Long Noncoding RNAs-Related Diseases, Cancers, and Drugs

Long noncoding RNA (lncRNA) function is described in terms of related gene expressions, diseases, and cancers as well as their polymorphisms. Potential modulators of lncRNA function, including clinical drugs, natural products, and derivatives, are discussed, and bioinformatic resources are summarized. The improving knowledge of the lncRNA regulatory network has implications not only in gene expression, diseases, and cancers, but also in the development of lncRNA-based pharmacology.


Introduction
Less than 2% of the mammalian genome is in proteinencoded regions, and the remainder is in noncoding RNAss (ncRNAs) [1]. Most long noncoding RNA (lncRNAs) are transcribed by RNA polymerase (Pol) II/Pol I, and some are transcribed by RNA Pol III [2]. The ncRNAs with nucleotide lengths of <200 and >200 are classified as short and long ncRNAs (lncRNAs), respectively. The lncRNAs can be further classified in terms of their orientation and location relative to neighboring genes as sense/antisense, divergent/convergent, and intronic/intergenic [3]. The lncRNAs function as chromatin scaffolds for complex assembly, as enhancers and decoys for improving and inhibiting transcription of target genes, and as cis-acting or trans-acting regulators of gene expression [4][5][6]. Cis-acting lncRNAs mediate local genes whereas trans-lncRNAs mediate multiple targets [6].
By dysregulating target gene expression, abnormal lncRNA expression causes cell dysfunction and disease progression. The official symbols of lncRNAs were designated by the HUGO Gene Nomenclature Committee [7].
In human cells, lncRNAs epigenetically regulate gene expression [25,26] through chromatin remodeling [27]. For example, the mouse lncRNA, namely, potassium voltagegated channel, KQT-like subfamily, member 1 (KCNQ1) overlapping transcript 1 (Kcnq1ot1) has a chromatin-interacting ability and can downregulate multiple genes in the Kcnq1 domain [28]. This gene silencing was reported to be mediated by DNA methylation at some target genes [29]. Other studies of cancer patients show that silenced tumor suppressor genes are often hypermethylated [30][31][32]. In the case of tumor suppressor genes, the epigenetic effect may have a role in carcinogenesis. In Hox antisense intergenic RNA (HOTAIR), long intergenic noncoding RNA (lincRNA), which is lncR-NAs transcribed from noncoding DNA regions between protein-coding genes [33], may function as scaffolds for assembly of histone modification machinery [34].
Some lncRNAs may function through repeat sequences. For example, some lncRNAs that contain Alu elements [35] may transactivate Staufen 1-(STAU1-) mediated mRNA decay (SMD) by base pairing of Alu elements within both lncRNAs and 3 untranslated region of the SMD target. These lncRNAs then downregulate several SMD targets [35].

The lncRNAs and Diseases
The functions of lncRNAs that are known to have roles in diseases have been reviewed previously [36,37]. Recent studies suggest that lncRNAs have roles in neurodegenerative disorders [38,39] and brain development [40]. In Huntington's disease, for example, neural lncRNAs are upregulated in taurine upregulated 1 (TUG1) and in nuclear paraspeckle assembly transcript 1 (NEAT1) but are downregulated in maternally expressed 3 (MEG3). The metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) lncRNA is reportedly highly upregulated in neurons. In cultured hippocampal neurons, synaptic density is reduced by MALAT1 depletion but rescued by MALAT1 overexpression. Studies of patients with alcohol addiction reveal upregulated MALAT1 in the cerebellum, hippocampus, and brain stem [41], which suggests that the lncRNA network may have key roles in neurodegenerative processes [42].
Studies of patients with facioscapulohumeral muscular dystrophy (FSHD) involving Polycomb/Trithorax epigenetic regulation show a deregulated copy number in D4Z4 repeat mapping to 4q35 [43]. A recent study of FSHD patients further showed that selective upregulation of DBE-T, a chromatin-associated lncRNA, reverses repression of 4q35 gene transcription [44]. These results suggest that lncRNAs derived from repetitive sequences may contribute to disease development through epigenetic regulation.
Recently, the single nucleotide polymorphisms (SNPs) of lncRNAs have been found to play important roles for disease association studies. For example, the SNP rs1333049 in the lncRNA, namely, antisense noncoding RNA in the INK4 locus (ANRIL) is reportedly associated with myocardial infarction as well as the pharmacogenomic evaluation in hypercholesterolemia [45]. SNP rs2383207 on lncRNA-ANRIL and SNP rs11066001 on protein-coded BRCA1 associated-protein (BRAP) gene were both associated with ankle-brachial index in a Taiwanese population [46]. Three SNPs (rs2067051, rs2251375, and rs4929984) located in 5 region of the H19 imprinted maternally expressed transcript (H19) genes were reportedly associated with birth weight [47]. Additionally, the rs2839698 TC genotype of H19 was reportedly associated with a low risk for nonmuscle-invasive disease [48].
The accumulating evidence of lncRNA involvement in carcinogenesis includes findings that downregulation of maternally expressed gene 3 (MEG3), an imprinted lncRNA, is associated with carcinogenesis of meningiomas [61] and bladder cancer [62]. The lncRNA, namely, ANRIL also contributes to the development of plexiform neurofibromas in neurofibromatosis type 1 [63]. The ANRIL downregulates tumor suppressor gene p15 (INK4B) expression by binding to and recruiting the suppressor of zeste 12 homolog (Drosophila) (SUZ12), a component of the Polycomb Repressive Complex 2 [64]. When DNA damage occurs, ANRIL is upregulated by the ATM-E2F1 signaling pathway [65].
In human colorectal cancer, lncRNA H19 and H19derived miR-675 are overexpressed in cell lines and primary tissues but not in adjacent noncancerous tissues [66]. Exogenous miR-675 expression also downregulates the tumor suppressor retinoblastoma, which is a direct target of miR-675 and increases tumor cell growth. Upregulation of H19 is also known to contribute to gastric cancer cell proliferation [67] and bladder cancer metastasis [68].
In lung cancer cells, downregulation of MALAT1 by siRNA decreases cell motility and downregulates motilityrelated genes [72], which suggests that MALAT1 promotes lung cancer metastasis. Similarly, MALAT1 is important in regulating cell proliferation, migration, and invasion of colorectal cancer metastasis [73]. In bladder cancer tissues, MALAT1 is overexpressed. Downregulation of MALAT1 by siRNA, the epithelial-to-mesenchymal transition-related genes, and cell migration of bladder cancer cells are inhibited [74]. After liver transplantation, MALAT1 is overexpressed The Scientific World Journal 3 in both cell lines and tissues of patients with hepatocellular carcinoma. Additionally, upregulated MALAT1 is associated with increased risk of liver tumor recurrence [75].
Similar to the disease association studies as described above, the accumulating evidence of SNPs in lncRNAs has been reported in cancer association studies. For example, SNP array-based study reported that several SNPs in lncR-NAs were associated with prostate cancer risk [84]. An lncRNA prostate cancer gene expression marker 1 (PCGEM1) is overexpressed in prostate cancer [85]. Two tagSNPs (rs6434568 and rs16834898) of the PCGEM1 were reported to be associated with prostate cancer [86]. Several lncRNAs contain SNPs such as rs7763881 in highly upregulated in liver cancer long noncoding RNA (HULC) and rs619586 in MALAT1 which are reportedly associated with decreased hepatocellular carcinoma risk [87].

