LncRNAs: Novel Biomarkers for Pancreatic Cancer

Pancreatic cancer is one of the most deadly neoplasms and the seventh major cause of cancer-related deaths among both males and females. This cancer has a poor prognosis due to the lack of appropriate methods for early detection of cancer. Long non-coding RNAs (lncRNAs) have been recently found to influence the progression and initiation of pancreatic cancer. MACC1-AS1, LINC00976, LINC00462, LINC01559, HOXA-AS2, LINC00152, TP73-AS1, XIST, SNHG12, LUCAT1, and UCA1 are among the oncogenic lncRNAs in pancreatic cancer. On the other hand, LINC01111, LINC01963, DGCR5, MEG3, GAS5, and LINC00261 are among tumor suppressor lncRNAs in this tissue. In the current review, we summarize the roles of these two classes of lncRNAs in pancreatic cancer and discuss their potential as attractive diagnostic and prognostic biomarkers for pancreatic cancer. We also identified that the low expression of MEG3, LINC01963, and LINC00261 and the high expression of MACC1-AS1, LINC00462, LINC01559, and UCA1 were significantly correlated with worse survival in pancreatic cancer patients. Further research on these lncRNAs will provide new clues that could potentially improve the early diagnosis, prognostic prediction, and personalized treatments of patients with pancreatic cancer.


Introduction
Pancreatic cancer is one of the most deadly neoplasms as the whole number of deaths from this neoplasm is nearly equal to the number of affected individuals. This cancer is among the major causes of cancer-related demises among both males and females [1]. In numerous countries, the records of new affected individuals and deaths from pancreatic cancer have remained constant or slightly increased, most likely due to an increase in the occurrence of lifestyle-related risk factors (such as obesity, diabetes, and alcohol) or improvements in diagnostic procedures and cancer registry programs [2]. The most common subtype of this neoplasm is pancreatic ductal adenocarcinoma (PDAC) as the malignant cells originate from the ductal epithelium of the exocrine pancreas. PDAC is an aggressive tumor with poor response to treatment options [3]. Furthermore, the diagnosis of PDAC is delayed due to a lack of early diagnostic strategies. Another challenging issue in the field of PDAC therapy is the genetic and phenotypic heterogeneity of this type of malignancy [3]. These issues necessitate the identification of molecular pathways that are involved in the initiation and progression of pancreatic cancer. Long non-coding RNAs (lncRNAs) as a group of transcripts with regulatory functions have been found to partake in the pathogenesis of almost all types of neoplasms [4]. LncRNAs are RNAs longer than 200 nucleotides and have no protein-coding capacity. They are mainly transcribed by RNA polymerase II, yet other RNA polymerases are involved in their transcription [5]. While a number of lncRNAs are transcribed from intergenic regions (lincRNAs), others can overlap with other genes in sense or antisense directions [5]. The presence of a 7-methyl guanosine cap at the 5 end, a polyadenylated tail at the 3 end and splicing are all characteristics shared by lncRNAs and mRNAs. In addition, enhancer RNAs and promoter upstream transcripts are other types of lncRNAs that are transcribed from enhancer and promoter regions of genes, respectively [5]. LncRNAs have been found to induce numerous central phenotypes of cancer cells through interacting with other biomolecules such as DNA, proteins, and RNAs. Several cancer-related lncRNAs have been functionally annotated and have been proposed as potential cancer therapeutic targets [4]. In pancreatic cancer cells, numerous lncRNAs have been found to be dysregulated in association with progression of cancer. Several lncRNAs have been annotated as oncogenic or tumor suppressor lncRNAs in pancreatic cancer. In the current review, we focus on the summarization of the roles and the potential clinical implications of lncRNAs in pancreatic cancer.

