Molecular mechanisms of long noncoding RNAs on gastric cancer

Long noncoding RNAs (lncRNAs) are non-protein coding transcripts longer than 200 nucleotides. Aberrant expression of lncRNAs has been found associated with gastric cancer, one of the most malignant tumors. By complementary base pairing with mRNAs or forming complexes with RNA binding proteins (RBPs), some lncRNAs including GHET1, MALAT1, and TINCR may mediate mRNA stability and splicing. Other lncRNAs, such as BC032469, GAPLINC, and HOTAIR, participate in the competing endogenous RNA (ceRNA) network. Under certain circumstances, ANRIL, GACAT3, H19, MEG3, and TUSC7 exhibit their biological roles by associating with microRNAs (miRNAs). By recruiting histone-modifying complexes, ANRIL, FENDRR, H19, HOTAIR, MALAT1, and PVT1 may inhibit the transcription of target genes in cis or trans. Through these mechanisms, lncRNAs form RNA-dsDNA triplex. CCAT1, GAPLINC, GAS5, H19, MEG3, and TUSC7 play oncogenic or tumor suppressor roles by correlated with tumor suppressor P53 or onco-protein c-Myc, respectively. In conclusion, interaction with DNA, RNA and proteins is involved in lncRNAs’ participation in gastric tumorigenesis and development.


INTRODUCTION
Generally defined as transcripts longer than 200 nucleotides, long noncoding RNAs (lncRNAs) are lack of significant open reading frames [1]. According to their location and orientation, lncRNAs are classified as intergenic lncRNAs (lincRNAs), genic and intragenic lncRNAs [2]. In nucleus, lncRNAs mainly modulate gene transcription and mRNA splicing; while they impact RNA stability and microRNA (miRNA) activity in cytoplasm [3].
In this article, we summarized the molecular mechanisms of lncRNAs on gastric cancer. By interacting with DNA, RNA, and proteins, lncRNAs play crucial roles in gastric tumorigenesis and development.

INTERACTION WITH DNA
LncRNAs may combine with histone-modifying complexes and then target on DNA [25]. For example, combining with histone-modifying complexes, forkhead box F1 (FOXF1) adjacent non-coding developmental regulatory RNA (FENDRR) anchors to targeted promoter fragments [26]. HOX transcript antisense RNA (HOTAIR) also occupies targeted double-strained DNA (dsDNA) [27]. Additionally, the lncRNA transcribed from the minor promoter of the human dihydrofolate reductase (DHFR) gene combines with the key regulatory region of its host gene in a triple helical model [28]. These triplex structures can be served as a specific recognition mechanism between lncRNA and genomic DNA. Theoretically, these are involved in specificities and affinities. Triplexes created by lncRNA and genomic DNA may decisively result in targeting specificity. Favorable chromatin conformation may contribute to the affinity [29].
(IGF2BP1) and c-Myc mRNA (Figure 1c). Albeit the short recognition element (CAUH, H = A, U, or C) of IGF2BP1 is extensively mapping to 8400 coding genes genomewide [38]. Remarkably, the motif CAUH is too short to format the secondary structure.
About a quarter of lncRNAs are consist of one or more Alu elements [30]. Inverted Alu repeats form long stable stem-loop structures and activate the post-transcription and translation [39,40]. Interestingly, the long inverted repeats flanking the mouse Sry gene enable the formation of a circular RNA [41]. Apart from the secondary structure in repeat elements, it is likely that the special architectures of lncRNAs accelerate their functions. Therefore, high order structures of lncRNAs may conduce to the specific recognization with proteins and nucleic acids. promoter loci. c. Through the formation of dsDNA/RNA triplex, HOTAIR recruits SUZ12 and EZH2 to the MiR-34A loci and then silences the transcription of miR-34a. HOTAIR also forms complex with LSD1. HOTAIR attenuates c-Met, Snail, N-cadherin and Vimentin protein level while upregulates E-cadherin protein level. All of them are targets of miR-34a. d. ANRIL suppresses miR-49a/449a expression by the recruitment of EZH2 and SUZ12. ANRIL also combines with CBX7. e. The P15/P16 is silenced by the occupancy of EZH2, which is recruited by PVT1. f. Using its complementary fragment, FENDRR anchors with target DNA and guides MLL and PRC2 to the targeted genes. Additionally, FENDRR decreases MMP2/MMP9 activity by reducing FN1. g. Combining with BRG1, UCA1 impairs BRG1's ability to bind with P21 promoter. Abbreviations: FN1, fironectin 1; MMP2/9, matrix metalloproteinase 2/9. www.impactjournals.com/oncotarget

Interaction with miRNAs
By assembly of RNA induced silencing complex (RISC), miRNAs lead mRNA degradation [42]. Studies showed that some lncRNAs are the precursors of miRNAs [43,44]. These mean that lncRNAs may affect mRNA degradation in a indirect way. miR-675, processed from its precursor (imprinted maternally expressed transcript, H19), has the properties of promoting proliferation and epithelial-mesenchymal transition (EMT) (Figure 1d). H19 is capable of enhancing gastric carcinogenesis [45]. Plasma H19 levels in patients with gastric cancer are significantly higher than those in healthy controls [46]. Besides, lncRNAs' expressions may be repressed by miRNAs [47][48][49]. The situations in gastric cancer are  (Figure 1g).
The examples about miRNAs' regulating lncRNAs via epigenetic modification, vice versa, can also be found in other parts of this article.

