Research progress on microRNAs as molecular markers in pancreatic cancer: a narrative review.

Background and Objective: The incidence and mortality of pancreatic cancer (PC) have increased in recent years. The current status of PC diagnosis and treatment remains grim in clinical practice because the commonly used early screening tools are not sufficient. Improving the early detection of PC and strengthening standardized comprehensive treatment remain the focus of PC research. Many studies have shown that micro RNAs (miRNAs) play an important role in the occurrence, development, and treatment of PC. It is expected that miRNAs will become new molecular markers of PC.
Methods: We extracted and compiled useful information from the PubMed database that met our criteria for analyzing PC diagnosis, treatment, and prognosis. Key Content and Findings: In this narrative review, we summarize the mechanism of some miRNAs in the occurrence and development of PC and review them as potential markers for the diagnosis, treatment, and prognosis of PC. The function of miRNAs in PC has great potential in studying the pathogenesis of PC. The discovery of many important oncogenic miRNAs and their downstream targets will bring new ideas and research paths for the diagnosis and targeted therapy of PC. Conclusions: MiRNAs are expected to provide novel ideas and research directions for the diagnosis and targeted treatment of PC. However, more patient data and clinical trials are needed before miRNAs can become novel molecular markers for PC. 2022 Journal of Gastrointestinal Oncology. All rights reserved.

Introduction

Pancreatic cancer (PC) has a high mortality rate (1). Despite its relatively low incidence (it is the 10th most common cancer among all cancers), it is the 4th most common cause of cancer-related death in most developed countries (1,2). Statistics from the China National Cancer Center have confirmed that PC is the 8th most common malignant tumor in Chinese men and the 6th leading cause of death from malignant tumors among people living in large cities in China (3,4). The clinical features of PC are not significant. PC has many symptoms, including jaundice, back/abdominal pain, loss of appetite, and weight loss (5). Additionally, PC is extremely aggressive, metastatic, and chemotherapy-resistant (6). Even at an early stage, local invasion and distant metastasis are often clinically observed. Surgery, radiation, chemotherapy, and some marginally effective targeted therapies are among the conventional treatment choices for people with PC.

Since the incidence and mortality of PC have increased in recent years, a better understanding of PC is urgently required. A greater knowledge of the disease’s pathophysiology will allow for the earlier identification and development of viable therapies. Micro RNAs (miRNAs) are endogenous short non-coding RNAs that control gene expression and play an important role in the onset, development, and therapy of different malignancies. Different levels of miRNAs expression can be detected in the blood of patients. A study has confirmed that abnormal expression of some miRNAs is not only related to the type of cancer, but also to its stage of development (7). These findings enable miRNAs to become non-invasive molecular biomarkers for PC detection, prediction and monitoring of therapeutic effect. Based on the structure and molecular characteristics of miRNAs, combined with the regulatory mechanism of miRNAs in PC, this paper describes the role of miRNAs in the pathogenesis, development, diagnosis, treatment and prognosis of PC. We believe that miRNAs are expected to be used in the diagnosis, prognosis and targeted therapy of PC through the analysis of miRNA differentiation and expression profile. We present the following article in accordance with the Narrative Review reporting checklist (available at https://jgo.amegroups.com/article/view/10.21037/jgo-22-577/rc).

Methods

We searched the PubMed database for literature on the miRNAs and molecular markers of PC published in the past 10 years, and selected classified, and summarized the relevant literature after discussions among the authors. The specific search strategy is listed in .

Table 1

The search strategy summary

Items	Specification
Date of search	January 1st, 2022
Databases and other sources searched	PubMed database
Search terms used	“Pancreatic cancer”; “MicroRNA”; “Molecular markers”; “Diagnosis”; “Treatment”; “Prognosis”
Timeframe	December 1, 1997 to September 26, 2021
Inclusion and exclusion criteria	Inclusion criteria—Study type: basic and clinical medical research. The articles were limited to full-text publications in English. No specific exclusion criteria
Selection process	The articles were independently selected by the first authors, Rui-Biao Fu and Xi Chen, and included in the review after agreement was reached following several consultations and discussions with the co-authors
Any additional considerations, if applicable	N/A