Oncogenic LncRNAs in Pancreatic Cancer
Expression of lncRNAs has been appraised in pancreatic cancer tissues and cell lines using lncRNA microarray and qRT-PCR methods. These methods have resulted in the identification of numerous differentially expressed lncRNAs between neoplastic and non-neoplastic tissues. For instance, MACC1-AS1 has been identified as the most over-expressed lncRNA in pancreatic cancer tissues in a study conducted by Qi C et al. [6]. Expression of MACC1-AS1 was particularly elevated in patients who had poor survival. MACC1-AS1 silencing suppresses the proliferation and metastatic ability of pancreatic cancer cells. Mechanistically, MACC1-AS1 enhances the expression of PAX8 protein, which, in turn, increases aerobic glycolysis and activates NOTCH1 signaling. In addition, the expression of PAX8 is increased in pancreatic cancer tissues in correlation with levels of MACC1-AS1 and the prognosis of patients with pancreatic cancer. Thus, the MACC1-AS1/PAX8/NOTCH1 axis has been suggested as a putative target for the treatment of pancreatic cancer [6]. The association between expression levels of LINC00976 and pancreatic cancer progression has been assessed using in situ hybridization (ISH) and qRT-PCR methods. LINC00976 is up-regulated in pancreatic cancer tissues and cell lines in correlation with poor survival of patients. LINC00976 silencing inhibits proliferation, migratory potential and invasiveness of pancreatic cancer in vivo and in vitro. LINC00976 has been found to target ovarian tumor proteases OTUD7B. This protein deubiquitinates EGFR and influences the activity of MAPK signaling. Further studies have shown the role of the LINC00976/miR-137/OTUD7B axis in the modulation of the proliferation of pancreatic cancer cells [7]. Expression of LINC00462 in pancreatic cancer cells is induced by OSM. Up-regulation of this lncRNA has been accompanied by enhancement of cell proliferation, acceleration of cell cycle progression, and inhibition of cell apoptosis and adhesion. Moreover, LINC00462 up-regulation increases the migration and invasiveness of pancreatic cancer cells through enhancement of epithelial-mesenchymal transition (EMT) and accelerated growth and metastasis of pancreatic cancer in vivo. Moreover, over-expression of this lncRNA in clinical samples has been associated with larger tumor dimension, poor tumor differentiation, advanced TNM stage, and higher probability of metastasis in patients. Notably, LINC00462 has been demonstrated to have interaction with miR-665. Upregulation of LINC00462 leads to the enhancement of expressions of TGFBR1 and TGFBR2 and the subsequent activation of the SMAD2/3 pathway in pancreatic cancer [8]. Table 1 shows the information on oncogenic lncRNAs in pancreatic cancer. Figure 1 illustrates the role of various lncRNAs in pancreatic cancer through regulating the TGF-β/SMAD signaling pathway.    Figure 1. A schematic diagram shows the role of various lncRNAs in modulating the TGF-β/SMAD signaling pathway in pancreatic cancer. According to this cascade, when bioavailable TGF-β binds a homodimer of TβRII, transphosphorylation of the TβRI can trigger the activation of kinase activity. SMAD proteins, the substrates for TβRI kinases, are downstream of the BMP-analogous ligand-receptor systems. SMAD1, SMAD2, SMAD3, SMAD5, and SMAD8 can bind to membranebound serine/threonine receptors and are up-regulated via their kinase function. As a co-factor, the Co-SMAD (SMAD4) can bind to the up-regulated R-SMAD to create a complex that translocates into the nucleus. Consequently, I-SMAD (SMAD7) can deactivate the impacts of R-SMADs [36,37]. Previous studies have authenticated that several lncRNAs can play an effective role in regulating the TGF-β/SMAD cascade in pancreatic cancer. LINC00462 can up-regulate expression levels of TGFBR1 and TGFBR2 and activate the SMAD2/3 pathway in pancreatic cancer cells through down-regulating miR-665 expression [8]. Furthermore, lncRNA XIST can promote TGF-β2 expression via inhibiting the expression of miR-141-3p, thus enhancing cell proliferation, migration, and invasion of PC cells [33]. Green arrows indicate the up-regulation of target genes by lncRNAs; red arrows depict the inhibitory effects of lncRNAs.

Tumor Suppressor LncRNAs in Pancreatic Cancer
LINC01111 is a newly identified lncRNA that is significantly down-regulated in tissue and plasma samples gathered from patients with pancreatic cancer. This lncRNA has been found to exert tumor-suppressive effects. Notably, expression levels of LINC01111 have been inversely correlated with the TNM stage but positively correlated with the survival rate of patients with pancreatic cancer. LINC01111 can suppress the proliferation, cell cycle progression, invasiveness, and migratory potential of pancreatic cancer cells in vitro. Moreover, it can suppress the tumorigenic potential and metastatic ability of Figure 1. A schematic diagram shows the role of various lncRNAs in modulating the TGF-β/SMAD signaling pathway in pancreatic cancer. According to this cascade, when bioavailable TGF-β binds a homodimer of TβRII, transphosphorylation of the TβRI can trigger the activation of kinase activity. SMAD proteins, the substrates for TβRI kinases, are downstream of the BMP-analogous ligand-receptor systems. SMAD1, SMAD2, SMAD3, SMAD5, and SMAD8 can bind to membranebound serine/threonine receptors and are up-regulated via their kinase function. As a co-factor, the Co-SMAD (SMAD4) can bind to the up-regulated R-SMAD to create a complex that translocates into the nucleus. Consequently, I-SMAD (SMAD7) can deactivate the impacts of R-SMADs [36,37]. Previous studies have authenticated that several lncRNAs can play an effective role in regulating the TGF-β/SMAD cascade in pancreatic cancer. LINC00462 can up-regulate expression levels of TGFBR1 and TGFBR2 and activate the SMAD2/3 pathway in pancreatic cancer cells through down-regulating miR-665 expression [8]. Furthermore, lncRNA XIST can promote TGF-β2 expression via inhibiting the expression of miR-141-3p, thus enhancing cell proliferation, migration, and invasion of PC cells [33]. Green arrows indicate the up-regulation of target genes by lncRNAs; red arrows depict the inhibitory effects of lncRNAs.

