Long Noncoding RNAs in Multiple Myeloma
Over the past 15 years, long non-coding RNAs (lncRNA) have emerged as an important class of regulatory molecules. The currently accepted definition is that lncRNA refers to RNA molecules with little or no protein-coding potential, and which are greater than 200 nucleotides in length, a size cut-off chosen largely to distinguish them from the more-extensively characterised group of small non-coding regulatory RNAs, which includes micro (mi)RNAs, small inhibitory (si)RNAs and PIWI-interacting (pi)RNAs . A recent compilation of annotations from diverse sources identified nearly 57,000 genes encoding lncRNAs in the human genome .
While the cellular role of the vast majority of these is still unknown, the picture that has emerged from characterisation of some important examples suggests that lncRNAs often act as regulatory molecules to control the expression of downstream target genes. Some lncRNAs regulate gene transcription, often by recruiting chromatin modifying proteins to control the epigenetic state of nearby or distal target genes, or by coordinating longrange chromosomal loops and regulatory domains [3-5]. Other lncRNAs influence post-transcriptional stages of gene expression, by affecting mRNA splicing, or by acting in the cytoplasm to sequester and dampen the activity of miRNAs and RNA binding proteins and thereby modify mRNA stability [3-5]. Cytoplasmic lncRNAs can also affect the translation and post-translational modifications of downstream proteins . In keeping with this emerging role as important regulators of gene expression, altered expression of lncRNAs is increasingly implicated in the onset and evolution of a variety of malignant diseases (for examples [6-9]). The altered expression of lncRNAs can lead to downstream changes in a variety of cancer associated pathways, including dysregulation of p53 and other cell signalling pathways, control of hypoxia and the Endothelial-Mesenchymal Transition (EMT) as well as epigenetic dysregulation .
One malignant disease in which several recent studies have revealed important roles of lncRNAs, is multiple myeloma (MM), a tumour of immunoglobulin-secreting plasma B cells in the bone marrow. MM is primarily a disease of the elderly (median age at diagnosis of 69 years) which, together with its pre-malignant precursor stage disease (monoclonal gammopathy of undetermined significance; MGUS) is the most common haematological malignancy, accounting for ~20% of all haematalogical cancer [11,12]. Although survival times are gradually increasing with recent advances in treatment regimes , disease relapse occurs frequently and MM is still considered essentially incurable [13,14]. Over the last five years, several microarray- and RNA-seq-based transcriptomic studies have documented widespread changes in the expression profile of lncRNAs in tumour plasma cells of MM patients relative to plasma cells from healthy individuals [15-19]. The expression of some lncRNAs was also dynamically regulated during the course of disease evolution , and upon acquisition of drug-resistance phenotypes by MM cell lines . Together these studies led to the identification of prognostic lncRNA expression signatures which were predictive of event free- or overall survival, indicating that lncRNAs exert an important effect on disease progression and outcome in MM.
Despite the accumulating evidence of their important role in MM, at present only a few of these lncRNAs have been individually investigated to determine to what extent and by what molecular pathways they influence MM cell growth, tumour progression, treatment-response and disease outcome. Among the few lncRNAs which have been extensively investigated in MM, important examples include the well-established oncogenic lncRNAs MALAT1, H19, TUG1, UCA1 as well as the known tumoursuppressor lncRNA MEG3 (for recent reviews see [21-23]). Importantly, these studies revealed that lncRNAs often affect MM cell growth and tumour progression by different pathways to those observed in other malignancies, suggesting that the molecular mechanisms involved are likely to be disease- and context-dependent.
