Non-coding RNA biomarkers in pancreatic ductal adenocarcinoma

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, which is usually diagnosed at an advanced stage. The late disease diagnosis, the limited availability of effective therapeutic interventions and lack of robust diagnostic biomarkers, are some of the primary reasons for the dismal 5-year survival rates (~8%) in patients with PDAC. The pancreatic cancer develops through accumulation of a series of genomic and epigenomic alterations which lead to the transformation of normal pancreatic epithelium into an invasive carcinoma – a process that can take up to 15-20 years to develop, from the occurrence of first initiating mutational event. These facts highlight a unique window of opportunity for the earlier detection of PDAC, which could allow timely disease interception and improvement in the overall survival outcomes in patients suffering from this fatal malignancy. Non-coding RNAs (ncRNAs) have been recognized to play a central role in PDAC pathogenesis and are emerging as attractive candidates for biomarker development in various cancers, including PDAC. More specifically, the ncRNAs play a pivotal role in PDAC biology as they affect tumor growth, migration, and invasion by regulating cellular processes including cell cycle, apoptosis, and epithelial-mesenchymal transition. In this review, we focus on three types of well-established ncRNAs — microRNAs (miRNAs), long noncoding RNAs (lncRNAs), and circular RNAs (circRNAs) — and discuss their potential as diagnostic, prognostic and predictive biomarkers in PDAC.

1. Introduction

Pancreatic ductal adenocarcinoma (PDAC) ranks as the 11^th most commonly diagnosed cancer in the United States, with an estimated 57,600 new cases in 2020 [1]. While the relative incidence of PDAC may not be as frequent, it remains one of the most lethal malignancies and the 3^rd leading cause of cancer-related deaths in the United States [2]. The rising mortality rates from this cancer has projected it to become the second leading cause of cancer-related mortality by the year 2030. This is partly because at initial diagnosis most patients with PDAC already are at an advanced stage, with only ~10-20% patients with a resectable disease. Not surprisingly, in spite of all the medical advances made in the last decade, the overall 5-year survival rates for metastatic PDAC remain only ~8% [2].

At present, surgical resection is the only curative treatment option available for patients with PDAC; however, 5-year survival rates following surgical resection alone still remain relatively low [3, 4]. As per the National Comprehensive Cancer Network (NCCN) guidelines, gemcitabine-based or fluorouracil (5-FU)-based adjuvant treatment is the mainstay for most PDAC patients with a resectable disease. However, due to an elevated risk of complications or poor performance status, some patients are not ideal candidates for receiving such adjuvant treatments. To overcome this clinical challenge, data gathered in the past decade has highlighted the potential therapeutic impact of neoadjuvant chemotherapy or chemoradiotherapy for improving patient survival and tumor resectability status, particularly in PDAC patients with a locally advanced disease [5–11]. In addition, while the introduction of newer gemcitabine-based adjuvant chemotherapy regimens referred to as modified FOLFIRINOX (mFOLFIRINOX) that include a combination of fluorouracil, leucovorin, irinotecan, and oxaliplatin, have somewhat improved the prognosis of patients with resectable PDAC, a significant number of patients still experience disease relapse and their prognosis remains relatively poor [4, 12]. Taken together, this highlights the underlying issue that for a complete cure or improved survival, early detection of cancer might have the greatest impact on patient outcomes. This certainly has been observed in several other cancers, including breast, colon, prostate, and cervical – in each instance, the survival outcomes were significantly improved due to earlier detection of the cancer [13–17]. However, this has not yet been possible in PDAC, because currently there is a lack of robust diagnostic modalities available for the early detection of this fatal disease.

While imaging-based methods such as computed tomography (CT) and magnetic resonance imaging (MRI) are routinely used for PDAC diagnosis, their diagnostic sensitivity and specificity remains quite poor for the detection of early-stage lesions [18]. Likewise, in terms of non-invasive markers, currently carbohydrate antigen 19-9 (CA19-9) and carcinoembryonic antigen (CEA) are the only tumor markers currently used in the clinic; however, these suffer from poor diagnostic accuracy [19]. The present challenges with the imaging-based approaches, and the issues with tumor markers as robust biomarkers for the early-detection of PDAC highlight the need for development of molecular biomarkers that are more robust and can be used in the clinic for the identification of patients at risk for developing this fatal disease.

One such molecular substrate that has garnered a lot of attention in the past several years is the development of various non-coding RNAs (ncRNAs) as biomarkers for early diagnosis and determining prognosis of patients with PDAC. The ncRNAs not only function as important epigenetic regulators in PDAC pathogenesis but have also emerged as attractive targets for their potential clinical use as diagnostic, prognostic, and predictive biomarkers in various human cancers including PDAC. In recent years, technological advancements such as genome-wide sequencing have discovered novel ncRNAs, and this list is continuously growing each day. In this review article, we highlight the importance of early detection in PDAC and discuss the accumulating evidence for ncRNAs in terms of their potential as diagnostic, prognostic, and predictive biomarkers in this malignancy.

2. Importance of early detection in PDAC

Late onset of symptoms and a rapid progression to death are hallmarks of PDAC. Due to the asymptomatic nature of the disease, most patients present with non-resectable, locally advanced or metastatic disease at initial diagnosis – at which time surgical resection is not possible; hence, limiting the availability of treatment options in these patients. To muddy the waters further, the currently available treatment options in PDAC are less effective when offered to patients with an advanced disease.

2.1. Pancreatic precursor lesions and progression to invasive carcinoma – a window of opportunity for early detection

As depicted in Figure 1, the development of PDAC from non-invasive stages is a slow and gradual process [20]. More specifically, invasive PDAC develops from precursor lesions that are classified into three different histological categories: pancreatic intraepithelial neoplasia (PanIN), intraductal pancreatic mucinous neoplasia (IPMN), and mucinous cystic neoplasia (MCN) [21, 22]. Among these, PanINs are the most common precursor lesions of PDAC. PanINs are microscopic lesions (often <5 mm in size) originating from the small pancreatic ducts and are composed of columnar and cuboidal cells with varying amounts of mucin [23, 24]. Based on a number of molecular and histological alterations that transform a normal pancreatic duct into PanINs, these can be classified into low-grade dysplasia (PanIN-IA and -IB) and high-grade dysplasia (PanIN-II and -III) [25]. Similar to PanINs, the IPMNs are mucin-producing epithelial neoplasms that can arise within the main duct of pancreas (MD-IPMN), in one of its side branch-ducts (BD-IPMN), or both (mixed-IPMN). Further classification of IPMN is based on the degree of dysplasia, which ranges from low-grade dysplasia (IPMN, low-grade), intermediate-grade dysplasia (IPMN, low grade) and IPMN with high-grade dysplasia or carcinoma in-situ (IPMN, high grade) [21, 26]. In terms of their neoplastic potential, the MD-IPMNs are associated with a higher risk of developing invasive disease (~63%) compared to BD-IPMNs (~15%) [27]. Lastly, MCNs have a distinct ovarian-type stroma and are more common in women than men [28, 29]. All types of cystic neoplasms of the pancreas carry a risk of malignant transformation, albeit to varying degrees; thus, finding biomarkers that can accurately predict which of the cystic neoplasms will progress to invasive carcinomas may improve surgical management and treatment decisions. From an early-detection standpoint, there is substantive evidence that it can take more than a decade for a normal pancreatic epithelial cell to progress into an invasive, metastatic pancreatic carcinoma from the time point of first genetic event acquired within the tumor-initiating cells [30]. This is particularly attractive from an early-detection viewpoint as this long timeframe provides a window of opportunity for the detection of early precursors and PDAC lesions, at which time therapeutic intervention could be employed to drastically improve the survival rates in patients suffering from this disease [31].