Tumor Suppressor LncRNAs in Pancreatic Cancer
LINC01111 is a newly identified lncRNA that is significantly down-regulated in tissue and plasma samples gathered from patients with pancreatic cancer. This lncRNA has been found to exert tumor-suppressive effects. Notably, expression levels of LINC01111 have been inversely correlated with the TNM stage but positively correlated with the survival rate of patients with pancreatic cancer. LINC01111 can suppress the proliferation, cell cycle progression, invasiveness, and migratory potential of pancreatic cancer cells in vitro. Moreover, it can suppress the tumorigenic potential and metastatic ability of neoplastic cells in vivo. LINC01111 over-expression leads to the up-regulation of DUSP1 through sponging miR-3924. These events result in the inhibition of phosphorylation of SAPK, therefore inactivating SAPK/JNK signaling in pancreatic cancer cells [38]. LINC01963 is another down-regulated lncRNA in clinical samples of pancreatic cancer and cell lines. Over-expression of LINC01963 leads to suppression of colony formation, attenuation of cell cycle progression, and inhibition of proliferation and invasion of pancreatic cancer cells while enhancing the apoptosis rate in these cells. More importantly, short hairpin RNA targeting LINC01963 increases the tumorigenicity of pancreatic cancer cells in vivo. Functionally, LINC01963 decreases the expression of miR-641, a miRNA that down-regulates TMEFF2. Thus, LINC01963 suppresses the progression of pancreatic cancer through the miR-641/TMEFF2 axis [39]. MEG3 is another down-regulated lncRNA in pancreatic cancer cells, the expression of which has been inversely correlated with the expression of PI3K. In clinical samples, expression of MEG3 has been inversely correlated with tumor dimension, organ metastasis, and vascular invasion in pancreatic cancer. Functionally, MEG3 can suppress the progression of pancreatic cancer through regulation of the activity of PI3K/AKT/Bcl-2/Bax/cyclin D1/P53 and PI3K/AKT/MMP-2/MMP-9 axes [40]. On the other hand, the lncRNA GAS5 has been found to suppress metastasis of pancreatic cancer via the regulation of the miR-32-5p/PTEN axis [41].
Several tumor suppressor lncRNAs exert prominent effects on cell apoptosis. For instance, DGCR5 functions as a molecular sponge for miR-27a-3p, a miRNA that regulates the expression of BNIP3. Forced up-regulation of DGCR5 in pancreatic cancer cells leads to the down-regulation of miR-27a-3p. Furthermore, DGCR5 regulates the expression of BNIP3 and the activity of p38 MAPK via sponging miR-27a-3p. The miR-27a-3p/BNIP3 axis has been found to be the main mediator of the pro-apoptotic effects of DGCR5 [42]. Table 2 shows the list of down-regulated lncRNAs in pancreatic cancer. Figure 2 shows the role of several lncRNAs in regulating the PI3K/AKT, MAPK/ERK, and JAK2/STAT3 cascades in pancreatic cancer.   Figure 2. A schematic representation shows that several lncRNAs regulate the PI3K/AKT, MAPK/ERK and JAK2/STAT3 pathways in pancreatic cancer. Growth factor-driven RTK (e.g., EGFR) or cytokine (e.g., IL-6) signaling can trigger the activation of PI3K/AKT, MAPK/ERK, and JAK2/STAT3 cascades. LncRNAs can affect the activity of these cascades. For instance, HOTAIR can trigger the activation of the JAK2/STAT3 pathway via down-regulating miR-34a expression, thus promoting invasion and migration of pancreatic ductal adenocarcinoma [12]. In addition, GAS5 can up-regulate PTEN expression by down-regulating the expression level of miR-32-5p, therefore inhibiting pancreatic cancer metastasis [41]. LINC01559, through sponging miR-1343-3p, can up-regulate RAF1 expression that can further activate the ERK signaling pathway, thereby enhancing pancreatic cancer progression and metastasis [15]. Green arrows indicate the up-regulation of target genes modulated via lncRNAs; red arrows depict the inhibitory effects.

