Summary:
Bladder cancer (BlCa) is the second most common malignancy of the male urinary system and affects men 3 to 4 times more than women. The recurrence rate that demonstrates, among the other types of cancers is considered as a unique feature. This characteristic underscores the clinical importance of this disease, and together with the high cost diagnostic and therapeutic procedures, such as cystoscopies and transurethral resections of tumors (TURBT), the huge economic burden for health care systems is clear. Therefore, the need of discovering novel biomarkers, which contribute in diagnosis, prognosis and surveillance of patients, is essential, in order to replace invasive approaches, that are considered as gold standards. Non coding RNAs (ncRNAs) could possibly function as biomarkers with huge contribution in the biomedical field, due to their crucial advantages, such as easy, reliable and rapid quantification. The current work emphasizes in a novel subset of ncRNAs, the tRNA-derived fragments (tRFs/t-halves (thalves ή tiRNAs)) and their correlation with oxidative stress in bladder cancer tissues, that exhibit certain clinicopathological features. In order to achieve those aims, theoretical and experimental design was performed.
Particularly, via bioinformatical analysis, online data from NGS experiments were used for fragments selection. In silico design of primers, pulverization of patients’ tissues, application and optimization of polyadenylation, reverse transcription protocols and qPCR molecular technique enabled the quantification of the selected tRNA-derived fragments. The linkage of ncRNAs with oxidative stress was approached by studying transcriptional levels of GSTP1, SOD2, NRF2 due to proper design of primers and Spearman correlation analysis. In the context of this work, 10 different types of tRFs/t-halves (thalves ή tiRNAs), which are encoded both from nuclear and mitochondrial genome, were identified in bladder cancer cell lines (RT112, T24) and bladder cancer tissues from patients (NMIBC, MIBC). The ncRNAs that were identified are the following: ts-36, 3 ́-half-His-GTG, 5 ́-half-Val-TAC, 5 ́-half- Met-CAT, 3 ́-tRF-Val-TAC, 5 ́-half-Val-AAC, i-Gln-CTG, i-Val-TAC, 5 ́-tRF-Gly-CCC and ts-46.
Three of those fragments were selected and studied in a cohort of 120 patients, 3 ́-half-His-GTG, 5 ́-half-Val-TAC and 5 ́-half-Met-CAT, because are exclusively encoded by the mitochondrial genome (mtDNA) and belong to t-halves (tiRNAs) (tiRNAs) category.
Bibliographic research led to the formulation of the following hypothesis: Non coding RNAs, tRFs/t-halves (thalves ή tiRNAs), boost their expression under stress conditions, that characterize cancer tumors, and in oxidative imbalance the role of mitochondria is crucial.
The expression levels of ncRNAs are elevated in high grade tumors (3 ́half-His-GTG, p-value =0.006) and in advanced stages of bladder cancer, especially in muscular invasive (T2-T4) tumors. Survival analysis suggests that increased levels of those ncRNAs are related to unfavorable prognosis of patients who were subjected to transurethral resection or radical cystectomy. Finally, the decreased levels of 5 ́half-Val-TAC and 5’half-Met-CAT, in conditions of high expression of GSTP1 and low expression of SOD2, probably suggest cellular stress correlates with t-halves (tiRNAs) production, which arise from mitochondrial tRNA encoded molecules.
The biological significance of the fragments and genes was estimated by using bioinformatic tools and data. Target prediction algorithms enabled the characterization of possible targets for tRNA fragments, using the assumption that function in a similar way as miRNAs, a fact which is extensively mentioned in literature. Also, according to philosophy of systems biology (Gene ontology analysis/ KEGG network analysis), network analysis was performed and ontology diagrams were designed in R programming environment. The Human Protein Atlas was used for the confirmation of protein products from GSTP1 and SOD2 in urothelial carcinoma.
The current study was based on the combination of bioinformatical, biostatistical and experimental procedures for the interpretation of results and concludes in further support of the theory that oxidative stress in cancer cells is linked to accumulation of different ncRNAs production, such as t-halves (tiRNAs) (tiRNAs).
