Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Molecular Diagnostics

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



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.


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.


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.


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.

Your institute does not have access to this article

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.


  1. Djebali S, Davis CA, Merkel A, Dobin A, Lassmann T, Mortazavi A, et al. Landscape of transcription in human cells. Nature. 2012;489:101–8.

    CAS  Article  Google Scholar 

  2. Iyer MK, Niknafs YS, Malik R, Singhal U, Sahu A, Hosono Y, et al. The landscape of long noncoding RNAs in the human transcriptome. Nat Genet. 2015;47:199–208.

    CAS  Article  Google Scholar 

  3. Chu C, Spitale RC, Chang HY. Technologies to probe functions and mechanisms of long noncoding RNAs. Nat Struct Mol Biol. 2015;22:29–35.

    CAS  Article  Google Scholar 

  4. Schmitt AM, Chang HY. Long noncoding RNAs in cancer pathways. Cancer Cell. 2016;29:452–63.

    CAS  Article  Google Scholar 

  5. Bassett AR, Akhtar A, Barlow DP, Bird AP, Brockdorff N, Duboule D, et al. Considerations when investigating lncRNA function in vivo. eLife. 2014;3:e03058.

    Article  Google Scholar 

  6. Choi HM, Chang JY, Trinh le A, Padilla JE, Fraser SE, Pierce NA. Programmable in situ amplification for multiplexed imaging of mRNA expression. Nat Biotechnol. 2010;28:1208–12.

    CAS  Article  Google Scholar 

  7. Choi HMT, Schwarzkopf M, Fornace ME, Acharya A, Artavanis G, Stegmaier J, et al. Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust. Development. 2018;145:dev165753.

  8. Choi HM, Beck VA, Pierce NA. Next-generation in situ hybridization chain reaction: higher gain, lower cost, greater durability. ACS Nano. 2014;8:4284–94.

    CAS  Article  Google Scholar 

  9. Sylwestrak EL, Rajasethupathy P, Wright MA, Jaffe A, Deisseroth K. Multiplexed intact-tissue transcriptional analysis at cellular resolution. Cell. 2016;164:792–804.

    CAS  Article  Google Scholar 

  10. Shen H, Luo G, Chen Q. Long noncoding RNAs as tumorigenic factors and therapeutic targets for renal cell carcinoma. Cancer Cell Int. 2021;21:110.

    CAS  Article  Google Scholar 

  11. Bridges MC, Daulagala AC, Kourtidis A. LNCcation: lncRNA localization and function. J Cell Biol. 2021;220:e202009045.

  12. Asano SM, Gao R, Wassie AT, Tillberg PW, Chen F, Boyden ES. Expansion microscopy: protocols for imaging proteins and RNA in cells and tissues. Curr Protoc Cell Biol. 2018;80:e56.

    Article  Google Scholar 

  13. Watanabe K, Kosaka T, Aimono E, Hongo H, Mikami S, Nishihara H, et al. Japanese case of enzalutamide-resistant prostate cancer harboring a SPOP mutation with scattered allelic imbalance: response to platinum-based therapy. Clin Genitourin Cancer. 2019;17:e897–e902.

    Article  Google Scholar 

  14. Takamatsu K, Tanaka N, Hakozaki K, Takahashi R, Teranishi Y, Murakami T, et al. Profiling the inhibitory receptors LAG-3, TIM-3, and TIGIT in renal cell carcinoma reveals malignancy. Nat Commun. 2021;12:5547.

    CAS  Article  Google Scholar 

  15. Győrffy B. Survival analysis across the entire transcriptome identifies biomarkers with the highest prognostic power in breast cancer. Computational Struct Biotechnol J. 2021;19:4101–9.

    Article  Google Scholar 

  16. Tumkur Sitaram R, Landström M, Roos G, Ljungberg B. Significance of PI3K signalling pathway in clear cell renal cell carcinoma in relation to VHL and HIF status. J Clin Pathol. 2021;74:216–22.