CRNDE: An Oncogenic lncRNA Implicated in the Progression and Outcome of MM
Within this context, findings from our group  and others  have recently shed new light on the role of the oncogenic lncRNA Colorectal Neoplasia Differentially Expressed (CRNDE) in the pathophysiology of MM. CRNDE was originally identified as an overexpressed transcript in colorectal carcinoma (CRC)  and has since been shown to be upregulated in a range of other solid and haematological tumours (including glioma, hepatocellular carcinoma, non-small-cell lung cancer, renal cell carcinoma, bladder cancer, breast cancer and acute myeloid leukemia), where high levels of expression correlate with poor prognosis (recently reviewed in [27,28]). CRNDE is expressed from a gene located at human chr16q12.2, which is comprised of 5 core exons, including a complex first exon resulting from the use of alternative transcription start sites, as well as an additional annotated exon (exon 3) which is infrequently used  (Figure 1A). The CRNDE transcript is subject to complex alternative splicing events, with at least 10 splice variants documented [26,30]. Incomplete splicing particularly affects the 3’ end of the transcript, and most often leads to the retention of intron 4, which contains a discrete region of ~600 nucleotides that has been strongly conserved [29-31] (Figure 1A). While the functional significance of the alternative CRNDE transcript isoforms remains unclear, fractionation experiments have revealed that different isoforms are located in distinct sub-cellular compartments, suggesting that they are likely to exert different molecular functions [24,31]. Observations in different malignancies suggest that CRNDE can influence tumour growth and disease evolution in a variety of ways, including effects on tumour cell proliferation and apoptosis, affecting adhesion, migration and invasion, by enhancing resistance to chemo- and radio-therapies, and even influencing inflammatory responses and tumour cell metabolism .
Consistent with these observations in other malignancies, we recently reported that expression of CRNDE is elevated in tumour plasma cells (CD138+) isolated from a cohort of newly-diagnosed MM patients, compared with plasma cells of healthy individuals . Within the patient group, higher levels of CRNDE correlated with poor disease outcome. Expression of CRNDE also increases during progression from early (MGUS) to late stages of the disease. Upregulation and prognostic significance of CRNDE has also been observed in an independent MM patient cohort . By using CRISPR-deletion and CRISPR-inhibition approaches to disrupt expression of the major CRNDE transcript isoforms, we found that depletion of CRNDE reduced the proliferation of MM cell lines and increased their sensitivity to dexamethasone, a therapeutic cornerstone of MM treatment regimes . Deletion of the CRNDE gene also reduced the tumorigenic potential of MM cells in a mouse xenograft model, confirming the relevance of this lncRNA for progression of the disease in an in vivo setting.
CRNDE Affects the IL6 Signalling Pathway in MM Cells
Transcriptomic profiling of CRNDE-deleted MM cells revealed an interesting link between this lncRNA and IL6 signalling, a pathway critical for MM cell proliferation and survival . Autocrine- and paracrine-IL6 signalling, which activates the downstream JAK/STAT3, p38/MAPK and PI3K/AKT/mTOR pathways , promotes survival of MM cells by inducing expression of the anti-apoptotic genes MCL1 and BCL-XL [34,35]. Due to its critical role in MM disease progression, drug resistance and relapse, the IL6 signalling pathway is frequently targeted by MM therapeutic strategies . Our transcriptomic analysis revealed that MM cells lacking CRNDE expressed reduced levels of IL6R, the gene encoding the gp80 subunit of the IL6 receptor complex, which correlated with a transcriptionally repressive epigenetic state at the IL6R promoter . Consistent with impaired IL6 signalling, CRNDE-deleted MM cells showed reduced phosphorylation of STAT3 and decreased proliferation in response to IL6 in vitro, together with gene expression suggesting perturbation of downstream JAK/STAT, PI3KAKT and MAPK pathways. Since IL6 signalling is known to protect MM cells against therapeutic agents, and in particular from the effects of glucocorticoids [37-39], it is likely that impaired IL6 signalling contributes to the Dexamethasone-sensitivity observed in the CRNDEdeleted MM cells. Expression of CRNDE also correlated with that of IL6R in our cohort of primary tumour plasma cells, supporting the idea that increased levels of CRNDE leads to transcriptional activation of the IL6R gene in MM patients . Together these observations indicate that CRNDE impacts upon MM progression and outcome by sensitizing malignant plasma cells to pro-tumorigenic IL6 signalling due to enhanced levels of the IL6 receptor (Figure 1B).