MiRNAs

In recent years, many studies have shown that miRNAs can be used as potential biomarkers of PC (7-9). MiRNAs are a class of non-coding single-stranded RNA molecules encoded by endogenous genes, about 22 nucleotides in length, which participate in the post-transcriptional regulation of gene expression and regulate cell growth, proliferation, differentiation, apoptosis and other important biological functions (4). In an organism, miRNAs are synthesized by miRNA genes through a complex process (see at ). The targeting characteristics of miRNAs are classified into groups depending on the identity of their extended seed region (miRNA nucleotides 2–8) (11). MiRNAs are a kind of non-coding RNA that control gene expression by degrading mRNA or inhibiting translation (12). MiRNAs play 2 major roles in carcinogenesis, and have inhibition and promotion effects (13). Normally, these 2 effects are in a state of balance; However, when this balance gets out of control, unregulated miRNAs (i.e., excessively upregulated or downregulated miRNAs) cause the body to lose control over the formation of specific tumors. This loss of control usually refers to the activation of oncogenes and the disorder of apoptosis (14).

Figure 1

The process of miRNA biogenesis. First, pri-miRNA is synthesized in the nucleus by the action of RNA polymerase II using miRNA genes as templates. Pri-miRNA is cleaved into miRNA double strands by Drosha RNase III and Dicer RNase III. Next, the RNA-induced silencing complex incorporates activated miRNA. After the above transcription and processing in the nucleus, the double-stranded miRNA moves to the cytoplasm and dissociates into mature single-stranded miRNA. Mature miRNAs play a role in mediating the silencing of target gene protein expression by binding to RNA inducers, thus controlling cell growth, development and apoptosis (10). miRNA, microRNA; TRBP, HIV-1 Tar RNA binding protein; Dicer, Dicer RNase III; RISC, RNA-induced silencing complex; FMRP, Fragile X mental retardation protein.

MiRNAs are widespread and stable in biological body fluids, so they are ideal molecular markers for the rapid, non-invasive, and inexpensive detection and diagnosis of diseases. A study has shown that some PC-related miRNAs present stably in tissues, blood, and feces, and can be quantified in very small sample sizes (15). PC tissues from various sources, such as fine-needle biopsies, fresh frozen tissues, and formalin-fixed paraffin-embedded tissues, are now being employed for miRNA mapping research. For example, a miRNA microarray examination of PC tissue collected through fine-needle aspiration biopsy revealed that 158 miRNAs were improperly expressed in PC tissue as compared to healthy pancreatic tissue. The researchers chose 8 miRNAs for further analysis, 5 of which were highly expressed (miR-21, miR-27a, miR-146a, miR-200a, and miR-196a), and 3 of which were found to be lowly expressed (miR-217, miR-20a, and miR-96) in PC tissues (16).

The findings on the miRNA mapping of PC tissues confirmed the importance of miRNAs in the development of PC. However, due to its invasive nature and high costs, fine-needle biopsy is a major impediment to the use of miRNAs as markers in the detection of PC. If aberrantly expressed miRNAs can be detected in the peripheral blood of patients with PC, miRNAs will have a better chance of becoming PC biomarkers. A study showed that serum miRNA can be used to distinguish PC patients from chronic pancreatitis patients and healthy individuals (17). Similarly, Schultz et al. used whole blood RNA samples in their study and found that 2 diagnostic panels of miRNAs had the potential to differentiate between PC patients and healthy controls (18). Hussein et al. detected the expression of miR-22-3p, miR-642B-3p, and miR-885-5p in the plasma of patients with PC and found that the levels of 3 kinds of miRNA in plasma were significantly higher than those in healthy controls, indicating that these related miRNAs in plasma may be used as potential biomarkers in the diagnosis of PC (19). These experiments revealed the possibility of using peripheral blood miRNAs as molecular markers of PC.