Figure 1:

The evolution and progression of PDAC: The PDAC develops gradually over a period of time in which a series of genomic, epigenomic and morphological alterations are initiated in the normal cells. Initial genetic and epigenomic changes occur slowly during the transformation of normal epithelium through premalignant lesions (PanIN I- III); where the changes are not visible at the organ level making detection of premalignant lesions difficult. The premalignant cells continue to grow leading to tumor formation which gradually invades nearby lymph nodes, ultimately enter the systemic circulation and metastasize to distant organ sites. Tumor cells that reach the blood circulation also release their cellular contents (e.g. DNA, RNA, proteins etc.), which are interrogated for the development of blood-based liquid biopsy assays for the early disease detection. PanIN – pancreatic intraepithelial neoplasia; PDAC – pancreatic ductal adenocarcinoma; lncRNA – long non-coding RNA; circRNA – circular RNA; miRNA – microRNA; RBC – Red blood cell, WBC – White blood cell

2.2. Limitations of current diagnostic approaches in PDAC – the need for development of robust molecular biomarkers for its early detection

Currently, various technical and molecular approaches are used for the diagnosis of PDAC (Table 1); with each of these modalities with their own inherent advantages and disadvantages. Imaging-based approaches that utilize multi-detector CT accompanied by 3D-reconstruction and MRI is frequently used to stage PDAC patients, prior to surgery [32, 33]. However, these imaging tools for diagnosing and staging PDAC patients lack adequate sensitivity, are beset with false negative results, and frequently fail to detect small and potentially curable pancreatic lesions [18]. Endoscopic ultrasound, which is another commonly used screening tool for high-risk PDAC patients, is an expensive and invasive modality [34–36]. In addition, due to an overall lower incidence rate of PDAC and high cost-to-benefit ratio, the existing methods for the screening of average-risk general population for PDAC are unlikely to have a substantial impact on patient outcomes.

Table 1.

A list of clinical and pre-clinical diagnostic approaches in patients with PDAC

Methods			Evidence	Sensitivity	Specificity	References
Imaging modalities	Endoscopic Ultrasound		Clinical (stage T1-T2)	72%	90%	[167]
			Clinical (stage T3-T4)	90%	72%
	Multi-detector CT		Clinical	76–92%	67%	[168–171]
	MRI		Clinical	78–100%	72–99%	[172–174]
	Molecular Imaging		Clinical	87–89%	70%	[175]
Tumor marker	CA19-9		Clinical	78.2%	82.8%	[176]
Mutational markers	TP53, SMAD4, PIK3CA, PTEN, AKT1, MUC3, MUC4		Clinical	32–79% 76–89%	96–100% 96–100%	[177–187]
ncRNA markers	miRNAs	miR-21	Pre-clinical	67–96%	61–100%	[64, 188–191]
		miR-155	Pre-clinical	53–93%	73–100%	[64, 190, 192]
		miR-196a	Pre-clinical	43–100%	84–90%	[64, 87]
		miR-196b	Pre-clinical	78–100%	78–100%	[64, 87]
	lncRNAs	HOTAIR	Pre-clinical	78–80%	86–90%	[193]
		PVT1	Pre-clinical	69–96%	64–95%	[193]
		MALAT1	Pre-clinical	66%	72%	[139]

In addition to imaging-based approaches, there are only two noninvasive, serological diagnostic biomarkers that are often used for PDAC diagnosis in the clinic – the CA19-9 and CEA; both of which suffer from limited sensitivity and specificity for PDAC. In addition, elevated expression of these biomarkers is usually associated with advanced disease stage but may also indicate the presence of diseases other than PDAC [37, 38]; highlighting their inadequacy for the early detection of PDAC. Moreover, 5-10% of patients who lack fucosyltransferase activity due to germline variants do not produce CA19-9 [39]. Various protein and DNA biomarkers have been reported that show diagnostic and prognostic potential in PDAC patients either alone or in combination with CA19-9 [39–44]. However, there are currently no available reliable liquid biopsy assays with high sensitivity and specificity to detect early pancreatic cancer.

In view of the limitations of existing methods described above, there has been unprecedented interest in developing novel diagnostic biomarkers for the detection of PDAC in its earliest stages. There is no question that detecting the disease early will have a direct impact on the management of PDAC and improving patient survival.

3. ncRNAS and their important role in PDAC

The ncRNAs are important functional components of the human genome that are transcribed from DNA but are not translated into proteins. While there is lack of clear consensus on the various categories of ncRNAs, based on size these can be classified into small ncRNAs (<200 nucleotides in length) and long non-coding RNAs (lncRNAs; size>200 nucleotides) [45]. While the nomenclature and discovery of various ncRNAs continue to evolve, at this time, small ncRNAs primarily encompass microRNAs (miRNAs), small inhibitory RNAs (siRNAs), PlWI-interacting RNAs (piRNAs), and small nucleolar RNAs (snoRNAs). In contrast, lncRNAs mostly consist of large intergenic non-coding RNAs (lincRNAs), transcribed ultraconserved regions (T-UCRs), and circular RNAs (circRNAs) [45]. Even though ncRNAs are not translated into proteins, they have been recognized to be critical regulators for various biological processes such as DNA replication, translation, RNA splicing, and epigenetic regulation (Figure 2). Deregulation of ncRNAs has been reported in many diseases, including cancer [45–47]. Additionally, with the advent of next-generation sequencing technologies, various categories of ncRNAs are continuously being discovered, characterized for their biological roles, and have been developed as potential disease biomarkers through their expression analysis not only in tissues, but as well as in other bodily fluids such as blood (plasma and serum), saliva, pancreatic juice, cerebrospinal fluid and urine – highlighting their promise as potential biomarkers for cancer diagnosis, prognosis and disease monitoring [48–51]. Moreover, some ncRNAs, specially miRNAs and lncRNAs have also shown therapeutic potential and different delivery systems for ncRNA-based therapeutics have also been previously described [52].

