RESUMO
Background: Bladder cancer (BLCA) is a common and deadly disease that results in a reduced quality of life for the patients and a significant economic burden on society. A better understanding of tumorigenesis is needed to improve clinical outcomes. Recent evidence places the RNA modification m1A and its regulatory proteins TRMT6/TRMT61A and ALKBH3 in BLCA pathogenesis. Methods: TRMT6/TRMT61A, ALKBH1, and ALKBH3 expression was examined in human BLCA cell lines and a normal urinary tract epithelium cell line through qRT-PCR and western blot analysis. Prestoblue Cell Viability Reagent, wound-healing assay, and live-cell imaging-based cell displacement analysis, were conducted to assess proliferation, migration, and displacement of this BLCA cell line panel. Cell survival was assessed after inducing cellular stress and activating the unfolded protein response (UPR) with tunicamycin. Moreover, siRNA-mediated gene silencing in two BLCA cell lines (5637 and HT1197) was conducted to investigate the biological roles of TRMT6/TRMT61A. Results: Heterogeneous morphology, proliferation, displacement, tunicamycin sensitivity, and expression levels of m1A regulators were observed among the panel of cell lines examined. In general, TRMT61A expression was increased in BLCA cell lines when compared to SV-HUC-1. Depletion of TRMT6/TRMT61A reduced proliferation capacity in both 5637 and HT1197 cell lines. The average cell displacement of 5637 was also reduced upon TRMT6/TRMT61A depletion. Interestingly, TRMT6/TRMT61A depletion decreased mRNA expression of targets associated with the ATF6-branch of the UPR in 5637 but not in HT1197. Moreover, cell survival after induction of cellular stress was compromised after TRMT6/TRMT61A knockdown in 5637 but not in HT1197 cells. Conclusion: The findings suggest that TRMT6/TRMT61A plays an oncogenic role in BLCA and is involved in desensitizing BLCA cells against cellular stress. Further investigation into the regulation of TRMT6/TRMT61A expression and its impact on cellular stress tolerance may provide insights for future BLCA treatment.
RESUMO
Background: Bladder cancer (BLCA) is one of the most common cancer types worldwide. The disease is responsible for about 200,000 deaths annually, thus improved diagnostics and therapy is needed. A large body of evidence reveal that small RNAs of less than 40 nucleotides may act as tumor suppressors, oncogenes, and disease biomarkers, with a major focus on microRNAs. However, the role of other families of small RNAs is not yet deciphered. Recent results suggest that small RNAs and their modification status, play a role in BLCA development and are promising biomarkers due to their high abundance in the exomes and body fluids (including urine). Moreover, free modified nucleosides have been detected at elevated levels from the urine of BLCA patients. A genome-wide view of small RNAs, and their modifications, will help pinpoint the molecules that could be used as biomarker or has important biology in BLCA development. Methods: BLCA tumor tissue specimens were obtained from 12 patients undergoing transurethral resection of non-muscle invasive papillary urothelial carcinomas. Genome-wide profiling of small RNAs less than 40 bases long was performed by a modified protocol with TGIRT (thermostable group II reverse transcriptase) to identify novel small RNAs and their modification status. Results: Comprehensive analysis identified not only microRNAs. Intriguingly, 57 ± 15% (mean ± S.D.) of sequencing reads mapped to non-microRNA-small RNAs including tRNA-derived fragments (tRFs), ribosomal RNA-derived fragments (rRFs) and YRNA-derived fragments (YRFs). Misincorporation (mismatch) sites identified potential base modification positions on the small RNAs, especially on tRFs, corresponding to m1A (N1-methyladenosine), m1G (N1-methylguanosine) and m2 2G (N2, N2-dimethylguanosine). We also detected mismatch sites on rRFs corresponding to known modifications on 28 and 18S rRNA. Conclusion: We found abundant non-microRNA-small RNAs in BLCA tumor samples. Small RNAs, especially tRFs and rRFs, contain modifications that can be captured as mismatch by TGIRT sequencing. Both the modifications and the non-microRNA-small RNAs should be explored as a biomarker for BLCA detection or follow-up.
RESUMO
RNA modifications are important regulatory elements of RNA functions. However, most genome-wide mapping of RNA modifications has focused on messenger RNAs and transfer RNAs, but such datasets have been lacking for small RNAs. Here we mapped N1-methyladenosine (m1A) in the cellular small RNA space. Benchmarked with synthetic m1A RNAs, our workflow identified specific groups of m1A-containing small RNAs, which are otherwise disproportionally under-represented. In particular, 22-nucleotides long 3' tRNA-fragments are highly enriched for TRMT6/61A-dependent m1A located within the seed region. TRMT6/61A-dependent m1A negatively affects gene silencing by tRF-3s. In urothelial carcinoma of the bladder, where TRMT6/61A is over-expressed, higher m1A modification on tRFs is detected, correlated with a dysregulation of tRF targetome. Lastly, TRMT6/61A regulates tRF-3 targets involved in unfolded protein response. Together, our results reveal a mechanism of regulating gene expression via base modification of small RNA.