Pathogenesis of miRNAs

Research has shown that cytokines and core signaling pathways are primarily involved in miRNA-mediated control (see ). Patients with PC had considerably higher levels of MiR-21 in their serum compared with healthy people (29). Abue et al. used quantitative real-time polymerase chain reaction (RT-PCR) to detect the expression of miR-21 in the plasma samples of 32 patients with pancreatic ductal adenocarcinoma (PDAC), 12 patients with intraductal papillary mucinous tumors, and 30 healthy controls, and found that the plasma expression levels of miR-21 of these patients were substantially higher than that of healthy controls, and that miR-21 expression was also related to staging, lymph node metastases, and a short survival time (29). Dong et al. studied the mechanism by which miR-21 affects the occurrence and development of PC. Specifically, Dong et al. selected PC cell line MIAPaca-2 as the research object to explore the mechanism of miR-21 on the crucial modulator of apoptosis, Bcl-2 factor, which mediates apoptosis. The Bcl-2 protein is the coding product of proto-oncogene Bcl-2, which can prevent the release of cytochrome C from mitochondria to the cytoplasm and thus inhibit cell apoptosis. Dong et al. found that miR-21 promotes the expression of Bcl-2 protein by binding to the 2 prime untranslated regions of Bcl-2mRNA, which inhibits the apoptosis of MIAPaca-2 PC cells (30). Research has also shown that the inhibition of miR-21 in PC cells decreases the proliferation rate of cancer cells, but increases apoptosis, and that the expression of the tumor suppressor genes programmed cell death 4 (PDCD4) and Phosphatase and tensin homolog (PTEN) is upregulated in PC patients, which suggests that both may be targets of miR-21 (18,19).

Table 2

A summary of the significant upregulated and downregulated miRNAs, and their relative roles in PDAC carcinogenesis

miRNA	Express status	Target genes/signaling pathways	Role in PC	Reference
miR-573	Downregulated	TSPAN1	Inhibition of PC cell proliferation, colony formation, migration, and invasion, as well as tumor growth suppression in vivo	(8)
miR-139	Downregulated	RalB	Suppression of PC cell growth and metastasis	(20)
miR-122-5p	Downregulated	CCNG1	Proliferation, migration, and invasion are decreased in vitro, and tumorigenesis is prevented in vivo	(21)
miR-505	Downregulated	HK2	Inhibition of cell proliferation, invasion, sphere formation, glucose consumption, and lactate production	(22)
miR-125b	Upregulated	Txnip	Promotion of proliferation and migration	(23)
miR-132-3p	Upregulated	Rb1	Promotion of PC cell growth	(24)
miR-543	Upregulated	STK31	Promotion of PC cell growth	(25)
miR-361-3p	Upregulated	B-cell Translocation Gene 2	Inhibition of cell proliferation, migration, and invasion, and stimulation of cell cycle arrest and apoptosis	(26)
miR-217	Upregulated	KRAS	The overexpression of miR-217 in a pancreatic tumor cell line lowers KRAS levels, inhibits cell proliferation, and diminishes the constitutive phosphorylation of the downstream signal transducer protein kinase B	(27)
miR-27a	Upregulated	Sprouty2	Promotion of PC cell growth	(28)

miRNA, micro RNA; PDAC, pancreatic ductal adenocarcinoma; PC, pancreatic cancer; KRAS, kirsten rat sarcoma viral oncogene.

Zhao et al. studied the biological activity of serum miR-192a in patients with PADC and evaluated its potential as a biomarker. They found 16 upregulated miRNAs, including miR-192, and 8 downregulated miRNAs in the miRNA expression profiles of human PDAC and neighboring normal pancreatic tissues. According to RT-PCR, the level of serum miR-192 in patients with PDAC was higher than that in PC patients and healthy controls. A further analysis showed that the sensitivity of serum miR-192 in detecting PDAC was 76% and the specificity was 55% (31). In PANC-1 PC cells, the ectopic expression of miR-192 can accelerate cell proliferation and migration, inhibit apoptosis, and shift the cell cycle from the G0/G1 to S phase. A western blot analysis showed that the forced expression of miR-192 in PANC-1 cells lowers the expression of Smad interacting protein 1 (SIP1) and modifies the expression of a collection of cell cycle associated genes (31). Thus, miR-192 overexpression aids in the growth and progression of PC.

The expression of miR-155 is upregulated in the blood of patients with PC, making it a potential marker of PC (32). Liu et al. studied the inhibitory effect of miR-155 on the tumor suppressor gene SEL1L (33). The messenger RNA (mRNA) of the SEL1L gene is highly expressed in healthy adult pancreatic tissues (34), and the gene has been shown to have a tumor inhibitory effect (35,36). The expression level of SEL1L mRNA was found to be negatively correlated with the expression of hsa-mir-143, hsa-mir-155 and hsa-mir-223. And after further analysis, it was concluded that hsa-mir-155 inhibited SEL1L in PDAC cells (33). Another study found that miR-155 expression was upregulated in PC tissues, while cytokine signal transduction inhibitor 1 (SOCS1) expression was lower in PC tissues and higher in paracrine tissues. Further, miR-155 expression levels are related to lymph node metastasis and clinical stage, which suggests that miR-155 may regulate PC cell invasion and migration by targeting the SOCS1 signal pathway (36).