Figure 2:

Overview of the biogenesis and functions of key ncRNAs: The biogenesis of miRNAs (left panel) involves the transcription of primary miRNA (Pri-miRNA) by RNA polymerase (II or III) from the miRNA gene. The Pri-miRNAs are long, RNA stem-loop structures, which are eventually processed by the DROSHA–DiGeorge syndrome critical region 8 (DGCR8) complex resulting in the cleavage of the Pri-miRNA and production of a smaller product called, the precursor miRNA (Pre-miRNA), which is approximately 60 nucleotides in length. The Pre-miRNA is exported to the cytoplasm from the nucleus by Exportin-5 protein. The Pre-miRNA is processed further in the cytoplasm by the ribonuclease DICER protein in conjunction with the RNA-binding protein transactivation response element RNA-binding protein (TRBP). The DICER-TRBP complex cleaves the Pre-miRNA to form a miRNA/miRNA duplex. One of the strands from this duplex is degraded while the other functional strand binds to the Argonaute 2 (AG02) protein and is incorporated into the RNA-induced silencing complex (RISC) involving DICER and TRBP. The miRNA strand guides the RISC complex to target mRNAs causing translational repression or degradation. The lncRNAs (middle panel) are transcribed by the RNA polymerase and are usually adenylated at the 3’ end and capped at 5’ end. Their expression is cell type- and cell state-specific and they can undergo alternative splicing leading to different isoforms. lncRNAs can regulate genes in many ways. For example, they can activate (enhancer RNAs) or inhibit the transcription of nearby genes by either directly interacting with the RNA polymerase or transcription factors. lncRNAs can also interact with DNA by virtue of their sequence complementarity to single stranded DNA mediating chromosomal looping. They can also induce changes in the chromatin structure and interact with nucleolar (paraspeckle) proteins forming paraspeckle assembly. Their other functions include acting as miRNA sponges, regulating mRNA stability, interaction with proteins or acting as a scaffold for proteins. The circRNAs (right panel) are also transcribed by the RNA polymerase similar to mRNAs however unlike mRNAs, circRNAs can be processed through alternative splicing of both, exons and introns of the pre-mRNA. The circular shape of circRNAs is the result of back-splicing which is described by lariat-driven circularization and intron-pairing-driven circularization. circRNAs can act as miRNA sponges due to the presence of multiple miRNA response elements in their sequence. Some circRNAs can also interact with proteins harboring RNA-binding sites. Additionally, they can also encode for and translate into various proteins.

Herein, we discuss the biomarker potential of three types of ncRNAs in PDAC – the miRNAs, lncRNAs, and circRNAs, and provide a succinct yet comprehensive state of literature for their emerging role as diagnostic, prognostic and predictive biomarkers.

4. miRNAs as potential biomarkers in PDAC

Among the ncRNAs, miRNAs are the most widely explored as biomarkers in malignant diseases. miRNAs are 18–25 nucleotides long and regulate gene expression at the post-transcriptional level [53, 54]. Abnormal expression levels of miRNAs have been implicated in the pathogenesis of PDAC [55, 56]. One of the most attractive features of miRNAs is their stability in bodily fluids (blood, urine, saliva), which makes them ideal targets for liquid biopsies.

4.1. Diagnostic significance of miRNAs in pancreatic cancer

The last decade has witnessed an explosion in the number of studies that have focused on the identification of miRNAs that are differentially expressed in clinical specimens from patients with PDAC compared to healthy controls (Table 2).

Table 2.

The potential miRNA biomarkers for the identification of patients with pancreatic precursor lesions and PDAC.

Pathologic condition	Source	miRNA	Expression	References
PDAC	Tissue	miR-21	Increased	[55, 57, 62, 63, 76, 86, 88, 90, 92, 96, 194]
		miR-31	Increased	[56]
		miR-96	Decreased	[56, 194, 195]
		miR-143	Increased	[56]
		miR-146a	Increased	[56, 194]
		miR-148a	Decreased	[56, 89, 196]
		miR-150	Increased	[56]
		miR-155	Increased	[56, 57, 62, 63, 88, 89, 92]
		miR-181a,b,d	Increased	[57]
		miR-196a,b	Increased	[56, 66, 92, 194, 197–199]
		miR-210	Increased	[56, 67, 200]
		miR-212	Increased	[55, 65]
		miR-216	Decreased	[20, 55, 56]
		miR-217	Decreased	[56, 89, 194]
		miR-222	Increased	[55, 57]
		miR-223	Increased	[56]
		miR-375	Decreased	[56]
		miR-483	Increased	[96]
		miR-494	Decreased	[56]
		miR-1290	Increased	[68]
	Blood (plasma or serum)	miR-16	Increased	[70]
		miR-18a	Increased	[201]
		miR-20a	Increased	[202, 203]
		miR-21	Increased	[64, 96, 203]
		miR-155	Increased	[64, 192]
		miR-185	Increased	[70, 202, 203]
		miR-191	Increased	[65, 203]
		miR-192	Increased	[65, 204]
		miR-194	Increased	[65]
		miR-196a,b	Increased	[64, 70, 198]
		miR-210	Increased	[205]
		miR-212	Increased	[65, 206]
		miR-483	Increased	[96]
		miR-492	Decreased	[207]
		miR-508	Decreased	[65]
		miR-513a	Decreased	[65]
		miR-602	Increased	[65]
		miR-630	Decreased	[65]
		miR-663a	Decreased	[207]
		miR-801	Increased	[65]
		miR-887	Decreased	[65]
		miR-923	Decreased	[65]
		miR-1246	Increased	[208]
		miR-3976	Increased	[208]
		miR-4306	Increased	[208]
		miR-4644	Increased	[208]
	Pancreatic Juice	miR-21	Increased	[62, 92]
		miR-155	Increased	[62, 92, 192]
		miR-196a,b	Increased	[92, 199]
		miR-210	Increased	[199]
		miR-1427	Increased	[199]
	Stool	miR-21	Increased	[200]
		miR-181a,b,d	Increased	[197]
		miR-210	Increased	[197, 200]
		miR-216	Decreased	[190, 200]
	Saliva	miR-940	Increased	[193]
		miR-3679	Decreased	[193]
PanIN	Tissue	miR-21	Increased	[86, 88, 90]
		miR-29b	Increased	[86]
		miR-148a	Decreased	[89]
		miR-155	Increased	[88–90]
		miR-182	Increased	[89]
		miR-196a,b	Increased	[66]
		miR-217	Decreased	[89]
		miR-425	Increased	[86]
		miR-708	Increased	[86]
		miR-874	Increased	[86]
	Blood (plasma or serum)	miR-21	Increased	[64, 96, 203]
		miR-196a,b	Increased	[70]
IPMN	Tissue	miR-21	Increased	[63, 86, 92]
		miR-99a,b	Decreased	[98]
		miR-155	Increased	[63, 92]
		miR-196a,b	Increased	[92]
	Blood (plasma or serum)	miR-21	Increased	[96]
		miR-212	Decreased	[206]
	Pancreatic juice	miR-21	Increased	[92]
		miR-92a	Increased	[206]
		miR-99a,b	Decreased	[206]
		miR-100	Decreased	[206]
		miR-125b	Increased	[206]
		miR-145	Increased	[206]
		miR-155	Increased	[92]
		miR-196a,b	Increased	[92]

