Plasma tsRNAs as novel diagnostic biomarkers for renal cell carcinoma

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L E T T E R T O T H E J O U R N A L Plasma tsRNAs as novel diagnostic biomarkers for renal cell carcinoma
Dear Editor, Early diagnosis greatly benefits renal cell carcinoma (RCC) patients with remarkable higher survival. 1Whereas, no appropriate liquid-biopsy biomarkers has been applied to RCC diagnosis in clinic so far. 2 Transfer RNA-derived small RNAs (tsRNAs), a class of newly discovered noncoding RNAs, are stable and abundant in circulation, promising in noninvasive early diagnosis of cancer. 3,4Here, we explore the diagnosis values and biological functions of plasma tsRNAs in RCC for the first time.
The study was evaluated and authorised by the Ethics Committee of Nanjing Drum Tower Hospital (2021-582-01).The research route diagram was shown in Figure S1.First, to explore the distinct plasma tsRNA expression profile in RCC patients, we performed small RNA sequencing using RNA extracted individually from plasma of five RCC patients and five healthy controls.The tsRNA expression profile in plasma of RCC patients were obviously changed (Figure 1A), and among 657 tsRNAs detected, 71 tsRNAs were significantly altered (Figure 1B).
Considering the tsRNAs expression levels (CPM > 10) and length (due to the length limitation, the length of tsRNAs should ≥ 17 nt when assessed by RT-qPCR), we selected 12 tsRNAs for further validation in an independent training set (30 healthy controls and 32 RCC patients) using RT-qPCR.The detection limits of RT-qPCR assay using tsRNA specific primers were evaluated by the standard curves developed with corresponding synthetic tsRNA oligonucleotides (Figure S2, Table S1).In this training set, 5 out of the 12 tsRNAs (tRF-19-DRMD5112, tRF-18-8R6Q46D2, tRF-17-884U1D2, tRF-17-8SOUPR2 and tRF-28-87R8WP9I1E0K) were significantly reduced in RCC patients, which was also consistent with the sequencing data (Figure 1C; Tables S2 and S3).Whereafter, the levels of these 5 tsRNAs were further confirmed in another larger validation set (99 healthy controls and 120 RCC patients), and showed a consistent trend (p < .0001for all tsRNAs; Figure 2A and Table S4).Combining the training and Meng Ding and Wanqing Zhou contributed equally.
the validation sets for analysis, the results were compatible (p < .0001for all tsRNAs; Figure S3).Therefore, these data confirmed the stability of the expression pattern.Patient characteristics in the training set and validation set were summarised in Table S5.Plasma tRF-28-87R8WP9I1E0K level was decreased in patients with higher TNM grading (Table S6), suggesting its potential biological function in RCC progression.
We then assessed the clinical usefulness of the five plasma tsRNAs for RCC.By ROC curve analysis, the AUC values for these tsRNAs ranged from 0.7487 to 0.8549 (p < .0001; Figure 2B).Among these tsRNAs, tRF-19-DRMD5112 showed the best performance (AUC: 0.8549, sensitivity: 80.92%, and specificity: 77.52%; Figure 2B).Furthermore, we assessed the values of these 5 plasma tsRNAs in early-stage RCC patients (stage I, 118 out of 152 RCC patients) diagnosis and figured out the range of AUC values from 0.7411 to 0.8650 (p < .0001; Figure S4).tRF-19-DRMD5112 also showed top performance (AUC: 0.8650, sensitivity: 80.51%, and specificity: 81.31%; Figure S4), which presented even higher diagnostic value in earlystage RCC patients compared to that in all RCC patients.To construct a tsRNA-based diagnostic model, we first performed LASSO-penalised logistic regression analysis using training and validation sets.Two tsRNAs, tRF-19-DRMD5112 and tRF-18-8R6Q46D2, were selected for the optimal model construction (Figure S5).However, in the following multivariate logistic regression analysis, tRF-19-DRMD5112 was the only tsRNA independently correlated with RCC (p < .001;Table S7).Taken together, the single expression level of tRF-19-DRMD5112 in plasma could be used as the diagnostic model for RCC.
In conclusion, we revealed plasma tsRNA expression patterns in RCC patients for the first time, and identified tRF-19-DRMD5112 with favourable diagnostic values for RCC (especially for early stage), providing novel potential biomarker for accurate RCC diagnosis.In addition, tRF-28-87R8WP9I1E0K is demonstrated to possess powerful anti-tumour function, which warrants further exploration to develop a novel tsRNA-based therapeutic strategy for RCC.