Effects of CRNDE on Interactions with the Bone Marrow Niche
The progression and evolution of MM is critically influenced by interactions between malignant plasma cells and the bone marrow microenvironment in which they reside [40-42]. The bone marrow milieu provides cytokines and chemokines which are critical for growth, survival and chemo-resistance of tumour plasma cells. Conversely, the tumour cells also influence the surrounding microenvironment, which is remodelled and adapted to establish a highly supportive neoplastic niche . Our studies of CRNDE-deleted MM cells suggest that, as well as regulating the IL6 signalling pathway, this lncRNA also affects disease progression and outcome by influencing the adhesion properties of MM tumour cells and their interactions with the bone marrow niche. MM cells lacking CRNDE were less adhesive to an extracellular matrix substrate in in vitro assays, and gene expression profiles revealed dysregulation of pathways related to cellcell and cell-substrate adhesion . CRNDE-deleted MM cells were also less able to induce IL6 secretion by bone marrow stromal cells in co-culture assays, suggesting that the influence of CRNDE on the adhesive properties of tumour cells could also feedforward to further enhance IL6 signalling. Of particular note, our gene expression profiling revealed that CRNDE activates expression of CDH2, encoding the cell adhesion molecule N-cadherin. It has previously been shown that CDH2 is a negative prognostic indicator  and is increased in plasma cells of approximately 50% of newly-diagnosed MM patients where it facilitates interaction with the cells of the bone marrow niche . Furthermore, knockdown or inhibition of CDH2 reduced both the adhesive properties and in vivo tumorigenicity of mouse MM cells . These observations therefore suggest that CDH2 could be another downstream target by which CRNDE impacts upon MM progression, by affecting the interaction between tumour plasma cells and their bone marrow niche (Figure 1B).
As in other diseases, the emerging importance of lncRNAs for the aetiology of MM has generated significant interest in these molecules both as prognostic markers, and as targets for the development of novel therapies. As discussed above, prognostically predictive signatures have been identified from lncRNA expression profiles in MM, and several individual lncRNAs, including CRNDE, are markers of poor disease outcome. Interestingly, for several of these lncRNAs, prognostically informative levels can be detected in circulating serum [46-48], suggesting the potential to develop lncRNA signatures as useful and easily accessible biomarkers of disease outcome and treatment response. Beyond their utility as biomarkers in malignant disease, there is expanding research interest in the potential to therapeutically target lncRNA activity, with several molecules already in the development pipeline within the pharmaceutical industry . While there has been little investigation to date of the therapeutic value of lncRNAs in the treatment of MM, the link between CRNDE and the critical IL6 signalling pathway in MM cells suggests that this lncRNA may present an interesting target to attenuate IL6 signalling and improve or prolong drug sensitivity in some MM patients.
To date, most strategies have focused on targeting pathogenic lncRNAs for degradation using small interfering (si)RNAs to trigger the RNA interference (RNAi) pathway. Indeed, knockdown of CRNDE by RNAi has been successfully carried out in pre-clinical models of diverse tumours, including MM [25,27,28]. However, since the RNAi machinery is located in the cytoplasm, these approaches are often less successful at degrading nuclear lncRNAs, such as the intron 4-retaining isoform of CRNDE. This is a serious limitation both in the laboratory and in the clinic. More recent approaches invoke the use of chemically modified antisense oligonucleotide (ASO) reagents, which bind to the target lncRNA by Watson- Crick base pairing and trigger endonucleolytic cleavage by endogenous RNAseH (reviewed in ). Since RNAseH is present in both the cytoplasm and the nucleus, ASO technology promises to be more generally effective against lncRNAs, many of which display a nuclear localisation [51,52]. ASO reagents have been used to inhibit the protumorigenic and metastatic potential of the nuclearlocated lncRNA MALAT1 in in vivo models of breast and lung cancer [53,54]. Nevertheless, several challenges remain in order to fully exploit the therapeutic potential of these molecules to inhibit lncRNAs in the clinic. These challenges include minimizing off-target effects, optimizing delivery technologies and strategies in order to limit on-target but off-tumour silencing of the lncRNA in healthy tissues, as well as improving the pharmacokinetics, stability and bioavailability of these reagents in the face of cellular nucleases and innate immune responses .