Diagnostic Role of LncRNAs in Pancreatic Cancer
The diagnostic role of lncRNAs in pancreatic cancer is appraised by depicting receiver operating characteristic (ROC) curves. The calculated values for the area under these curves (AUC values) for a number of these lncRNAs are more than 0.8, suggesting the potential of these lncRNAs as diagnostic markers for pancreatic cancer (Table 3). For instance, the expression of LINC00675 is firstly assessed in a small cohort of PDAC tissues Figure 2. A schematic representation shows that several lncRNAs regulate the PI3K/AKT, MAPK/ERK and JAK2/STAT3 pathways in pancreatic cancer. Growth factor-driven RTK (e.g., EGFR) or cytokine (e.g., IL-6) signaling can trigger the activation of PI3K/AKT, MAPK/ERK, and JAK2/STAT3 cascades. LncRNAs can affect the activity of these cascades. For instance, HOTAIR can trigger the activation of the JAK2/STAT3 pathway via down-regulating miR-34a expression, thus promoting invasion and migration of pancreatic ductal adenocarcinoma [12]. In addition, GAS5 can up-regulate PTEN expression by down-regulating the expression level of miR-32-5p, therefore inhibiting pancreatic cancer metastasis [41]. LINC01559, through sponging miR-1343-3p, can up-regulate RAF1 expression that can further activate the ERK signaling pathway, thereby enhancing pancreatic cancer progression and metastasis [15]. Green arrows indicate the up-regulation of target genes modulated via lncRNAs; red arrows depict the inhibitory effects.

Diagnostic Role of LncRNAs in Pancreatic Cancer
The diagnostic role of lncRNAs in pancreatic cancer is appraised by depicting receiver operating characteristic (ROC) curves. The calculated values for the area under these curves (AUC values) for a number of these lncRNAs are more than 0.8, suggesting the potential of these lncRNAs as diagnostic markers for pancreatic cancer (Table 3). For instance, the expression of LINC00675 is firstly assessed in a small cohort of PDAC tissues and chronic pancreatitis tissues through microarray screening. At the next step, these results are validated in larger cohorts of patients using the qRT-PCR method. Over-expression of LINC00675 is significantly correlated with lymph node metastasis, perineural invasion, and poor clinical outcome of patients with pancreatic cancer. Notably, this lncRNA has a 0.893 AUC value for predicting the progression of pancreatic cancer within one year. Moreover, the AUC value for the prediction of tumor progression within six months is 0.928. Thus, LINC00675 is a potential diagnostic marker for the prediction of recurrence in PDAC patients following radical surgical resection [45]. C9orf139 is another up-regulated lncRNA in the tissues and sera of patients with pancreatic cancer that has diagnostic value in clinical settings since the AUC value of this lncRNA has been estimated to be 0.923. Over-expression of this lncRNA has been associated with a higher possibility of cancer progression to advanced stages, lymph node metastasis, and poor differentiation [46].
Moreover, serum levels of UFC1 expression are relatively higher in patients with pancreatic cancer compared with healthy controls. ROC curve analyses have shown that the serum levels of this lncRNA can separate pancreatic cancer patients from healthy subjects with an AUC value of 0.810. Moreover, serum levels of UFC1 have been associated with lymph nodes involvement, metastases to distant organs, and clinical stage [47].

Prognostic Role of LncRNAs in Pancreatic Cancer
The prognostic role of lncRNAs in pancreatic cancer has been validated in several investigations (Table 4). Many lncRNAs with potential application as diagnostic markers have also been demonstrated to have prognostic potential. For instance, UFC1 not only serves as a diagnostic marker but also facilitates the prediction of the prognosis of pancreatic cancer. Based on the results of the Kaplan-Meier analysis, over-expression of UFC1 has been associated with shorter progression-free survival and overall survival rates. Multivariate analyses have also shown the potential of UFC1 expression levels as an independent prognostic factor for pancreatic cancer [47]. UNX1-IT1 is another lncRNA whose expression levels have been correlated with differentiation grade of tumors, lymph node involvement, and clinical stage. Over-expression of RUNX1-IT1 has also been correlated with a significant reduction in overall survival. Based on univariate and multivariate Cox regression analyses, over-expression of RUNX1-IT1 has been identified as a factor that increases the risk of mortality from pancreatic cancer [52]. ENSG00000254041.1 is another novel lncRNA whose expression is particularly elevated in pancreatic cancer samples with high EMT signature scores. Multivariate analyses have proposed ENSG00000254041.1 as an independent factor for pancreatic cancer [53]. Previous survival analyses performed on pancreatic cancer patients revealed that patients with low MEG3 expression had a worse prognosis [54]. Consistent with this result, our Kaplan-Meier analysis by the KM-plotter database has shown that MEG3 expression is positively correlated with longer overall survival in pancreatic cancer patients (Figure 3). Our survival analysis also indicated that an increased expression of lncRNA LINC01963 and LINC00261 were significantly associated with better overall survival in pancreatic cancer patients (Figure 3). In contrast, upregulation of lncRNA MACC1-AS1, LINC00462, LINC01559, and UCA1 predicted shorter overall survival in pancreatic cancer patients (Figure 4).