4.1.1. Diagnostic potential in PDAC

Due to the presence of thick stromal layer and persistent inflammation in patients with chronic pancreatitis (CP) and PDAC, the diagnostic utility of imaging-based approaches has been clinically challenging. In an effort to overcome this issue for improved discrimination between CP and PDAC, several studies have focused on the interrogation of miRNA expression profiles. In one of the first such studies, Bloomston et al. identified a panel of 25 miRNAs that were significantly deregulated in PDAC compared to adjacent healthy tissues [57]. Of these, miR-196a, miR-196b, miR-203, miR-210, miR-217, miR-222,, and miR-375 were dysregulated only in PDAC, whereas miR-29c, miR-96, miR-143, miR-145, miR-148b, and miR-150 were abnormally expressed in both CP and PDAC. These findings suggested that the latter group of miRNAs were likely responsible for causing a desmoplastic reaction, as opposed to tumorigenesis. More specifically, several studies have noted significant downregulation of miR-92, miR-132, miR-148a, miR-216a, and miR-217 in PDAC [56, 58–61]. In contrast, miR-31, miR-143, miR-145, miR-146a, miR-150, miR-155, miR-194, miR-196a, miR-196b, miR-210, miR-222, and miR-223 were observed to be markedly upregulated in PDAC compared to healthy specimens [55–57, 62–67].

These initial studies for the observed deregulation of miRNA expression became the basis for subsequent studies that began evaluating their biomarker potential. For example, Lee et al. identified upregulation of miR-21, miR-301, and miR-376a in PDAC tissues compared to normal pancreatic tissues, indicating their potential to detect patients with PDAC from healthy individuals [55]. Later studies reported elevated expression of miR-1290 in patients with early-stage PDAC patients, which also exhibited significantly superior diagnostic potential compared to the classic tumor marker, CA19-9, in differentiating early-stage PDAC from controls [68]. Significant upregulation of miR-135b was observed in PDAC vs. non-diseased control specimens, and could also distinguish patients with PDAC from those with CP with relatively high sensitivity and specificity [69].

Following the observation of encouraging results in tissues, researchers started to explore the feasibility of translating these miRNA biomarkers in blood (serum or plasma) specimens. In one such effort, the previously identified miRNAs miR-21, miR-155, and miR-196a were also found to be upregulated in sera from patients with PDAC compared with healthy subjects [70]. While individual miRNAs have shown potential to detect PDAC, a combination of circulating miRNAs often results in an increased diagnostic accuracy. For instance, a combination of miR-196a and miR-217 expression levels discriminated PDAC from healthy controls and CP cases [56]. More intriguingly, diagnostic performance of circulating miRNAs together with tumor markers such as CA19-9 has also been analyzed. In this context, plasma levels of miR-16 and miR-196a combined with CA19-9 offered a significantly superior diagnosis of early stage PDAC [70].

As the enthusiasm for miRNA-based biomarkers in PDAC continues to build, various researchers also began to systematically examine the functional significance of specific miRNAs, their downstream target genes (mRNAs), the signaling pathways and cellular processes controlled by individual miRNAs in PDAC (Figure 3). For example, miR-217, which has been found to be significantly downregulated in PDAC tissues and cell lines, was revealed to target KRAS mRNA [71]. Subsequent studies supported this notion and illustrated that miR-217 acts as a tumor suppressor by inhibiting KRAS, thereby reducing the constitutive phosphorylation of the downstream signal transducer AKT and eventually inhibiting cell proliferation. Kent et al. characterized miRNA expression profiles in multiple experimental model systems in which KRAS was constitutively activated due to the presence of a KRAS^G12D mutation [72]. These studies revealed that activated KRAS signaling led to the repression of the miR-143/145 cluster, which is required to maintain the tumorigenic potential of pancreatic cancer cells. Using a dual-reporter luciferase assay, it was demonstrated that downregulation of miR-143/145 was achieved by binding of the KRAS-responsive element-binding protein (RREB1) to the miR-143/145 promoter region. Likewise, miR-375, which is upstream of PI3K/AKT signaling, acts as a tumor suppressor by inhibiting the growth of PDAC cells through AKT signaling pathway [73, 74]. Specific attention has been placed on the role of miR-21 in PDAC, as it is implicated in tumorigenesis, tumor cell invasion, and metastasis of pancreatic tumor cells, as well as desmoplastic reaction and has been consistently observed to be overexpressed in PDAC compared to healthy and/or CP samples [75–78]. Phosphatase and tensin homolog (PTEN), a tumor suppressor gene known to suppress PI3K-AKT-mTOR signaling and control various cellular processes, is targeted by miR-21, miR-221, and miR-181a [79–81]. One of the downregulated miRNAs, miR-216a, targets JAK2 mRNA and inhibits its expression, lead to reduction in the tumor volume in an animal model by increasing tumor cell apoptosis and decreasing cell proliferation [82]. Furthermore, transfection with miR-216a inhibited the growth of pancreatic cells and the transcription of the survivin gene and other apoptotic genes located downstream of the JAK/STAT pathway [83]. miR-155 is also associated with the JAK/STAT pathway, and pancreatic cells with its knockdown resulted in increased expression of suppressor of cytokine signaling 1 (SOCS1) – a tumor suppressor protein that negatively regulates the JAK/STAT3 signaling pathway [84]. Collectively, these data provide a strong evidence supporting the importance of miRNAs as potential biomarkers for the diagnosis of PDAC.

Figure 3:

Mechanistic role of the miRNAs in PDAC: miRNAs can act both as tumor suppressors and oncogenes by regulating different key downstream gene targets that mediate cellular growth signaling pathways. For example, miR-217 which is often found downregulated in PDAC, acts as a tumor suppressor and targets KRAS oncogene which endows proliferation, survival and invasion properties onto cancer cells through the activation of several downstream effector pathways such as the PI3K/AKT and the RAF/ERK pathway. On the other hand, miR-21, which is usually found upregulated in PDAC, targets the tumor suppressor genes PTEN and PDCD4. miR-21 mediated inhibition of PTEN leads into activated downstream signaling of PI3K/AKT pathway. PDCD4, a tumor suppressor gene involved in apoptosis, invasion is inhibited by miR-21 and is potentially involved in PI3K/AKT signaling pathway. miRNAs can also induce transcriptional repression or activation by modulating chromatin structure through the targeting of epigenetic regulatory genes. For example, miR-342-3p can act as a tumor suppressor by inhibiting cancer cell proliferation and invasion through targeting DNMT1.