An alternative approach to impede the activity of lncRNAs is to inhibit the interactions between lncRNAs and the partner proteins through which they exert their biological activity. High-throughput molecular screens have successfully identified small molecules able to block the interaction between two lncRNAs (BDNF-AS and HOTAIR) and EZH2, a histone methyltransferase component of the Polycomb Repressive Complex 2 (PRC2), thereby upregulating expression of target genes normally repressed by these lncRNAs . As for many other lncRNAs, one of the principle bottlenecks for this approach with respect to CRNDE is a current lack of understanding of its mechanisms of action and partner proteins. While several studies have described a mechanism of action in which CRNDE acts as a sponge to inhibit the activity of various miRNAs [27,28], miRNA sponging by CRNDE appears to be highly cell-type specific and is unlikely to account for the full range of its observed effects. Other evidence suggests that CRNDE (also called lincIRX5) can physically interact with members of the PRC2 complex to repress downstream target genes [56,57]. However, in MM cells, CRNDE is a transcriptional activator of IL6R and CDH2, suggesting that it interacts with other partner proteins in some circumstances. To fully understand the cellular functions of CRNDE, it will be important to extend beyond candidate approaches by carrying out holistic profiling of its effects on the miRnome, and using unbiased screens to identify the full range of CRNDE-interacting proteins. As well as mass spectrometry approaches to identify proteins co-purified with a lncRNA of interest , other screening approaches, such as that reported recently which detects in-cell interactions using lncRNAs that have been tagged with the MS2 coat protein, promise to improve the sensitivity and resolution of these analyses .
In summary, CRNDE adds to a growing list of lncRNAs implicated in MM, with recent studies furthering our understanding of the important role played by CRNDE in this currently incurable malignancy. As a result of rapid evolution in the field of antisense-based therapeutics, as well as the improved characterisation of lncRNA partners and molecular function, there is a clear potential for the development of new therapeutic molecules targeting lncRNAs that could complement existing treatment regimes. Further, the link between CRNDE and the IL6 signalling pathway observed in MM may have more general implications for other malignancies in which CRNDE is involved.
Research in our laboratory on lncRNAs is supported by the Association pour la Recherche sur le Cancer (Fondation ARC, France), the Fondation Française pour la Recherche contre le Myélome et les Gammapathies (FFRMG, France) and the Initiative d’excellence Program (IDEX, Université de Paris).
2. Volders PJ, Anckaert J, Verheggen K, Nuytens J, Martens L, Mestdagh P, et al. LNCipedia 5: towards a reference set of human long non-coding RNAs. Nucleic Acids Research. 2019 Jan 8;47(D1):D135-9.
3. Sun Q, Hao Q, Prasanth KV. Nuclear long noncoding RNAs: key regulators of gene expression. Trends in Genetics. 2018 Feb 1;34(2):142-57.
4. Zhang X, Wang W, Zhu W, Dong J, Cheng Y, Yin Z, et al. Mechanisms and functions of long non-coding RNAs at multiple regulatory levels. International Journal of Molecular Sciences. 2019 Jan;20(22):5573.
5. Yao RW, Wang Y, Chen LL. Cellular functions of long noncoding RNAs. Nature cell biology. 2019 May;21(5):542-51.
6. Sun J, Cheng L, Shi H, Zhang Z, Zhao H, Wang Z,et al. A potential panel of six-long non-coding RNA signature to improve survival prediction of diffuse large-B-cell lymphoma. Scientific Reports. 2016 Jun 13;6(1):1-0.
7. Beck D, Thoms JA, Palu C, Herold T, Shah A, Olivier J, et al. A four-gene LincRNA expression signature predicts risk in multiple cohorts of acute myeloid leukemia patients. Leukemia. 2018 Feb;32(2):263-72.
8. Yang F, Song Y, Ge L, Zhao G, Liu C, Ma L. Long non-coding RNAs as prognostic biomarkers in papillary renal cell carcinoma. Oncology Letters. 2019 Oct 1;18(4):3691-7.