Discussion
Non-coding RNAs have important roles in the pathoetiology of human disorders [5 LncRNAs can be classified based on their genomic locations into four classes, name intergenic lncRNAs (such as LINC00462 and LINC00958), which are transcribed fro

Discussion
Non-coding RNAs have important roles in the pathoetiology of human disorders [59]. LncRNAs can be classified based on their genomic locations into four classes, namely, intergenic lncRNAs (such as LINC00462 and LINC00958), which are transcribed from intergenic regions of either sense or antisense strands; intronic lncRNAs, which are transcribed totally from the intronic regions of protein-coding genes; sense lncRNAs, which are transcribed from the sense strand of protein-coding genes and encompass exons; and antisense lncRNAs (such as MACC1-AS1 and HOXA-AS2), which are transcribed from the antisense strand of protein-coding genes [60]. These transcripts have a crucial impact on gene expression through different mechanisms, namely, transcriptional interference, chromatin remodeling, regulation of splicing events, modulation of translation through binding to translation factors or ribosomes, acting as competing endogenous RNAs for miRNAs, altering localization of proteins, modulation of telomere replication, and RNA interference [60,61]. Based on these diverse roles in cellular and biochemical processes, lncRNAs have been suggested as disease biomarkers and therapeutic targets. Suppression of expression of over-expressed lncRNAs has been suggested as a therapeutic option for human diseases. This aim has been accomplished through the application of RNAi methods, degradation of lncRNAs by RNase H, antisense oligonucleotides, or application of the genome-editing strategy CRISPR/Cas9 [62].
LncRNAs can affect the growth, migration, and invasion of pancreatic cancer [63]. These transcripts have been suggested as a key class of pervasive genes participating in tumorigenesis and metastasis [64]. They have been demonstrated to partake in the pathobiology of pancreatic cancer via different mechanisms, among which is the modulation of cancer-related pathways such as JAK2/STAT3, EGFR/MAPK, ERK, NOTCH, and PTEN pathways. The EMT process is an important process for the progression of cancer metastasis and is affected by several lncRNAs in pancreatic cancer. LINC00462, LINC00958, SNHG12, and OIP5-AS1 are among lncRNAs whose roles in the progression of EMT have been validated in pancreatic cancer.
Several lncRNAs, including lncRNA-UFC1, RP11-263F15.1, ABHD11-AS1, LINC00675, HULC, and C9orf139, have been shown to have the potential to be included in diagnostic approaches for pancreatic cancer. Therefore, combinations of expression amounts of these lncRNAs might enhance the efficiency of diagnosis of pancreatic cancer, particularly using non-invasive methods conducted on biofluids obtained from patients.
Oncogenic lncRNAs can be targeted by strategies such as shRNAs or antisense oligonucleotides [67]. Another promising strategy in this regard is the use of emerging routes of genome editing such as the CRISPR system [68]. However, several biosafety and bioavailability issues should be solved before the wide application of these techniques in clinical settings. Moreover, the context-dependent functions of lncRNAs should be completely clarified to avoid any detrimental effects when applying anti-lncRNA therapeutic modalities.
In brief, lncRNAs have pivotal roles in the pathogenesis of pancreatic cancer and have applications in diagnostic and prognostic approaches to this cancer. Based on the results of in vitro and in vivo experiments, modulation of expression of lncRNAs can be regarded as an appropriate strategy for the treatment of pancreatic cancer. The lncRNAs that have been previously reported in the literature and those lncRNAs identified in this study have the potential to serve as novel diagnostic, prognostic, and individualized treatment-predictive biomarkers for pancreatic cancer.

Conflicts of Interest:
The authors declare no conflict of interest.