LncRNAs acting as ceRNAs
Emerging evidences suggest that lncRNAs may participate in competitive endogenous RNA (ceRNA) network [52], in which lncRNAs cross talk with other RNAs by sharing miRNAs.
HOTAIR is the first demonstrated lncRNA coordinating gene silencing via assembly of PRC2 [69]. The structural domains of HOTAIR formed in its 5' region and 3' region are bound to enhancer of zeste 2 (EZH2, PRC2 subunit) and lysine specific demethylase 1 (LSD1), respectively [70]. HOTAIR preferentially occupies a GA-rich DNA motif to enable the formation of RNA:dsDNA triplex. This occurs independently of EZH2 [27]. Simultaneously, HOTAIR is required for the occupancy of suppressor of zeste 12 homolog (SUZ12, PRC2 subunit) on MIR-34A loci (Figure 3c). With the metastasis potential, H19 and HOTAIR epigenetically modify their targeted genes including E-cadherin ( Figure  3b, c). Remarkably, the 1062 nt region at the 5' end of H19 is indispensable [71].

Interaction with P53 and c-Myc
Among the transcription factors (TFs) activating carcinogenesis, P53 and c-Myc represent potent inducers. The proliferative subtype is a well-defined subtype of gastric cancer characterized by p53 mutations, DNA hypermethylation, as well as activated E2F, Myc and Ras oncogenic pathways [73]. H19 is concordantly stimulated by tumor suppressor P53 [74] and onco-protein c-Myc [75].
Myc protein contains a basic DNA binding domain that binds to E-box DNA recognition fragment (CACGTG) [82]. CpG islands and pre-acetylated state of chromatin enable high affinity of sites bound to c-Myc [83]. c-Myc is overexpressed in 43% gastric cancer patients [84]. Microarray results indicated that 1244 lncRNAs are directly activated by c-Myc [85]. Remarkably, two lncRNAs, H19 (Figure 4f) and colon cancer associated transcript 1 (CCAT1) (Figure 4g) are transcriptionally activated by c-Myc in gastric cancer [75,86]. In addition, the active regulatory region of the CCAT1 site occurs physically interaction with c-Myc enhancer region [87].

Interaction with other proteins
With the exception of the direct interaction with proteins, how do lncRNAs regulate the levels of proteins is arresting. GAS5 positively influences YBX1 protein stability without increasing its transcription [81]. The putative stem-loop structure formed by exon 12 of GAS5 is responsible for its interplay with YBX1 ( Figure  2d). YBX1 possesses the capacity of complexing with IGF2BP1 [88], which combines with GHET1 and prevents mRNA degradation [33]. The exon 12 is a GAS5's predominant structural domain for mimicking binding domain of glucocorticoid receptor (GR) [89]. GAS5, GR, YBX1, and P53 may collaborate as complex to achieve cell cycle regulation [90] (Figure 2d).
H19 is found bound with Isthmin 1 (ISM1) [45] and positively regulates its expression (Figure 2e). ISM1 is a factor promoting endothelial cell survival and cell death synchronously [91]. This may explain why H19's role converts between multiple cancers. H19's high expression level enforces the oncogenic progress of gastric cancer cells; while in hepatocellular carcinoma (HCC), it is recognized as tumor suppressor [92]. Additionally, H19derived miR-675 may succeed in causing the alterations of ISM1 [45].
There are also inhibitory effects of lncRNAs on the binding proteins, such as nc886 on double stranded RNAdependent protein kinase (PKR) (Figure 2f) and HOTAIR on poly(C) binding protein 1 (PCBP1) (Figure 2g). PKR recognizes single strand nucleotide sequence in the central region of nc886 [93], which deviates from canonical PKR ligands with abundant hairpin structures [94]. In this way, nc886 displays its anti-apoptotic ability in a cell type dependent manner [95] (Figure 2f). PCBP1 inactivates the AKT pathway to against metastatic progression [96]. This finding per se matches the potential of HOTAIR in metastasis in gastric cancer.

CONCLUSIONS
LncRNAs are characterized by the complexity of their mechanisms. We summarized lncRNAs' interaction with DNA, RNA, and proteins in gastric cancer occurrences.
It is tempting to speculate that a multitude of lncRNAs may interrupt definitive steps in numerous tumor suppressive and oncogenic pathways. The uncovering of the underlying mechanisms of lncRNAs may benefit our understanding of gastric cancer's pathogenesis.

CONFLICTS OF INTEREST
The authors declare no conflict of interests.