The lncRNAs and Their Potential Modulators
Chemically engineered oligonucleotides that have proven effectivess for targeting endogenous miRNAs in mice [88] have potential applications in lncRNAs. For example, antisense oligonucleotides targeted at the mouse lncRNA Malat1 correct RNA gain-of-function effects of myotonic dystrophy [89]. Using siRNA treatment to lncRNA, the lncRNA, namely, antidifferentiation ncRNA (ANCR) is downregulated to promote osteoblast differentiation [90]. Similarly, siRNA-based downregulation of lncRNA associated with liver regeneration (LALR1) inhibits hepatocyte proliferation and cell cycle progression during liver regeneration [91]. Data obtained by a recent systematic transcriptome-wide analysis of lncRNA-miRNA interactions [92] may reveal additional regulators of lncRNA expression such as miRNAs that contribute to lncRNA degeneration. For example, in some lncRNAs targeted by breast cancer-related miRNAs, changes in gene expressions differ between women with and without breast tumors [93].
Inhibitors that modulate lncRNA function have also been identified. For example, small molecules such as diazobenzene-related compounds are now known to inhibit the function of miR-21 [94], a polyadenylated lncRNA [95]. 5-aza-2 -deoxycytidine (5-aza-dC), a methylation inhibitor, inhibits the methylation of putative imprinted control region (ICR) of H19 gene and leads to the downregulation of the H19 mRNA expression in blastocysts derived from vitrified twocell embryos [96]. This finding suggests that epigenetic agents may be the modulators for lncRNA expression as well as their related targeting signals.
The hypothesis that environmental exposures are another cause of ncRNA alterations [97] was tested by exposing aquatic midges to xenobiotics, which revealed upregulation of lncRNAs derived from repetitive sequences [98]. Additionally, telomeric and centromeric ncRNA can be activated by bisphenol A, a synthetic chemical with estrogen-like effects [98]. Based on these findings, some drugs may also modulate lncRNA expression. Therefore, many natural products and their derivatives are likely to prove suitable for screening and identifying these modulators in lncRNAs.

Long Noncoding RNA and Bioinformatics Resources
Computational methods for predicting lncRNA function have been well reviewed [99]. Recently, consistently improving computational capability enabled rapid development of functional analyses and bioinformatics resources for lncR-NAs [100]. Except for NRED [8], ncFANs [9], and lncRNAdb [10], we summarize the update progression of bioinformatics resources for lncRNAs during 2012-2013 as shown in Table 1.
For example, the NRED [8] database of lncRNA expression includes both microarray and in situ hybridization data for human and mouse lncRNAs. The noncoding RNA Function Anotation server (ncFANs) [9], a web server for functional anotation of lncRNAs, includes ten reannotated human and mouse microarray datasets. The lncRNAdb [10] is a comprehensive database of eukaryotic lncRNA anotations. The data contained in the lncRNAdb include sequences, structures, genomic contexts, expressions, and subcellular distributions. Most (∼75%) lncRNAs in the database were collected from mammals.

Conclusion
Various lncRNA functions are essential for regulating gene expression. This study focused on lncRNA dysregulation associated with disease progression and carcinogenesis and on the development of drugs for modulating lncRNA function. Since lncRNA is rarely studied in natural products, the resources mentioned in the paper may provide helpful information for researchers studying natural products.