«International Journal of Molecular Sciences Review Novel Insights into the Role of Long Noncoding RNA in Ocular Diseases Fang Li †, Xuyang Wen † ...»
2.5. Role of lncRNAs in Choroidal Neovascularization Choroidal neovascularization (CNV) is a hallmark of neovascular age-related macular degeneration (AMD), a leading cause of visual impairment in elderly individuals . Xu et al.  found that the expression of VEGF and two lncRNAs, Vax2os1 and Vax2os2, were signiﬁcantly up-regulated in the aqueous humor of CNV patients, making them predictive biomarkers for the diagnosis of ocular neovascular diseases . Vax2os1 and Vax2os2, which are antisense transcripts of the Vax2 gene, are highly expressed in the choroid and retinal vasculature. The strong RNA-protein interactions between Vax2os1 and C1D and between Vax2os2 and PATL2 play important roles in the mechanism underlying the pathogenesis of CNV because C1D and PATL2 are important for regulating Int. J. Mol. Sci. 2016, 17, 478 5 of 11 the stability of chromatin structure [60–62]. Increased information about these two lncRNAs will facilitate a greater understanding of CNV pathogenesis. Provided that each lncRNA regulates speciﬁc facets of protein activity, a more reﬁned and less toxic drug targeting a lncRNA may be employed for CNV treatment.
2.6. Roles of lncRNAs in Ocular Tumors
2.6.1. Retinoblastoma Retinoblastoma is a rare malignancy of the retina that usually appears before the age of ﬁve years, threatening the vision and survival of children if timely detection and treatment are not achieved. Therefore, to preserve vision, salvage the eye, and save the child’s life, elucidating the molecular mechanisms of retinoblastoma and identifying speciﬁc biomarkers for tumor progression are of utmost importance. However, only two lncRNAs, BANCR and MEG3, have been associated with retinoblastoma.
The involvement of BRAF-activated noncoding RNA (BANCR), a 693-bp lncRNA encoded on chromosome 9, in the proliferation and metastasis of malignant melanoma and lung cancer via the MAPK pathway has been reported [63–65]. BANCR has been shown to play a key role in gastric cancer cells via regulation of NF-κB1 . In retinoblastoma tissues and cell lines, recent evidence has shown that BANCR is over-expressed and is highly associated with tumor size, choroidal invasion, and optic nerve invasion. Knockdown of BANCR signiﬁcantly suppresses the proliferation, migration, and invasion of retinoblastoma cells in vitro, thus implying a better prognosis .
Maternally-expressed gene 3 (MEG3), an imprinted gene located on chromosome 14q32 , is considered to act as a tumor suppressor lncRNA. The loss of MEG3 expression in various human tumors has been well documented. Re-expression of MEG3 inhibits proliferation, induces apoptosis, and inhibits the anchorage-independent growth of human tumor cells . In retinoblastoma samples, Gao et al.  found that MEG3 is signiﬁcantly down-regulated and that the reduced expression is associated with a poor prognosis among retinoblastoma patients. Studies have shown that MEG3 suppresses retinoblastoma progression by negatively regulating the Wnt/β-catenin pathway. Studies have also shown that pancreatic cancer cell proliferation could be inhibited via MEG3-mediated p53 activation , implying that MEG3 is a potential molecular therapeutic target.
2.6.2. Uveal Melanoma Uveal melanoma is the most common eye malignancy in adults; it causes severe visual morbidity and is fatal to approximately 50% of patients. Fan et al.  found that the lncRNA ROR (retinoid-related orphan nuclear receptor) and its target gene TESC were both highly expressed relative to normal cells or adjacent normal tissues in three malignant ocular melanoma cell lines and in 20 ocular melanoma tissues. ROR acts as an oncogenic lncRNA, activating the TESC promoter by repelling the histone G9A methyltransferase and promoting the release of histone H3K9 methylation.
Suppression of ROR could reduce tumor growth and metastasis.
SF3B1 mutations are associated with a good prognosis for uveal melanoma. Recently, an RNA-seq analysis showed that mutations in SF3B1 are associated with cryptic alternative splicing within exon 4 of CRNDE, indicating that this lncRNA has potential importance for determining how alternative splicing affects cellular function . Evidence has shown that CRNDE can promote glioma cell growth and invasion through mTOR signaling, thereby highlighting the potential of CRNDE as a novel therapeutic target for the treatment of glioma . In addition, several lines of evidence have shown that CRNDE exerts its effects on RNA transcripts primarily via epigenetic mechanisms, particularly through histone methylation or demethylation by the PRC2 or CoREST complexes, respectively .
The detailed mechanism of how lncRNA is involved in uveal melanoma remains to be studied;
however, the results will undoubtedly contribute to knowledge of the uveal melanoma tumorigenesis and suggest new therapeutic strategies.