9. Zhang L, Chen S, Wang B, Su Y, Li S, Liu G, et al. An eight-long noncoding RNA expression signature for colorectal cancer patients’ prognosis. Journal of Cellular Biochemistry. 2019 Apr;120(4):5636-43.
10. Evans JR, Feng FY, Chinnaiyan AM. The bright side of dark matter: lncRNAs in cancer. The Journal of Clinical Investigation. 2016 Aug 1;126(8):2775-82.
11. Network THMR. Incidence. https://www.hmrn.org/ statistics/incidence.
12. National Cancer Institute. SEER Cancer Stat Facts: Myeloma. https://seer.cancer.gov/statfacts/html/mulmy. html.
13. Ribatti D. A historical perspective on milestones in multiple myeloma research. European Journal of Haematology. 2018 Mar;100(3):221-8.
14. Kumar SK, Rajkumar V, Kyle RA, van Duin M, Sonneveld P, Mateos MV. Multiple myeloma. vol. 3. Nat Rev Dis Prim. 2017:17046.
15. Zhou M, Zhao H, Wang Z, Cheng L, Yang L, Shi H, et al. Identification and validation of potential prognostic lncRNA biomarkers for predicting survival in patients with multiple myeloma. Journal of Experimental & Clinical Cancer Research. 2015 Dec;34(1):1-4.
16. Ronchetti D, Agnelli L, Taiana E, Galletti S, Manzoni M, Todoerti K, et al. Distinct lncRNA transcriptional fingerprints characterize progressive stages of multiple myeloma. Oncotarget. 2016 Mar 22;7(12):14814.
17. Hu AX, Huang ZY, Zhang L, Shen J. Potential prognostic long non-coding RNA identification and their validation in predicting survival of patients with multiple myeloma. Tumor Biology. 2017 Apr;39(4):1010428317694563.
18. Ronchetti D, Agnelli L, Pietrelli A, Todoerti K, Manzoni M, Taiana E, et al. A compendium of long non-coding RNAs transcriptional fingerprint in multiple myeloma. Scientific Reports. 2018 Apr 26;8(1):1-9.
19. Samur MK, Minvielle S, Gulla A, Fulciniti M, Cleynen A, Samur AA, et al. Long intergenic non-coding RNAs have an independent impact on survival in multiple myeloma. Leukemia. 2018 Dec;32(12):2626-35.
20. Malek E, Kim BG, Driscoll JJ. Identification of long non-coding RNAs deregulated in multiple myeloma cells resistant to proteasome inhibitors. Genes. 2016 Oct;7(10):84.
21. Nobili L, Ronchetti D, Agnelli L, Taiana E, Vinci C, Neri A. Long non-coding RNAs in multiple myeloma. Genes. 2018 Feb;9(2):69.
22. Cui YS, Song YP, Fang BJ. The role of long noncoding RNAs in multiple myeloma. European Journal of Haematology. 2019 Jul;103(1):3-9.
23. Butova R, Vychytilova-Faltejskova P, Souckova A, Sevcikova S, Hajek R. Long non-coding RNAs in multiple myeloma. Non-coding RNA. 2019 Mar;5(1):13.
24. David A, Zocchi S, Talbot A, Choisy C, Ohnona A, Lion J, et al. The long non-coding RNA CRNDE regulates growth of multiple myeloma cells via an effect on IL6 signalling. Leukemia. 2020 Sep 3:1-2.
25. Meng YB, He X, Huang YF, Wu QN, Zhou YC, Hao DJ. Long Noncoding RNA CRNDE Promotes Multiple Myeloma Cell Growth by Suppressing miR-451. Oncology Research. 2017 Mar 9;25(7):1207-14.
26. Graham LD, Pedersen SK, Brown GS, Ho T, Kassir Z, Moynihan AT, et al. Colorectal neoplasia differentially expressed (CRNDE), a novel gene with elevated expression in colorectal adenomas and adenocarcinomas. Genes & Cancer. 2011 Aug;2(8):829-40.