4.1.2. Diagnostic potential in PanINs

There exist histological and molecular differences between PDAC and its precursor lesions, as well as between different types of precursor lesions – PanINs and IPMNs [85]. Moreover, based on the idea that the transformation of normal pancreatic tissue into an invasive PDAC manifests over several years through the accumulation of a series of genetic and epigenetic changes, miRNA expression profiles have been evaluated in low- and high-grade PanINs to identify biomarkers that can detect the disease in the earliest stages of neoplastic transformation.

In a PanIN progression model based on miRNA expression profiles, 735 human miRNAs were interrogated in PanINs, PDAC, and healthy tissues which led to the identification of specific miRNAs that discriminated PanINs from healthy and PDAC [86]. This report also revealed that the expression profiles of specific miRNAs correlated with different stages of premalignant lesions. Thirteen miRNAs (miR-21, miR-29b, miR-146a, miR-182, miR-193a-3p, miR-193b, miR-200a, miR-200b, miR-200c, miR-425, miR-486-3p, miR-708, and miR-874) were significantly upregulated in PanIN-II & III lesions compared to PanIN-I lesions and normal pancreatic tissues. More specifically, miR-196b was significantly upregulated in PanIN-III compared to normal pancreatic tissues [86]. In a subsequent effort, Slater et al. studied miRNA expression profiles in serum specimens from patients with PDAC and PanINs, as well as healthy controls [66, 87]. Among the five miRNAs evaluated (miR-21, miR-155, miR-196a, miR-196b, and miR-210), only miR-196a and miR-196b showed significantly increased expression in patients with PDAC or PanIN-II & III lesions compared to those with PanIN-I lesions or healthy controls. Of note, a combination of miR-196a and miR-196b expression levels demonstrated a perfect sensitivity and specificity of 100% for discriminating between patients with PanIN-II & III lesions and healthy controls [66, 87]. Ryu et al. compared the relative expression of three candidate miRNAs (miR-21, miR-155, and miR-221, all reported to be overexpressed in PDAC) between PanIN lesions of various histological grades and non-neoplastic pancreatic ductal epithelium [88]. These researcher found that miR-21 was marginally upregulated in PanIN-III, while miR-155 was significantly overexpressed in PanIN-II & III cases compared to PanIN-I or healthy controls; highlighting that miR-155 overexpression is likely an early event in the multi-step progression model of PDAC [88]. Several follow-up studies have since then corroborated the upregulated expression of miR-21 and miR-155 in PanIN lesions [89, 90]. Increased expression of other miRNAs (miR-10b, miR-200, miR-205, miR-221, and miR-222) has also been reported during the progression from PanIN to PDAC [89, 90].

In a detailed functional study, miR-145 expression was shown to decrease with increasing stages of PanINs [91]. While investigating the biological mechanism underlying this phenomenon, it was reported that miR-145 regulated the expression of MUC13 and acted as a tumor suppressor. Ectopic expression of miR-145 in cultured cells and animal models resulted in decreased cellular invasion and reduced tumor growth, respectively, highlighting the role of miR-145 in the development and progression of pancreatic cancer [91]. Likewise, expression of miR-148 was also found to inversely correlate with disease progression, with higher expression in normal tissue compared to PanINs and PDAC [89] – all of which support the role of distinct miRNAs involved in neoplastic disease progression and their biomarker potential.

4.1.3. Diagnostic potential in IPMNs

The first study of miRNA expression profiles in IPMNs was conducted in 2009 [92]. In a small cohort of 15 IPMN tissues, the expression of a panel of 12 miRNAs was measured, which led to the identification that 10 of the 12 miRNAs were significantly overexpressed in IPMNs compared to normal pancreatic tissues. The most promising of these miRNAs were miR-21 and miR-155 – and the elevated expression of these two miRNAs also correlated with greater cellular atypia in the IPMNs [92]. As described earlier, miR-21 has been implicated in repressing the activity of the tumor suppressor genes PTEN and programmed cell death 4 (PDCD4), resulting in the activation of the AKT signaling pathway and increased cellular transformation and metastases, respectively. Similarly, miR-155 was reported to repress a pro-apoptotic gene, tumor protein 53-induced nuclear protein 1 (TP53INP1), and increased tumorigenicity in an animal model [93].

In a larger cohort study comprising of 65 invasive IPMNs, 16 non-invasive IPMNs and 5 normal pancreatic ductal tissues, miR-21 and miR-155 were yet again confirmed to be overexpressed in surgically resected IPMN specimens [63]. Furthermore, the overexpression of these miRNAs was more prominent in invasive vs. non-invasive IPMNs. The same study also reported downregulation of miR-101 in invasive IPMNs [63]. Subsequently, Nakahara et al. confirmed these results and demonstrated that from a mechanistic viewpoint, miR-101 targets oncogenic enhancer of zeste 2 homolog (EZH2). The authors proposed that downregulation of miR-101 leads to the overexpression of EZH2, which could promote the transformation of premalignant IPMNs into PDAC [94].

A subsequent study analyzed pancreatic juice specimens and FFPE tissues for the alterations in expression of miR-196a in specific subtypes (intestinal vs. non-intestinal) of IPMNs. This study reported that miR-196a was specifically upregulated in intestinal-type IPMNs and showed a diagnostic performance, with an area under the curve (AUC) value of 0.85 for the receiver operating characteristic (ROC) curve analysis [95]. The expression of circulating miR-483-3p has also been shown to differentiate IPMNs from PDAC and healthy controls [96]. Combining the expression of miR-483-3p with miR-21 further increased the diagnostic potential for discriminating patients with IPMNs from those with PDAC and healthy controls, and was comparable to the traditional markers CA19-9 and CEA [96]. The miR-483-3p is located within intron 2 of the IGF2 locus and its overexpression has been reported to suppress the expression of the transcription factor DPC4/Smad4 in PDAC tissues [97]. DPC4/Smad4 act as mediators of the TGF-β signaling pathway and are involved in TGF-β-induced epithelial-mesenchymal transition (EMT) [97].

In a genome-wide miRNA expression profiling effort, miR-99a, miR-99b, miR-100, miR-126, miR-130a, and miR-342-3p were found to be under expressed in high-risk vs. low-risk IPMNs [98]. In addition, low levels of miR-99b in cystic fluid from patients with IPMNs were associated with main duct involvement and hence associated with an increased risk for developing into malignant neoplasms. The authors also evaluated expression of the downstream target mRNAs of these downregulated miRNAs and found that some of them, such as IRS-1 (miR-126 target), ATG2B and MEOX2 (miR-130a targets), and DNMT1 (miR-342-3p target), were upregulated in high-risk vs. low-risk IPMNs [98]. More recently, miRNA expression profiles were shown to differentiate pancreatic cystic neoplasms, wherein miR-31-5p, miR-99a-5p, miR-375, and miR-4830-5p were characteristic of serous cyst adenomas (SCAs) and distinguished SCAs from MD- and BD-IPMNs [99].

