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Molecular Diagnostics

Hybridisation chain reaction-based visualisation and screening for lncRNA profiles in clear-cell renal-cell carcinoma

Abstract

Background

Analysis of long noncoding RNA (lncRNA) localisation at both the tissue and subcellular levels can provide important insights into the cell types that are important for their function.

Methods

By applying new fluorescent in situ hybridisation technique called hybridisation chain reaction (HCR), we achieved a high-throughput lncRNA visualisation and evaluation of clinical samples.

Results

Assessing 1728 pairs of 16 lncRNAs and clear-cell renal-cell carcinoma (ccRCC) specimens, three lncRNAs (TUG1, HOTAIR and CDKN2B-AS1) were associated with ccRCC prognosis. Furthermore, we derived a new lncRNA risk group of ccRCC prognosis by combining the expression levels of these three lncRNAs. Examining genomic alterations underlying this classification revealed prominent features of tumours that could serve as potential biomarkers for targeting lncRNAs. We then derived combination of HCR with expansion microscopy and visualised nanoscale-resolution HCR signals in cell nuclei, uncovering intracellular colocalization of three lncRNA (TUG1, HOTAIR and CDKN2B-AS1) signals such as those located intra- or out of the nucleus or nucleolus in cancer cells.

Conclusion

LncRNAs are expected to be desirable noncoding targets for cancer diagnosis or treatments. HCR involves plural probes consisting of small DNA oligonucleotides, clinically enabling us to detect cancerous lncRNA signals simply and rapidly at a lower cost.

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Fig. 1: The sixteen lncRNAs in this study.
Fig. 2: Assessment of hybridisation chain reaction.
Fig. 3: Relationship between expression levels of 16 lncRNAs and outcome of prognosis in ccRCC patients.
Fig. 4: Relationship between the lncRNA risk classification, outcome of prognosis and genetic alterations in ccRCC patients.
Fig. 5: The HCR-ExFISH reveals intracellular colocalization of lncRNAs.

Data availability

All data supporting the findings of this study are included within the article and its Supplementary Information files (and Reporting summary). Also, the data will be shared upon reasonable request to the corresponding author from colleagues who want to analyse in deep our findings.

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Funding

This study was supported by Grants-in-Aid for Scientific Research (KAKENHI 19K18598 and 21K09356 to RK; 19H03792, 21K19414, and 22H03217 to NT; and 18H02939 to MO) and grants from the Kobayashi Foundation for Cancer Research (to NT), the SGH Foundation for Cancer Research (to NT), the JUA Research Grant (to NT), the Princess Takamatsu Cancer Research Fund (to NT), and the Keio Gijuku Academic Development Funds (to NT).

Author information

Authors and Affiliations

Authors

Contributions

RK, NT and MO designed the study. RK, KT and EA performed the experiments. YY, TT, KM, SM, TK, HN and RM provided conceptual advice. RK and NT wrote the manuscript.

Corresponding author

Correspondence to Nobuyuki Tanaka.

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Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

All procedures were performed in approval of the Research Ethics Committee of Keio University (Approval Nos.: 20180098 and 20190059) and in compliance with the 1964 Helsinki Declaration and present ethical standards. Both written informed consent and passive (opt-out) informed consent procedures have been applied to the experimental use of human samples. Opt-out informed consent from patients was obtained by exhibiting the research information on our department's website (Department of Urology, Keio University Hospital, Tokyo, Japan). The need to obtain written informed consent was waived if patients had finished their follow-up or had died, due to the study’s observational nature and the urgent need for cancer patient care. This was approved and reviewed by the Research Ethics Committee of Keio University, in accordance with the ethical guidelines for Medical and Health Research Involving Human Subjects (Public Notice of the Ministry of Education, Culture, Sports, Science and Technology and the Ministry of Health, Labor and Welfare as of July 2018; https://www.lifescience.mext.go.jp/files/pdf/n2181_01.pdf).

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Kufukihara, R., Tanaka, N., Takamatsu, K. et al. Hybridisation chain reaction-based visualisation and screening for lncRNA profiles in clear-cell renal-cell carcinoma. Br J Cancer (2022). https://doi.org/10.1038/s41416-022-01895-3

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