Int. J. Mol. Sci. 2016, 17, 478 6 of 11
2.7. Roles of lncRNAs in Other Ocular Disease MOMO syndrome, short for macrosomia, obesity, macrocephaly, and ocular abnormalities, is an extremely rare syndrome. The main features of ocular abnormalities include retinal coloboma, nystagmus, and downward-slanting palpebral ﬁssures. Recently, one MOMO patient showed a homozygous, balanced, reciprocal translocation (16; 20) (q21; p11.23) that was inherited from healthy consanguineous parents. The breakpoint at 16q21 did not disrupt any known or predicted gene, whereas the chromosome 20 breakpoint disrupted a new lncRNA at 20p11.23 named LINC00237.
Compared with control individuals, the expression of LINC00237 was reduced by approximately 50% in patients’ lymphoblasts. This disruption causes gene inactivation that results in the loss of complete transcript production . However, the function of this candidate gene and the consequences of its haploinsufﬁciency remain to be characterized.
The maintenance of corneal-speciﬁc epithelial qualities plays an important role in maintaining corneal transparency and preventing vision loss. PNN is a nuclear protein that is associated with the splicing apparatus within the nuclei of epithelial cells, and it appears to play a key role in the establishment and maintenance of epithelial phenotypes . Joo et al.  studied the lncRNAs of the corneal epithelium by focusing on a small subset of lncRNAs that exhibit splicing changes in response to PNN knockdown. The results showed that the lncRNAs SPACA6P, HAS2-AS1, RPARP-AS1, RP11-295G20.2, and NUTM2a-AS1 exhibited signiﬁcant and reproducible expression changes and RNA processing after the perturbation of PNN expression. Although the ﬁndings are incomplete, they provide the ﬁrst glimpse into the complexity and potential relevance of lncRNAs in the maintenance of epithelial cells, paving the way for further investigations into the roles of lncRNAs in cornea. In addition, Hoang et al.  performed an RNA-seq analysis and identiﬁed 86 differentially-expressed lncRNAs between lens epithelial cells and lens ﬁber cells; they included RP23-237H8.2, AC135859.1, AL663030.1, AC128663.1, and AC100730.1. Although the functional signiﬁcance of these lncRNAs in lens development or physiology remains unknown, this comprehensive transcriptome analysis provides a valuable resource for the study of lens development, ﬁber differentiation, and lens pathogenesis .
3. Conclusions To date, several lncRNAs have been implicated in eye development, includingVax2os1, RNCR2, Six3OS, Tug1, and MALAT1. These lncRNAs are recognized as important regulators of various processes, such as photoreceptor progenitor progression and retinal cell fate speciﬁcation [14,59,76–78].
However, the roles of lncRNAs in the pathogenesis of ocular diseases are far from understood. Most of the lncRNAs mentioned in our review were identiﬁed by consulting relevant studies about diseases that share the same etiology or pathogenesis. For example, BANCR is involved in malignant melanoma and lung cancer; thus, researchers explored its role in retinoblastoma. ANRIL is signiﬁcantly associated with increased susceptibility to type 2 diabetes ; thus, it is no surprise that this abnormally-expressed lncRNA may be relevant to the molecular mechanisms underlying diabetes complications. In addition, microarray analysis and RNA sequencing provide convenient but also comprehensive ways to identify aberrantly expressed lncRNAs. Undoubtedly, high-throughput RNA sequencing and computational analyses will substantially improve the characterization of noncoding RNAs to a much broader level than that of previous work.
As discussed above, many lncRNAs regulate speciﬁc facets of protein activity, thus, they may represent potential targets for drugs that are more reﬁned and less toxic than conventional protein-targeting drugs. For instance, oligonucleotide antagonists speciﬁcally block the binding of oncogenic PRC2 to lncRNA, thereby inhibiting the repression of tumor suppressor genes.
Oligonucleotides for the knockdown of deleterious lncRNAs have already been studied [21,80].
Although promising, this approach also has challenges, such as how to demonstrate efﬁcient delivery accompanied by long-lasting effects on abnormal cells and how to evaluate toxicity, stability, and Int. J. Mol. Sci. 2016, 17, 478 7 of 11 efﬁcient targeting. Additional studies focusing on the druggability of known lncRNAs remain to be conducted.
Systematic identiﬁcation of lncRNAs and a better understanding of their mechanisms of action can pave the way for early diagnosis and the design of better therapeutics. New candidate lncRNA genes and their molecular mechanisms remain to be explored. Focused studies will surely provide useful insights for understanding disease pathogenesis and identifying new disease mechanisms. Intensive research will inspire new hypotheses about pathogenesis and will lead to novel clinical applications.
Acknowledgments: This work was supported by the Scientiﬁc Research Program of the National Health and Family Planning Commission of China (201402014), the National Natural Science Foundation of China (grant 31470757), the Program for Professors of Special Appointment (Eastern Scholar) at the Shanghai Institutions of Higher Learning (1410000159), SMC-ChenXing Yong Scholar Program (2014, Class B), and the Science and Technology Commission of Shanghai (grants 14JC1404100, 14JC1404200, and 14430723100). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Author Contributions: Fang Li and Xuyang Wen wrote the manuscript, and collaborate in literature collection;
He Zhang and Xianqun Fan critically revised the manuscript, provided funding and proofed of manuscript.