Taken together, these studies underscore the importance of miRNAs for their clinical significance as diagnostic biomarkers for differentiating patients with IPMNs from healthy controls, as well for discriminating between various subtypes of pancreatic lesions – thereby potentially improving the diagnosis and management of these patients.

4.2. Prognostic significance of miRNAs in PDAC

In addition to early detection, identifying prognostic biomarkers that offer improved prediction of patient survival are also of critical importance. Several studies have found associations between miRNA expression and patient outcomes in PDAC [100]. Not only higher expression of miR-21 was observed in PDAC compared to healthy tissues, it was also a superior predictor of poorer outcomes in patients with PDAC [76, 101]. High expression of miR-21 also led to reduced gemcitabine sensitivity and apoptosis in PDAC cells [77]. Further downstream analysis revealed that overexpression of miR-21 potentially downregulated expression of PTEN and activated the PI3K/AKT signaling pathway.

It was reported that expression of a panel of six miRNAs (miR-30a-3p, miR-105, miR-127, miR-187, miR-452, and miR-518a-2) was significantly associated with improved prognosis in patients with PDAC [57]. In a study of 225 PDAC patients, high expression of miR-212 and miR-675 and low expression of miR-148a, miR-187, and let-7g were independent predictors of poor prognosis in PDAC [102]. Recently, Liang et al. conducted a TCGA database study to evaluate the predictive value of several miRNAs [103]. These researchers reported that a 5-miRNA signature consisting of miR-125a, miR-328, miR-376b, miR-376c, and miR-1301 had the highest prognostic potential, with a corresponding hazard ratio (HR) of 0.139 (95% CI, 0.043–0.443; P<0.001). To better understand the underlying molecular mechanisms of these five miRNAs, the authors performed gene set enrichment and gene ontology analyses. They discovered that the target genes of the candidate miRNAs were involved in a variety of critical biological processes including developmental process, cell differentiation and anatomical structure morphogenesis [103]. Finally, miR-15a miR-155, miR-200c, miR-203, miR-210, miR-222, miR-302, and miR-506 were also shown to correlate with the prognosis in patients with PDAC [104–107]; emphasizing the clinical significance of miRNAs as prognostic biomarkers in PDAC.

5. LncRNAs as Biomarkers in PDAC

The lncRNAs are >200 nucleotides long and are transcribed by RNA polymerase II. The lncRNAs play important roles in an array of diverse biological, developmental, and pathological processes, as they are involved in RNA regulatory mechanisms and control the expression of their downstream target genes. As illustrated in Figure 4, in addition, they mediate a diverse array of cellular processes through chromatin reprogramming, cis or trans regulation at neighboring genes, and post-transcriptional regulation of mRNA processing [108–110]. Dysregulation of lncRNAs has been implicated in several human diseases including cancer [111–113]. As shown in Table 3, increasing evidence supports that aberrant expression of lncRNAs plays an oncogenic or tumor-suppressive role in various cancers including PDAC [114–120]. As a result, there is great degree of interest in the identification of lncRNAs that can be developed for their clinical application as cancer biomarkers. Additionally, the lncRNAs are highly tissue- and disease-specific, which makes them attractive candidates for development as disease biomarkers.

Figure 4:

Functional roles of lncRNAs and circRNAs in PDAC: The lncRNAs and circRNAs implicated in PDAC are shown. The left half of the circle shows the lncRNAs and their downstream targets, cellular phenotype and target genes or pathways affected. For example, HOTAIR interacts with EZH2 and binds to the promoter region of miR-34a. This binding leads to chromatin modification due to hypermethylation of miR-34a promoter repressing its expression. The lncRNA ENST00000480739 acts as an enhancer for OS9, increasing the expression of OS9. OS9 potentially binds to the ubiquitination complex comprised of prolyl hydroxylase (PHD) and other proteins which lead to the degradation of HIF-1α protein changing the hypoxic condition in the cells. The lncRNA PVT1 interacts with the SMAD2/3 proteins of the TGF-β/SMAD pathway, activating the expression of vimentin and inducing EMT changes in pancreatic cancer cells. The lncRNA MALAT1 interacts with EZH2 which in turn leads to the hypermethylation of E-cadherin promoter inhibiting its expression and increasing invasive potential of pancreatic cancer cells. The lncRNA HOTTIP regulate the expression of HOXA9, activating the Wnt-β-catenin pathway and increasing cancer stem cell properties of the pancreatic cancer cells. CircRNAs and their target miRNAs are shown in the right half of the circle. For example, circLDLRAD3 sequester miR-137-3p. In the absence of miR-137-3p, pleiotrophin (PTN) is expressed and leads to increased proliferation and invasion of pancreatic cancer cells. Circ0030235 inhibits the expression of miR-1253 and miR-1294 and leads to increased cell progression. Circ0007534 acts as a sponge for miR-625 and miR-892b and affects apoptosis. Circ-PDE8A targets miR-338 activating the MET/AKT pathway and increase invasive potential of pancreatic cancer cells. Circ-IARS increases the expression of RhoA by inhibiting miR-122 increasing the permeability and invasive potential of pancreatic cancer cells.

Table 3.

A list of candidates lncRNAs and circRNAs implicated in PDAC.

LncRNA	Expression	Targets	Pathway	Effect on biological process	References
HOTAIR	Upregulated	GDF15 NOTCH 3 DR5 miR-34	WNT	Increased proliferation Increased invasion Decreased apoptosis	[126, 127]
HOTTIP	Upregulated	PD-L1 HOXA13	WNT/ β-Catenin	Increased H3K27 trimethylation Increased cancer stem cell proliferation Increased cell survival Increased migration	[140–142]
MALAT-1	Upregulated	E-cadherin promoters NORG-1 CCND, MAPK8 VEGFA	Hippo-YAP PI3K-AKT NF-κb mTOR MAPK WNT	Increased growth  increased invasion Increased EMT	[135, 139]
ENST00000480739	Down regulated	HIF-1α  OS-9	HIF	Increased invasion	[129]
PVT1	Upregulated	MYC promoters	TGF- β/Smad	Increased cytoprotective autophagy Increased EMT	[126]
CircRNA	Expression	Target miRNA	Pathway	Effect on biological process	References
circ-LDLRAD3	Upregulated	miR-137-3p	PTN	Increased invasion Increased metastasis	[156, 158]
circ_0030235	Upregulated	miR-1253 miR-1294	-	Increased invasion Increased migration	[159]
circ_0007534	Upregulated	miR-625 and miR-892b	-	Increased proliferation Increased invasion  Decreased apoptosis	[162]
circ-PDE8A	Upregulated	miR-338	MACC/MET/ERK	Increased invasion	[163]
circ-IARS	Upregulated	miR-122	-	Increased metastasis	[164]
ciRS-7	Upregulated	miR-7	EGFR/STAT3	Increased proliferation Increased metastasis	[157]