    Article  Google Scholar 

  17. D’Avella C, Abbosh P, Pal SK, Geynisman DM. Mutations in renal cell carcinoma. Urologic Oncol. 2020;38:763–73.

    Article  Google Scholar 

  18. Wang Y, Li Z, Li W, Zhou L, Jiang Y. Prognostic significance of long non-coding RNAs in clear cell renal cell carcinoma: a meta-analysis. Medicine 2019;98:e17276.

    CAS  Article  Google Scholar 

  19. Cabili MN, Trapnell C, Goff L, Koziol M, Tazon-Vega B, Regev A, et al. Integrative annotation of human large intergenic noncoding RNAs reveals global properties and specific subclasses. Genes Dev. 2011;25:1915–27.

    CAS  Article  Google Scholar 

  20. Polovic M, Dittmar S, Hennemeier I, Humpf HU, Seliger B, Fornara P, et al. Identification of a novel lncRNA induced by the nephrotoxin ochratoxin A and expressed in human renal tumor tissue. Cell Mol Life Sci: CMLS. 2018;75:2241–56.

    CAS  Article  Google Scholar 

  21. Shi H, Sun Y, He M, Yang X, Hamada M, Fukunaga T, et al. Targeting the TR4 nuclear receptor-mediated lncTASR/AXL signaling with tretinoin increases the sunitinib sensitivity to better suppress the RCC progression. Oncogene. 2020;39:530–45.

    CAS  Article  Google Scholar 

  22. Miranda-Castro R, de-Los-Santos-Álvarez N, Lobo-Castañón MJ. Long noncoding RNAs: from genomic junk to rising stars in the early detection of cancer. Anal Bioanal Chem. 2019;411:4265–75.

    CAS  Article  Google Scholar 

  23. Qi P, Zhou XY, Du X. Circulating long non-coding RNAs in cancer: current status and future perspectives. Mol Cancer. 2016;15:39.

    Article  Google Scholar 

  24. Katayama H, Tamai K, Shibuya R, Nakamura M, Mochizuki M, Yamaguchi K, et al. Long non-coding RNA HOTAIR promotes cell migration by upregulating insulin growth factor-binding protein 2 in renal cell carcinoma. Sci Rep. 2017;7:12016.

    Article  Google Scholar 

  25. Wang PQ, Wu YX, Zhong XD, Liu B, Qiao G. Prognostic significance of overexpressed long non-coding RNA TUG1 in patients with clear cell renal cell carcinoma. Eur Rev Med Pharmacol Sci. 2017;21:82–6.

    PubMed  Google Scholar 

  26. Xie X, Lin J, Fan X, Zhong Y, Chen Y, Liu K, et al. LncRNA CDKN2B-AS1 stabilized by IGF2BP3 drives the malignancy of renal clear cell carcinoma through epigenetically activating NUF2 transcription. Cell Death Dis. 2021;12:201.

    CAS  Article  Google Scholar 

  27. Zhang K, Shi ZM, Chang YN, Hu ZM, Qi HX, Hong W. The ways of action of long non-coding RNAs in cytoplasm and nucleus. Gene. 2014;547:1–9.

    CAS  Article  Google Scholar 

  28. Lennox KA, Behlke MA. Cellular localization of long non-coding RNAs affects silencing by RNAi more than by antisense oligonucleotides. Nucleic Acids Res. 2016;44:863–77.

    CAS  Article  Google Scholar 

  29. Goldfarb KC, Cech TR. Targeted CRISPR disruption reveals a role for RNase MRP RNA in human preribosomal RNA processing. Genes Dev. 2017;31:59–71.

    CAS  Article  Google Scholar 

  30. Noh JH, Kim KM, Abdelmohsen K, Yoon JH, Panda AC, Munk R, et al. HuR and GRSF1 modulate the nuclear export and mitochondrial localization of the lncRNA RMRP. Genes Dev. 2016;30:1224–39.

    CAS  Article  Google Scholar 

Download references


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



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.

Ethics declarations

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;

Consent to publish

Not applicable.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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).

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • DOI:


Quick links