MiRNAs as diagnostic markers

Currently radical resection is the only possible cure for PC, and early operation provides patients with a better chance of cure. However, due to the low rate of early diagnosis of PC, only a very small number of patients obtain radical resection at the early stage. The key to changing this situation lies in the use of more sensitive molecular markers of PC and in finding therapeutic approaches other than surgery. As a result, laboratories around the world are searching for molecular markers that can detect PC early and enable a larger proportion of patients to be treated.

According to a study, using miR-27a-3p expression levels in peripheral blood monocytes (PBMCs) to differentiate PC from benign pancreatic/peripancreatic diseases (BPD had a sensitivity of 82.2% and a specificity of 76.7%, while the combined diagnosis of PBMC miR-27a-3p and serum CA19-9 levels is more accurate, with a sensitivity of 85.3% and a specificity of 81.6% (37). Hussein et al. showed that the abnormal expression of plasma miR-22-3p, miR-642b-3p and miR-885-5p easily distinguished PC patients from healthy controls (19). Slater et al. examined the role of miRNA in screening individuals at risk for familial pancreatitis. Specifically, they analyzed miRNA-21,-155,196a, 196b, and 210 in the serum of transgenic LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mice to determine whether they could differentiate between different grades of pancreatic intraepithelial neoplasia (mPanIN1-3) or PC, and concluded that the combination of miR-196a and 196b might be a potential biomarker for identifying people at risk of familial pancreatitis (38). Zou et al. discovered that mir-192-5p, mir-19a-3p, mir-19b-3p, mir-223-3p, and mir-25-3p were significantly upregulated in serum PC samples, and suggested that they might be used for the early non-invasive detection of PC (39).

MiRNAs as therapeutic markers

In addition to being potential PC biomarkers, miRNAs have shown potential in PC therapy (28,37). Studies have shown the effectiveness of miRNAs in PC treatment. For example, a study showed that the abnormal expression of miRNA is closely related to the occurrence and development of PC. As a result, the reversal of aberrant miRNA expression might be a viable technique for PC prevention and treatment (40). MiRNA-21, a miRNA that has one of the greatest effects on pancreatic ductal carcinoma, has been shown to be significantly increased in patients with PC (41). Zhao et al. found that both miRNA-21 and miRNA-221 upregulate and participate in the proliferation, apoptosis, and chemotherapy resistance of PC in small groups of cells with stem cell-like characteristics, and the combination of antisense oligodeoxynucleotides targeting miRNA-21 and miRNA-221 significantly inhibits the growth and metastasis of primary tumors (42). In another study on miRNA-21, researchers used an animal model of pancreatic ductal carcinoma for the non-invasive tracking of tumor growth. Ultimately, they found that invasive tumors within the animal model stopped growing after miRNA-21 expression in tumor cells was suppressed. Thus, they suggested that the therapeutic administration of miRNA-21 antagonists would be beneficial for PC treatment (43). Nalls et al. discovered that the re-expression of miR-34a in human PC stem cells and human PC cell lines after treatment with the demethylation drug 5-aza-29-deoxcytidine (5-aza-DC) and the histone deacetylase inhibitor Vorinostat (SAHA) strongly inhibited cell proliferation, cell cycle progression, self-renewal, epithelial-to-mesenchymal transformation, and invasion (44). Passadouro et al. developed a novel treatment method that employed anti-miRNA oligonucleotides (AMOs) against overexpressed miRNAs in PDAC, mediated by a lipid-based nano system, with considerable anti-cancer effectiveness (45). Further, when this anti-cancer gene therapy technique that employed AMOs was combined with modest doses of chemotherapeutic drugs, it had a synergistic and substantial antitumor effect in PDAC cell lines (46).

