasma membrane cell adhesion molecules, transport, ERK1 and ERK2 cascade, positive regulation of transcription from RNA polymerase II promoter, small GTPase-mediated signal transduction, brain development, ER to Golgi vesicle-mediated transport, and cell proliferation (Figure 6A). The CC comprised intracellular, nucleus, cis-Golgi network, Golgi membrane, receptor complex, chromatin, early endosome, and actin cytoskeleton (Figure 6B), while MF included protein binding, metal ion binding, nucleic acid binding, transcription factor activity, sequence-specific DNA binding, DNA binding, RNA polymerase II core promoter proximal region sequence-specific DNA binding, chromatin binding, protein serine/threonine kinase activity, and transcription corepressor activity (Figure 6C).
The target mRNA of tiRNA-Lys-CTT-003 was enriched in BP, involved in transcription regulation, DNA-templated, regulation of cell cycle, DNA damage response, signal transduction by p53 class mediator resulting in cell cycle arrest, transcription from RNA polymerase II promoter, megakaryocyte development, platelet alpha granule organisation (Figure 6D), in CC, comprising the nucleus (Figure 6E), and MF, in RNA polymerase II core promoter proximal region sequence-specific DNA binding, metal ion binding, transcriptional activator activity, RNA polymerase II core promoter proximal region sequence-specific binding (Figure 6F).
The target mRNA of tRF-Ser-TGA-007 was enriched in BP involved in negative regulation of transcription from RNA polymerase II promoter, positive regulation of transcription from RNA polymerase II promoter, negative regulation of vascular endothelial growth factor receptor signalling pathway, positive regulation of IRE1-mediated unfolded protein response, activation of signalling protein activity involved in unfolded protein response, negative regulation of protein serine/threonine kinase activity, cellular response to vascular endothelial growth factor stimulus (Figure 6G). The CC comprised cerebellar mossy fibre, cell-cell adherens junction (Figure 6H), and MF in transcription factor activity, RNA polymerase II distal enhancer sequence-specific binding, transcription factor activity, RNA polymerase II distal enhancer sequence-specific binding, and protein phosphatase 2A binding (Figure 6I).
KEGG analysis revealed that the target mRNA of tiRNA-Glu-TTC-001 was particularly enriched in pathways in cancer, chemokine signalling, cAMP signalling, Rap1 signalling, endocytosis, Ras signalling, cGMP-PKG signalling, retrograde endocannabinoid signalling, FoxO signalling, adrenergic signalling in cardiomyocytes, and inflammatory mediator regulation of TRP channels (Table 5), while no KEGG pathway was associated with the target mRNAs of tiRNA-Lys-CTT-003 and tRF-Ser-TGA-007 (P < 0.05).
Hub gene analysis
The top ten genes of each sub-plugin in the PPI network of the target mRNAs of tiRNA-Glu-TTC-001, tiRNA-Lys-CTT-003, and tRF-Ser-TGA-007 were identified using the cytoHubba plugin, respectively (Table 6). The ten genes with the highest frequency in all sub-plugins were classified as hub genes. KEGG analysis of the hub genes showed their involvement in the insulin signalling pathway (hsa04910), chemokine signalling pathway (hsa04062), regulation of lipolysis in adipocytes (hsa04923), bacterial invasion of epithelial cells (hsa05100), neurotrophin signalling pathway (hsa04722), AMPK signalling pathway (hsa04152), platelet activation (hsa04611), FoxO signalling pathway (hsa04068), cAMP signalling pathway (hsa04024), proteoglycans in cancer (hsa05205), and pentose phosphate pathway (hsa00030).
We used AUC analysis to evaluate their potential for diagnosing sarcoidosis using the expression data from the GSE19314 database. The hub genes associated with tiRNA-Glu-TTC-001 were all expressed in the serum. The AUCs of CLTC, APP, PRKACB, RAP1B, ARRB2, IRS1, ACTR2, and PIK3R3 were all greater than 0.5, of which APP, PRKACB, and ARRB2 were 0.707, 0.728, and 0.714, respectively (Figure 7A). Except for KIAA0101, the hub genes associated with tiRNA-Lys-CTT-003 were all expressed in the serum of patients with sarcoidosis. The AUCs of nine genes were all greater than 0.5 (Figure 7B). The hub gene associated with tRF-Ser-TGA-007 was expressed in the serum. The AUCs of NR5A1, SPI1, TKTL2, FMN1, MNDA, and NCOR2 were all greater than 0.5, of which NR5A1 and MNDA were 0.712 and 0.726, respectively (Figure 7C). The AUCs >0.5 were considered statistically significant. These genes have higher diagnostic potential since they were all greater than 0.70.
RNAhybrid software was used to construct a base complementary pairing model between tiRNA-Glu-TTC-001 and APP, PRKACB, and ARRB2 (Figure 8A-C), and between tRF-Ser-TGA-007 and NR5A1 and MNDA, respectively. There was no base complementary pairing model between tRF -Ser-TGA-007 and MNDA (Figure 8D).