Characterization of novel small non-coding RNAs and their modifications in bladder cancer using an updated small
Zhangli Su1,2, Ida Monshaugen3,4,5, Arne Klungland3,6
1Department of Genetics, University of Alabama at Birmingham, Birmingham, AL, United States.
Abstract:
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.
Insights
Non-microRNA small RNAs, including tRNA and ribosomal RNA fragments, are abundant in bladder cancer (BLCA) and show modifications. These small RNAs and their modifications are promising biomarkers for BLCA detection and follow-up.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Bladder cancer (BLCA) is a prevalent malignancy with significant mortality, necessitating improved diagnostic and therapeutic strategies.
- Small non-coding RNAs, particularly microRNAs, are implicated in cancer, but other small RNA families' roles in BLCA remain underexplored.
- Elevated levels of free modified nucleosides in urine suggest small RNAs and their modifications may serve as BLCA biomarkers.
Purpose of the Study:
- To perform a genome-wide analysis of small RNAs (<40 nucleotides) and their modification status in BLCA.
- To identify novel small RNA molecules and potential biomarkers for BLCA detection and management.
Main Methods:
- Small RNA profiling (<40 bases) was conducted on 12 non-muscle invasive papillary urothelial carcinoma (BLCA) tumor specimens.
- A modified protocol using thermostable group II reverse transcriptase (TGIRT) was employed for enhanced small RNA identification and modification analysis.
- Analysis focused on identifying non-microRNA small RNAs and detecting potential base modifications via mismatch sites.
Main Results:
- A significant portion (57 ± 15%) of sequencing reads mapped to non-microRNA small RNAs, including tRNA-derived fragments (tRFs), ribosomal RNA-derived fragments (rRFs), and YRNA-derived fragments (YRFs).
- Potential base modifications, such as N1-methyladenosine (m1A), N1-methylguanosine (m1G), and N2,N2-dimethylguanosine (m22G), were identified on tRFs.
- Mismatch sites on rRFs corresponded to known modifications on 28S and 18S ribosomal RNA.
Conclusions:
- Non-microRNA small RNAs, particularly tRFs and rRFs, are abundant in BLCA tumor tissues.
- TGIRT sequencing can capture small RNA modifications as mismatch sites, indicating potential biological relevance.
- Both non-microRNA small RNAs and their modifications warrant further investigation as potential biomarkers for BLCA diagnosis and monitoring.
