NORAD2: an enhancer RNA that strongly activates DCBLD2 gene transcription upon drug treatment


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Andrei Zheltukhin1,Dmitry Karpov1,Pavel Spirin1,Olga Zinovieva1,Evgenia Grineva1,George Krasnov1,Tamara Mashkova1,Nikolai Lisitsyn1
1Engelhardt Institute of Molecular Biology

Abstract

Background

Prediction of tumor progression and relapse depends on identification of novel prognostic cancer biomarkers, which are suitable for monitoring of the response of various cancer types to the action of chemotherapeutic drugs.

Method

CRISPR knockout, ChIRP-MS, lentiviral transduction, Cignal Finder analysis of signaling pathways and Western blotting, RNA-Seq, RT-qPCR and bioinformatics.

Results

We have discovered that the abundance of the long non-coding RNA LINC00973 is increased up to one hundred times upon treatment of various types of cancer cells with a wide spectrum of chemotherapeutic drugs at different times and durations, both in vitro and in vivo. Functional studies demonstrated that this RNA, which we termed NORAD2 (i.e. non-coding RNA activated by DNA damage, number 2), is an enhancer RNA that strongly activates transcription of the DCBLD2 gene. This gene encodes receptor tyrosine kinase discoidin 2, which activates EGFR signaling pathway in response to chemotherapeutic drugs. Besides, ChIRP-MS and lentiviral transduction data demonstrated that NORAD2 also function in trans, as a scaffolding RNA, which takes part in the formation of the NORAD2-activated ribonucleoprotein complex that posts histone repressive marks in active chromatin. We have also found that NORAD2 transcription is regulated by p53 in response to DNA damage, as follows from comparative analysis of p53wt and p53 -/- cancer cells, as well as of cells that contain p53 gain-of-function mutations.

Conclusion

Since both NORAD2 and DCBLD2 RNAs are at the moment on the top of the list of the most informative biomarkers for colon cancer prognosis and relapse, further identification and analysis of other NORADs provides a new avenue in search for valuable cancer biomarkers and putative targets for cancer chemotherapy.