Conﬂicts of Interest: The authors declare no conﬂict of interest.
1. McCusker, S.; Koola, M.M. Association of ophthalmologic disorders and depression in the elderly: A review of the literature. Prim. Care Companion CNS Disord. 2015, 17. [CrossRef] [PubMed]
2. Price, E.A.; Price, K.; Kolkiewicz, K.; Hack, S.; Reddy, M.A.; Hungerford, J.L.; Kingston, J.E.; Onadim, Z.
Spectrum of RB1 mutations identiﬁed in 403 retinoblastoma patients. J. Med. Genet. 2014, 51, 208–214.
3. Dommering, C.J.; van der Hout, A.H.; Meijers-Heijboer, H.; Marees, T.; Moll, A.C. IVF and retinoblastoma revisited. Fertil. Steril. 2012, 97, 79–81. [CrossRef] [PubMed]
4. Johansson, P.; Aoude, L.G.; Wadt, K.; Glasson, W.J.; Warrier, S.K.; Hewitt, A.W.; Kiilgaard, J.F.; Heegaard, S.;
Isaacs, T.; Franchina, M.; et al. Deep sequencing of uveal melanoma identiﬁes a recurrent mutation in PLCB4.
Oncotarget 2016, 7, 4624–4631. [PubMed]
5. Van Raamsdonk, C.D.; Bezrookove, V.; Green, G.; Bauer, J.; Gaugler, L.; O’Brien, J.M.; Simpson, E.M.;
Barsh, G.S.; Bastian, B.C. Frequent somatic mutations of GNAQ in uveal melanoma and blue naevi. Nature 2009, 457, 599–602. [CrossRef] [PubMed]
6. Van Raamsdonk, C.D.; Griewank, K.G.; Crosby, M.B.; Garrido, M.C.; Vemula, S.; Wiesner, T.; Obenauf, A.C.;
Wackernagel, W.; Green, G.; Bouvier, N.; et al. Mutations in GNA11 in uveal melanoma. N. Engl. J. Med. 2010, 363, 2191–2199. [CrossRef] [PubMed]
7. Harbour, J.W.; Onken, M.D.; Roberson, E.D.; Duan, S.; Cao, L.; Worley, L.A.; Council, M.L.; Matatall, K.A.;
Helms, C.; Bowcock, A.M. Frequent mutation of BAP1 in metastasizing uveal melanomas. Science 2010, 330, 1410–1413. [CrossRef] [PubMed]
8. Kapranov, P.; Cheng, J.; Dike, S.; Nix, D.A.; Duttagupta, R.; Willingham, A.T.; Stadler, P.F.; Hertel, J.;
Hackermuller, J.; Hofacker, I.L.; et al. RNA maps reveal new RNA classes and a possible function for pervasive transcription. Science 2007, 316, 1484–1488. [CrossRef] [PubMed]
9. Devaux, Y.; Zangrando, J.; Schroen, B.; Creemers, E.E.; Pedrazzini, T.; Chang, C.P.; Dorn, G.W., 2nd; Thum, T.;
Heymans, S.; Cardiolinc, N. Long noncoding RNAs in cardiac development and ageing. Nat. Rev. Cardiol.
2015, 12, 415–425. [CrossRef] [PubMed]
10. Panzeri, I.; Rossetti, G.; Abrignani, S.; Pagani, M. Long intergenic non-coding RNAs: Novel drivers of human lymphocyte differentiation. Front. Immunol. 2015, 6. [CrossRef] [PubMed]
11. Fatica, A.; Bozzoni, I. Long non-coding RNAs: New players in cell differentiation and development.
Nat. Rev. Genet. 2014, 15, 7–21. [CrossRef] [PubMed] Ilott, N.E.; Ponting, C.P. Predicting long non-coding RNAs using RNA sequencing. Methods 2013, 63, 50–59.
13. Memczak, S.; Jens, M.; Elefsinioti, A.; Torti, F.; Krueger, J.; Rybak, A.; Maier, L.; Mackowiak, S.D.;
Gregersen, L.H.; Munschauer, M.; et al. Circular RNAs are a large class of animal RNAs with regulatory potency. Nature 2013, 495, 333–338. [CrossRef] [PubMed] Int. J. Mol. Sci. 2016, 17, 478 8 of 11
14. Rapicavoli, N.A.; Poth, E.M.; Blackshaw, S. The long noncoding RNA RNCR2 directs mouse retinal cell speciﬁcation. BMC Dev. Biol. 2010, 10. [CrossRef] [PubMed]
15. Guttman, M.; Donaghey, J.; Carey, B.W.; Garber, M.; Grenier, J.K.; Munson, G.; Young, G.; Lucas, A.B.; Ach, R.;
Bruhn, L.; et al. lincRNAs act in the circuitry controlling pluripotency and differentiation. Nature 2011, 477, 295–300. [CrossRef] [PubMed]
16. Ramos, A.D.; Diaz, A.; Nellore, A.; Delgado, R.N.; Park, K.-Y.; Gonzales-Roybal, G.; Oldham, M.C.; Song, J.S.;