27. Dai M, Li S, Qin X. Colorectal neoplasia differentially expressed: a long noncoding RNA with an imperative role in cancer. OncoTargets and Therapy. 2018;11:3755.
28. Lu Y, Sha H, Sun X, Zhang Y, Wu Y, Zhang J, et al. CRNDE: an oncogenic long non-coding RNA in cancers. Cancer Cell International. 2020 Dec;20:1-0.
29. Ellis BC, Molloy PL, Graham LD. CRNDE: a long non-coding RNA involved in cancer, neurobiology, and development. Frontiers in Genetics. 2012 Nov 29;3:270.
30. Sciences IRCfIM. FANTOM CAT Browser. https:// fantom.gsc.riken.jp/.
31. Ellis BC, Graham LD, Molloy PL. CRNDE, a long noncoding RNA responsive to insulin/IGF signaling, regulates genes involved in central metabolism. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research. 2014 Feb 1;1843(2):372-86.
32. Rosean TR, Tompkins VS, Tricot G, Holman CJ, Olivier AK, Zhan F, et al. Preclinical validation of interleukin 6 as a therapeutic target in multiple myeloma. Immunologic Research. 2014 Aug 1;59(1-3):188-202.
33. Heinrich PC, Behrmann I, Haan S, Hermanns HM, Müller-Newen G, Schaper F. Principles of interleukin (IL)- 6-type cytokine signalling and its regulation. Biochemical Journal. 2003 Aug 15;374(1):1-20.
34. Puthier D, Bataille R, Amiot M. IL-6 up-regulates MCL-1 in human myeloma cells through JAK/STAT rather than RAS/MAP kinase pathway. European Journal of Immunology. 1999 Dec;29(12):3945-50.
35. Gupta VA, Matulis SM, Conage-Pough JE, Nooka AK, Kaufman JL, Lonial S, et al. Bone marrow microenvironment–derived signals induce Mcl-1 dependence in multiple myeloma. Blood, The Journal of the American Society of Hematology. 2017 Apr 6;129(14):1969-79.
36. Matthes T, Manfroi B, Huard B. Revisiting IL-6 antagonism in multiple myeloma. Critical Reviews in Oncology/Hematology. 2016 Sep 1;105:1-4.
37. Hardin J, MacLeod S, Grigorieva I, Chang R, Barlogie B, Xiao H, et al. Interleukin-6 prevents dexamethasoneinduced myeloma cell death. Blood. 1994 Nov 1;84(9):3063-70.
38. Hönemann D, Chatterjee M, Savino R, Bommert K, Burger R, Gramatzki M, et al. The IL-6 receptor antagonist SANT-7 overcomes bone marrow stromal cell–mediated drug resistance of multiple myeloma cells. International Journal of Cancer. 2001 Sep 1;93(5):674-80.
39. Voorhees PM, Chen Q, Small GW, Kuhn DJ, Hunsucker SA, Nemeth JA, et al. Targeted inhibition of interleukin-6 with CNTO 328 sensitizes pre-clinical models of multiple myeloma to dexamethasone-mediated cell death. British Journal of Haematology. 2009 May;145(4):481-90.
40. Hideshima T, Mitsiades C, Tonon G, Richardson PG, Anderson KC. Understanding multiple myeloma pathogenesis in the bone marrow to identify new therapeutic targets. Nature Reviews Cancer. 2007 Aug;7(8):585-98.
41. Manier S, Sacco A, Leleu X, Ghobrial IM, Roccaro AM. Bone marrow microenvironment in multiple myeloma progression. Journal of Biomedicine and Biotechnology. 2012 Oct 3;2012.
42. Lomas OC, Tahri S, Ghobrial IM. The microenvironment in myeloma. Current Opinion in Oncology. 2020 Mar 1;32(2):170-5.
43. Vandyke K, Chow AW, Williams SA, To LB, Zannettino AC. Circulating N-cadherin levels are a negative prognostic indicator in patients with multiple myeloma. British Journal of Haematology. 2013 May;161(4):499-507.