MiRNAs as prognostic markers

Some miRNAs have been shown to be associated with the aggressiveness of PC and patient survival. In a meta-analysis of 1,471 patients in 17 studies, the upregulation of miRNA-21 had a strong correlation with poor overall survival (OS), disease-free survival (DFS), and progression-free survival, and for patients undergoing adjuvant chemotherapy, the overexpression of miRNA-21 had a considerably great prognostic value and has also been linked to a greater probability of metastatic lymph node involvement and poorly differentiated malignancies (47). Namkung et al. performed a microarray analysis on pancreatic tumors in Korea, and obtained 1,733 miRNA expression profiles. MiRNA-574-5p, miRNA-1244, and miRNA-4474-5p were upregulated in expression. MiRNA-574-5p, miRNA-1244, miRNA-145, miRNA-328, miRNA-26b, and miRNA-4321 were correlated with total OS and DFS. Thus, these markers may be used as biomarkers to predict the prognosis of PC patients (48).

Additionally, miRNAs may also play a potential role in predicting the efficacy of the chemotherapeutic drug gemcitabine (48). Another study found that miR-7 was considerably downregulated in PC patients, and that low levels of miR-7 expression were associated with a poor prognosis. Ohuchida et al. found that patients with a high expression of miR-142-5p and miR-204 had significantly longer survival than patients with a low expression of miR-142-5p (P=0.0077) and miR-204 (P=0.0054) (49). Similarly, another study found that the postoperative survival rate of patients with elevated miRNA-200c expression was higher than that of patients with lower miRNA-200c expression (50). By combining paired miRNA/mRNA expression data, Vrahatis et al. developed an excellent method for capturing a miRNA-mediated signaling pathway (51). Liu et al. extracted 10 miRNA-mRNA modules associated with PC on this basis. Ultimately, 6 miRNA-mRNA modules were discovered to have value in predicting PC patient survival (52). Zhang et al. established and validated a miRNA set-based pathway predictive signature for PC (miPPSPC) using a gene set variation analysis. The miPPSPC showed that 4 metabolic pathways and 1 oxidative stress route were linked to PC patient survival based on tissue microarray results (53). The cell experiments of Wu et al. showed that Mir-221-3p promoted the proliferation, migration, invasion and drug resistance of PC cells, but inhibited apoptosis, which reduced the survival rate of PC patients (54).

Discussion

Based on previous research, the role of miRNAs in PC may help us to reveal the mechanism of pancreatic carcinogenesis in the future. For example, cancer-associated fibroblasts (CAFs) play an important role in the development and progression of PC (55). Studies have shown that miR-15a, miR-16, miR-31, miR-155, and miR-124 can promote the proliferation of CAFs (9). CAFs can provide PDAC cells with metabolic support, including amino acids and lipids, as well as paracrine signaling to promote growth and survival (56). The regulation of miRNAs in PC was a network. In PC, the decreased expression level of miRNAs regulating oncogene formation leads to their overproduction, while the increased level of other miRNAs involved in the regulation of inhibiting gene expression inhibits the formation of these anticancer genes (57). The network promotes PC formation, or is able to restore normal gene expression profiles to halt PC development. MiRNAs have shown great potential in the diagnosis, treatment and prognosis of PC. MiRNAs have proved that they can be used in the diagnosis and prognosis of PC by regulating proto-oncogenes and tumor suppressor genes if we obtain enough MiRNA expression profiles from cancer and normal tissues. By identifying the differentiation and expression profile of miRNA, we can determine the degree of tumor development and provide the possibility of using corresponding therapeutic approaches. Additionally, some key oncogenic miRNAs have been identified and their downstream targets will provide new ideas and research directions for the diagnosis and targeted therapy of PC. In terms of diagnostic markers, the early diagnosis rate of PC via detection with a single marker is still not sufficiently high; however, the combination of multiple markers improves the accuracy of early diagnosis. In terms of treatment, the existing research results have mainly relied on animal models; thus, there is still a lack of clinical trials. Additionally, to use miRNAs as a mature treatment of PC, we also need to overcome a number of problems, including finding an efficient and accurate precise delivery vector, and the high treatment costs (43). In the field of PC research in the future, the research on miRNAs will focus on miRNA-mediated cytokines and core signaling pathways, as well as the specific mechanism of miRNA promoting drug resistance in PC.

Conclusions

Currently, more extensive patient data, relevant clinical experience, and further studies are needed to verify whether miRNAs can serve as meaningful molecular markers for the early diagnosis, treatment, and prognosis of most PC patients.

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