5.1. HOTAIR

The HOX antisense transcript intergenic RNA (HOTAIR) is a well-characterized lncRNA whose aberrant expression has been documented in several cancer types. It is transcribed from the homeobox C (HOXC) locus located on chromosome 12 [121, 122]. In PDAC, HOTAIR promotes cellular proliferation and metastasis [123]. HOTAIR interacts with human EZH2, a component of the polycomb repressive complex 2 (PRC2) and binds to the promoter region of miR-34. This binding leads to histone 3 lysine 27 (H3K27) trimethylation and eventually to the transcriptional repression of miR-34, an increase in cellular proliferation, and a decrease in apoptosis [123]. Kim et al. reported that HOTAIR targeted and bound the tumor suppressor gene GDF15, repressing its expression, which led to increased proliferation of pancreatic cancer cells [124]. These researchers also used a publicly-available database to illustrate that HOTAIR was overexpressed in PDAC specimens compared to normal pancreatic tissues, and in more aggressive tumors characterized by higher tumor and lymph node staging [124].

In a large study involving 900 PDAC and equal number of control specimens, a functional single nucleotide polymorphism (SNP) located within the 3’ UTR region of the HOTAIR gene was associated with a significantly higher risk for developing pancreatic cancer and higher HOTAIR expression [125]. Xie and colleagues noted that expression of HOTAIR was significantly higher in pancreatic tumor tissues [126], subsequently they validated their findings in salivary samples and noted that HOTAIR expression was also significantly higher in saliva from patients with PDAC vs. patients with benign tumors [126]. In another study, HOTAIR expression exhibited impressive diagnostic potential which was superior than the conventional CA19-9 marker for discriminating PDAC from benign pancreatic lesions and healthy controls. Increased levels of HOTAIR in serum also correlated with advanced PDAC stages [127]. HOTAIR was also shown to regulate the expression of the death receptor 5 (DR5) gene through epigenetic modulation and to contribute to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance – suggesting it to serve as a potential target for sensitization of pancreatic cancer cells resistant to TRAIL therapy [128].

5.2. lncRNA ENST00000480739

The lncRNA ENST00000480739 functions as a tumor suppressor. In a study comprising of 35 patients with PDAC, expression of ENST00000480739 was lower in PDAC specimens compared to the adjacent normal tissues [129]. In addition, its expression negatively correlated with tumor stage and lymph node metastasis status. The authors demonstrated that in cultured cells, ENST00000480739 directly increased the expression of the osteosarcoma amplified 9 (OS-9) gene, both at the mRNA and protein level by activating its promoter region. Further knockdown experiments demonstrated that lncRNA ENST00000480739 negatively regulated HIF-1α by upregulating OS-9, thereby leading to a reduction in PDAC cell invasion [129].

5.3. PVT1

The lncRNA plasmacytoma variant translocation 1 (PVT1) is located at the 8q24.21 locus, and has been shown to interact with MYC gene promoter [130]. Several functions of PVT1 have been reported in pancreatic cancer cells, including promotion of EMT via the TGF- β/Smad pathway [131], acting as a miR-448 sponge to promote proliferation and migration [132], and modulating cytoprotective autophagy to promote PDAC development [133]. Xie et al. reported that PVT1 expression was higher in PDAC tissue and serum specimens compared to healthy specimens, and could serve as a diagnostic biomarker in this malignancy [126]. In comparison to the tumor marker CA19-9, PVT1 expression demonstrated higher sensitivity and specificity. In addition, the progression-free survival of patients with locally advanced or advanced PDAC treated with gemcitabine as first-line treatment was significantly higher in patients with low expression of PVT1; highlighting its potential as a prognostic biomarker in patients with PDAC treated with first-line gemcitabine-based treatment [134].

5.4. MALAT-1

The metastasis-associated lung adenocarcinoma transcript-1 (MALAT-1) is another lncRNA that is frequently overexpressed in PDAC. Expression of MALAT-1 also positively correlates with tumor size, clinical stage, lymph node metastasis, distant metastasis, and prognosis [135]. A negative correlation was observed in PDAC patients with higher MALAT-1 expression levels and disease-free survival [136]. Various transcriptional targets of MALAT-1 have been identified, including the promoter of E-cadherin [137], N-myc downregulated gene-1 (NDRG-1) [138], cyclin D1 (CCND1), RAF-mitogen-activated kinase 8 (MAPK8), and vascular endothelial growth factor A (VEGFA) [139]. Through RNA immunoprecipitation assays, MALAT1 was demonstrated to physically bind to EZH2 and negatively correlate with E-cadherin expression. Further investigations revealed that silencing of EZH2 increased E-cadherin expression in pancreatic cancer cells suggesting that MALAT1 potentially promotes pancreatic cancer cell migration and invasion through the repression of tumor suppressor gene E-cadherin [137]. Altogether, the findings suggest that MALAT1 plays an important role in PDAC pathogenesis, warranting further investigation.

5.5. HOTTIP

The HOXA distal transcript antisense RNA (HOTTIP) lncRNA is a HOX-related lncRNA. Similar to HOTAIR, the HOTTIP lncRNA is associated with chromatin-modification complexes and enhances H3K27 trimethylation to repress the expression of multiple HOXA genes [140]. In the context of PDAC, the HOTTIP lncRNA seems to regulate HOX genes i.e. HOXA9, HOXA10 and HOXA11. The HOXA9 gene has been shown to promote cancer stem cell proliferation through the Wnt/β-catenin signaling pathway [141, 142]. Likewise, the HOXA10 and HOXA11 genes regulate expression of matrix metalloproteinase 3 and 2 genes which promote invasion in pancreatic cancer cells [142]. Increased expression of HOTTIP has been documented in various pancreatic cancer cell lines as well as tissue specimens [143]. The same study also demonstrated that downregulation of HOTTIP expression increased the anti-tumor effects of gemcitabine in cultured cells and animal models.

6. CircRNAs as Biomarkers in PDAC

CircRNAs are another class of ncRNAs that are currently a popular research topic. circRNAs are circular in shape and lack a 5’ cap or 3’ Poly-A tail terminal ends. These ncRNAs are more stable than other endogenous mRNAs due to their circular shape, which protects them from traditional RNA-mediated degradation [144]. circRNAs have been proposed to function as miRNA sponges that suppress the ability of miRNAs to bind to their target mRNAs [145,146]. The role of miRNAs in various cellular processes and diseases are well established. Considering that circRNAs interact with miRNAs, their association is also implicated in various diseases, including cancer [147–151]. The role of circRNAs in cancer and the regulation of key cellular processes by circRNAs are reviewed elsewhere [152].