44. Groen RW, de Rooij MF, Kocemba KA, Reijmers RM, de Haan-Kramer A, Overdijk MB, et al. N-cadherinmediated interaction with multiple myeloma cells inhibits osteoblast differentiation. Haematologica. 2011 Nov;96(11):1653.
45. Mrozik KM, Cheong CM, Hewett D, Chow AW, Blaschuk OW, Zannettino AC, et al.Therapeutic targeting of N-cadherin is an effective treatment for multiple myeloma. British Journal of Haematology. 2015 Nov;171(3):387-99.
46. Pan Y, Chen H, Shen X, Wang X, Ju S, Lu M, et al. Serum level of long noncoding RNA H19 as a diagnostic biomarker of multiple myeloma. Clinica Chimica Acta. 2018 May 1;480:199-205.
47. Sedlarikova L, Bollova B, Radova L, Brozova L, Jarkovsky J, Almasi M, et al. Circulating exosomal long noncoding RNA PRINS—First findings in monoclonal gammopathies. Hematological Oncology. 2018 Dec;36(5):786-91.
48. Shen X, Zhang Y, Wu X, Guo Y, Shi W, Qi J, et al. Upregulated lncRNA-PCAT1 is closely related to clinical diagnosis of multiple myeloma as a predictive biomarker in serum. Cancer Biomarkers. 2017 Jan 1;18(3):257-63.
49. Slaby O, Laga R, Sedlacek O. Therapeutic targeting of non-coding RNAs in cancer. Biochemical Journal. 2017 Dec 15;474(24):4219-51.
50. Arun G, Diermeier SD, Spector DL. Therapeutic targeting of long non-coding RNAs in cancer. Trends in Molecular Medicine. 2018 Mar 1;24(3):257-77.
51. Cabili MN, Dunagin MC, McClanahan PD, Biaesch A, Padovan-Merhar O, Regev A, et al. Localization and abundance analysis of human lncRNAs at single-cell and single-molecule resolution. Genome Biology. 2015 Dec;16(1):1-6.
52. Derrien T, Johnson R, Bussotti G, Tanzer A, Djebali S, Tilgner H, et al. The GENCODE v7 catalog of human long noncoding RNAs: analysis of their gene structure, evolution, and expression. Genome Research. 2012 Sep 1;22(9):1775-89.
53. Arun G, Diermeier S, Akerman M, Chang KC, Wilkinson JE, Hearn S, et al. Differentiation of mammary tumors and reduction in metastasis upon Malat1 lncRNA loss. Genes & Development. 2016 Jan 1;30(1):34-51.
54. Gutschner T, Hämmerle M, Eißmann M, Hsu J, Kim Y, Hung G, Revenko A, et al. The noncoding RNA MALAT1 is a critical regulator of the metastasis phenotype of lung cancer cells. Cancer Research. 2013 Feb 1;73(3):1180-9.
55. Pedram Fatemi R, Salah-Uddin S, Modarresi F, Khoury N, Wahlestedt C, Faghihi MA. Screening for smallmolecule modulators of long noncoding RNA-protein interactions using AlphaScreen. Journal of Biomolecular Screening. 2015 Oct;20(9):1132-41.
56. Khalil AM, Guttman M, Huarte M, Garber M, Raj A, Morales DR, et al. Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression. Proceedings of the National Academy of Sciences. 2009 Jul 14;106(28):11667-72.
57. Zhang M, Gao C, Yang Y, Li G, Dong J, Ai Y, et al. Long noncoding RNA CRNDE/PRC2 participated in the radiotherapy resistance of human lung adenocarcinoma through targeting p21 expression. Oncology Research. 2018 Sep 14;26(8):1245-55.
58. McHugh CA, Guttman M. RAP-MS: a method to identify proteins that interact directly with a specific RNA molecule in cells. In RNA Detection 2018 (pp. 473-488). Humana Press, New York, NY.
59. Graindorge A, Pinheiro I, Nawrocka A, Mallory AC, Tsvetkov P, Gil N, et al. In-cell identification and measurement of RNA-protein interactions. Nature Communications. 2019 Nov 22;10(1):1-1.