A U T H O R C O N T R I B U T I O N S
MD, WD and HG conceived and designed the study.MD, WD and HG wrote the manuscript.MD and WD performed the quantification experiments.MD, WM and XG performed cell experiments.MD, WD and WM performed the bioinformatic analysis and statistical analysis.WZ, WC, XG, YL, CJ and GL contributed to sample collection.All authors read and approved the final manuscript.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare they have no conflicts of interest.

F U N D I N G
This work was supported by grants from the National Natural Science Foundation of China (81902571 to M.D., 82173160 to W.D., and 81972388 to H.G.), Nanjing Medical Science and technology development Foundation (ZKX22024 to M.D.).

D ATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used in this study are available upon reasonable request from the corresponding author.

E T H I C S S TAT E M E N T
The study was evaluated and authorised by the Ethics Committee of Nanjing Drum Tower Hospital (2021-582-01), and all participants voluntarily signed an informed consent.
Meng Ding 1 Wanqing Zhou 2 Wenyuan Chen 1,3 Wenjing Mo 1,3 Xinyue Guo 1,3 Yuhang Li 1,3 Changwei Ji 1 Guangxiang Liu 1 Wenli Diao 1 Hongqian Guo Clin.Transl.Med.2024;14:e1575.wileyonlinelibrary.com/journal/ctm2 1 of 7 https://doi.org/10.1002/ctm2.1575F I G U R E 1 Identification of differentially expressed plasma tsRNAs in RCC patients.(A) Heat map of the expression profile of plasma tsRNAs in 5 RCC patients and 5 healthy controls.(B) Scatter plots of differentially expressed plasma tsRNAs.The red dots indicate upregulated tsRNAs, the blue dots are for downregulated tsRNAs, and the grey dots are for nondifferentially expressed tsRNAs.The grouping criteria is fold-change ≥ 1.5, p < .05. (C) The relative expression levels of 12 plasma tsRNAs in 32 RCC patients and 30 healthy controls using RT-qPCR.*p < .05;**p < .01;***p < .001.F I G U R E 2 Validation and diagnosis value of differentially expressed plasma tsRNAs in RCC patients.(A) The relative expression levels of 5 plasma tsRNAs in 120 RCC patients and 99 healthy controls using RT-qPCR.(B) ROC curves for the abilities of 5 individual plasma tsRNAs to discriminate RCC patients from healthy controls in a total of 152 RCC patients and 129 healthy controls.(C) The relative expression levels of tRF-19-DRMD5112 in testing set (43 RCC patients and 31 healthy controls) using RT-qPCR.(D) ROC curve for the ability-of plasma tRF-19-DRMD5112 level to discriminate RCC patients from healthy controls in testing set.***p < .001.F I G U R E 3 Biology functions of these 5 tsRNAs in RCC.(A) Relative expression levels of the 5 tsRNAs in RCC tissues and paired normal kidney tissues by RT-qPCR, N = 21.(B) Representative images (Left) and histogram statistics (Right) from EdU assay of RCC cells transfected with individual tsRNA mimics or negative controls, scale bar = 50 µm.(C) Representative images (Left) and histogram statistics (Right) from colony formation assay of RCC cells transfected with individual tsRNA mimics or negative controls, scale bar = 0.4 cm.(D) Representative images (Left) and histogram statistics (Right) from sphere formation assay of RCC cells transfected with individual tsRNA mimics or negative controls, scale bar = 100 µm.(E) Representative images (Left) and histogram statistics (Right) from transwell migration assay of RCC cells transfected with individual tsRNA mimics or negative controls, scale bar = 200 µm.(F) Representative images (Left) and histogram statistics (Right) from transwell invasion assay of RCC cells transfected with individual tsRNA mimics or negative controls, scale bar = 200 µm.Cell experiment was repeated three times independently, *p < .05;**p < .01;***p < .001.F I G U R E 4 Enrichment analysis of predicted target genes of tRF-28-87R8WP9I1E0K. (A) Secondary structure prediction of tRF-28-87R8WP9I1E0K. (B) GO analysis of the predicted targets of tRF-28-87R8WP9I1E0K. (C) Pathway analysis of the predicted targets of tRF-28-87R8WP9I1E0K.

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Department of Urology, Nanjing Drum Tower Hospital Clinical College of Nanjing University of Chinese Medicine, Nanjing, China Correspondence Hongqian Guo and Wenli Diao, Department of Urology, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, 321 Zhongshan Road, Nanjing 210008, Jiangsu Province, China.Email: dr.ghq@nju.edu.cn;diaowl@126.comO R C I D Hongqian Guo https://orcid.org/0000-0002-3121-5157R E F E R E N C E S

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Department of Urology, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Institute of Urology, Nanjing University, Nanjing, China 2 Department of Laboratory Medicine, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing University, Nanjing, China