A number of studies have reported aberrant expression of circRNAs in PDAC (Table 3). Li et al. performed one of the very first studies to investigate the expression of circRNAs in PDAC, using a microarray expression approach in six pairs of PDAC samples and matched adjacent normal tissues [153]. They identified a number of up- and downregulated circRNAs between PDAC and adjacent normal tissues, among which they further validated seven circRNAs using quantitative real-time PCR assays [153]. A subsequent study identified 115 upregulated and 141 downregulated circRNAs in PDAC compared to adjacent normal mucosa specimens, through the analysis of publicly-available microarray datasets [154]. These authors also considered potential circRNA:miRNA interactions and performed pathway analysis to discover that the B-Raf proto-oncogene, serine/threonine kinase (BRAF) and Dual specificity mitogen-activated protein kinase kinase 2 (MAP2K2) interacted with the most number of pathways, indicating the involvement of MAPK signaling pathway. Li et al. investigated circRNA expression in extracellular vesicles derived from the plasma of patients with PDAC and uncovered 453 circRNAs that were differentially expressed between patients and healthy controls [155]. As illustrated in Figure 4, several studies have also shown the aberrant expression of circRNAs in PDAC and their association with pancreatic cancer cell proliferation and metastasis [156, 157].

In view of their stable expression, circRNAs represent potentially viable biomarkers for PDAC. Yang et al. found circ-LDLRAD3 was upregulated in PDAC tissues, plasma, and PDAC cell lines. They also reported a positive correlation between the expression of circ-LDLRAD3 and clinicopathological factors, such as venous and lymphatic invasion, in the patient specimens [158]. Moreover, to investigate the suitability of circ-LDLRAD3 as a PDAC diagnostic biomarker, the authors also found that when combined with CA19-9, circ-LDLRAD3 exhibited superior diagnostic sensitivity and specificity vs. CA19-9 [158]. The circ-LDLRAD3 was shown to directly target miR-137-3p and to regulate proliferation, migration, and invasion in pancreatic cancer cells through the miR-137-3p/pleiotrophin (PTN) axis [156].

Likewise, the circ_0030235 is another circRNA overexpressed in PDAC tissues and cell lines [153, 159]. Functional studies revealed that overexpression of circ_0030235 associated with advanced tumor stage and positive lymph node metastasis in patients with PDAC, suggesting that it may function as an oncogene. Additionally, circ_0030235 is thought to act as a molecular sponge for miR-1253 and miR-1294, as the two miRNAs were found to be significantly downregulated in its presence [159]. These two miRNAs have been shown to act as tumor suppressors in several other cancers as well. The miR-1253 inhibits cell growth and invasion by targeting WNT5A [160] while miR-1294 has been shown to inhibit cell proliferation by targeting c-Myc [161]. In a similar fashion, circ_0007534 was found to be upregulated in PDAC tissues and correlated with aggressive phenotypes [162]. Furthermore, circ_0007534 was shown to promote the oncogenic potential of cells by enhancing cell proliferation, migration, and invasion in PDAC cell lines; and miR-625 and miR-892b were identified as the targets of circ_0007534 [162].

The stable nature of circRNAs in serum provides a rationale for investigating circulating circRNAs as cancer biomarkers [163, 164]. In this context, circ-PDE8A was identified in exosomes derived from the plasma of patients with PDAC, and it was shown that its high expression associated with disease progression and poor prognosis. Further mechanistic investigations revealed that circ-PDE8A could activate the MET tyrosine kinase receptor and act as a sponge for miR-338 [163]. The circ-IARS was also found to be highly expressed in the exosomes of patients with PDAC and positively correlated with tumor and lymph node metastasis status [164]. Functional assays revealed that circ-IARS increases the activity of RhoA by absorbing miR-122, resulting in an increased permeability of the cells promoting metastasis [164].

Another circRNA that is implicated in PDAC is ciRS-7. Previously, ciRS-7 was shown to act as a miR-7 sponge in colorectal and hepatocellular cancer [165, 166]. In pancreatic cancer, ciRS-7 was found to be upregulated in PDAC tissues as compared to normal tissues. Using pancreatic cancer cells, ciRS-7 was shown to act as a miR-7 sponge, and ciRS-7 knockdown led to a decrease in EGFR and STAT3 expression suppressing proliferation and reducing invasion of PDAC cells [157].

Although only a limited number of studies have investigated the role of circRNAs in PDAC to date, these studies suggest that aberrantly expressed circRNAs might be involved in the regulation of PDAC and could potentially serve as diagnostic makers.

7. Conclusion and Future Perspectives

The only chance of a complete cure in PDAC is highly dependent on early disease diagnosis, upon which curative treatments can be implemented. Thus, there remains an unmet clinical need for the identification and development of highly sensitive and specific diagnostic biomarkers that can detect PDAC and its precursor lesions at their earliest stage. Given that the effective treatment options in PDAC patients are still limited, availability of improved prognostic and predictive biomarkers are also desirable in PDAC – which can help adequate risk-stratification and selection of patients for specific treatment regimens. Various studies have revealed that ncRNAs are important regulators of a multitude of cancer-associated mechanisms in PDAC, including cellular proliferation, invasion and apoptotic deregulation – making them attractive candidates for diagnostic, predictive, and prognostic biomarkers.

In spite of the substantial enthusiasm for miRNAs as robust biomarkers in PDAC, to date, not a single miRNA biomarker has made it to the clinic for the diagnosis of patients with PDAC. This can be attributed to various limitations of the existing studies, including lack of a systematic biomarker discovery and validation approach, analysis of well-defined clinical cohorts with adequate statistical considerations, and the use of inconsistent analytical and biomarker normalization approaches. In the case of cell-free miRNAs, their heterogenous origin is also a potential limiting factor. With regards to lncRNAs, while the data to date seems promising, further studies are needed to systematically dissect the full potential of lncRNAs as biomarkers in PDAC. Finally, circRNAs are an exciting and emerging concept; however, the research in this regard is still in its infancy. As it stands currently, more sophisticated bioinformatic approaches are needed to identify the entire compendium of circRNAs in PDAC, followed by their functional characterization – which eventually will provide the springboard for their development into diagnostic, prognostic, and predictive biomarkers in PDAC.

Taken together, the expression profiles of various ncRNAs offer an exciting opportunity for development into biomarkers for PDAC. However, before we get to that point, substantial challenges must be overcome, including careful planning of large-scale prospective human studies to validate the clinical significance of most promising PDAC-associated ncRNA biomarkers. In addition, concurrent functional studies to unravel the functional underpinnings for their mechanistic role will lend further credence to their biomarker potential; which collectively will provide the much needed rationale and confidence for their translation into the clinic as we usher into the exciting era of precision oncology.