Introduction

Cancer has become a global health problem resulting in a shortened life and lowering its quality. Among all gastrointestinal cancers, two of them come to the fore: colorectal cancer, due to high incidence concerning environmental factors, and pancreatic ductal adenocarcinoma (PDAC) because of poor prognosis [1]. The last one may be confused with chronic pancreatitis (CP) due to similarity in the clinical course and imaging studies.

Chronic pancreatitis is a disease that, due to its slow oligosymptomatic course in an early phase, causes many diagnostic problems. In 2016, a new mechanistic definition of CP was proposed, which was accepted by the majority of international gastroenterological societies. According to this definition, CP is a pathologic fibro-inflammatory syndrome of the pancreas with genetic, environmental, and/or other risk factors. It leads to parenchymal injury or stress. As a consequence of injury, exocrine and, in the latest stage, endocrine insufficiency develops [2].

Chronic pancreatitis is also associated with the risk of pancreatic ductal adenocarcinoma (PDAC), higher than in the general population. Patients with CP have a nearly 8-fold increased risk of developing pancreatic cancer five years after diagnosis [3]. Until now little is known about this relationship although some in vivo studies indicated a significant role of interleukin-22 (IL-22) in the promotion of PDAC development [4].

Pancreatic ductal adenocarcinoma is a highly aggressive disease with a poor prognosis and rising incidence and with an average 5-year survival rate of less than 10% [5]. Enlargement of the pancreas (tumor-like mass) with inflammation in the course of CP may mimic PDAC at imaging, which precludes pre-operative diagnosis and may lead to unnecessary surgical intervention [6]. Moreover, these two pathologies show similar biochemical parameters and clinical manifestations [7]. For these reasons, there is an urgent need to identify non-invasive markers that help distinguish PDAC from CP because all available serological and imaging examinations are non-specific for these diseases.

Colorectal cancer (CRC) is now the third most common cancer in the western world. According to the World Health Organization, 1.8 million new cases of CRC in 2018 were diagnosed, and 862 000 patients died from CRC [8]. Although we have screening tools, most notably colonoscopy, many colorectal cancers are diagnosed in advanced stages. A better understanding of pathological and molecular mechanisms of CRC may provide new perspectives for cancer prevention and care.

The non-coding microRNAs (miRNAs) seem to be promising and valuable markers of cancer development. These molecular players, 18–25 nucleotides in length, are involved in various biological processes, including cell proliferation, apoptosis, differentiation, and metabolism. MicroRNAs function is post-transcriptional regulation of gene expression complementary linkage to sequences within mRNA molecules (most often to untranslated regions). As a result, these mRNAs are silenced, and the expression of the proteins they encode decreases. Therefore, miRNAs could act as oncogenes if they reduce the expression of tumor suppressor genes or as tumor suppressors if they reduce the expression of oncogenes. Moreover, the same miRNAs can have a dual function, having the ability to bind to different mRNA molecules [9]. MiRNAs are present in stable forms in body fluids, such as plasma or serum, so their expression profiles are tightly related to the pathological conditions inside the cells [10, 11].

This study is focused on miRNA-17-5p, miR-NA-93--5p, miRNA-320a, miRNA-519d-3p, miR-NA-526b-3p, and miRNA-5590-3p, which according to the reviewed literature, are associated with inflammation and carcinogenesis. MiRNA-17-5p was up-regulated in pancreatic adenocarcinoma and directly targeted retinoblastoma-like protein 2 (RBL2). High levels of miR-17-5p and low levels of RBL2 protein are associated with poor prognosis [12]. The summary of molecular targets for miRNAs is in Table 1 [12–25]. Moreover, miRNA-93-5p is involved in gemcitabine resistance in pancreatic cancer via targeting the PTEN-mediated PI3K/Akt signaling pathway [13]. There is an indication that miRNA-320a takes part in promoting 5-fluorouracil (5-FU) resistance of human pancreatic cancer cells by targeting the programmed cell death 4 (PDCD4) transcript and is involved in proliferation, invasion, metastasis, drug-resistance characteristics, and the epithelial-to-mesenchymal transition of pancreatic cancer [14]. Moreover, the expression of miR-320a is considered a predictive marker for chemotherapy in pancreatic cancer patients [14]. The miRNA-519d-3p is a suppressor molecule whose downregulation in pancreatic cancer was observed with the simultaneously high level of ribosomal protein S15a (RPS15A), a gene that regulates expression of b-catenin and activity of the Wnt signaling pathway [15] Up-regulation of miRNA-519d-3p could suppress proliferation of pancreatic cancer cells and activity of Wnt/b-catenin, imitating the impact of RPS15A silencing [15]. MiRNA-526b-3p is considered a tumor suppressor. MiRNA-526b-3p directly targets the 3’UTR (untranslated region) of E2F transcription factor 1 (E2F1), decreasing its expression. Overexpression of miRNA-526b-3p inhibited the proliferation of CRC cells by reducing the level of E2F1 [16]. In glioma, miRNA-526b-3p regulates the tumor process through WEE1 (WEE1 G2 checkpoint kinase), and it is reported as a prognostic factor for this neoplasm [17]. MiRNA-5590 is considered a tumor suppressor molecule that prevents excessive cell proliferation and migration; its importance has been evaluated in human gastric cancer and breast cancer [18, 19].

In this pilot study, we have examined the expression of the above-mentioned miRNAs in the plasma of CP, PDAC, and CRC patients and correlated them with available clinical and demographic data and with markers of carcinogenesis: CA19-9 and carcinoembryonic antigen (CEA).

Our study aimed to investigate the potential of selected molecules as diagnostic biomarkers. Additionally, we checked whether the examination of miRNA expression could help differentiate between CP and PDAC.

Materials and methods

Studied group

The study was approved by the Research Ethics Committee of the Medical University of Lublin (approval no. KE 0254-/54/2015) and conducted in conformity with the Declaration of Helsinki.

The study of plasma miRNA expression was performed in 88 patients. Blood samples were taken at the moment of diagnosis. The study population included 40 (45%) patients with chronic pancreatitis, 20 (23%) patients with pancreatic cancer (PC), and 28 (32%) patients with colorectal cancer (CRC). Fifty-nine (67%) male and 29 (33%) female patients were in the examined group [median age and standard deviations (SD): 63.5 ± 16.2 years, range 27–96 years]. The clinical and demographic data are presented in Table 2. The control group consisted of 31 healthy participants (median age and SD: 45 ± 11.8 years, range 29–67 years). The control group did not differ significantly in terms of age and sex from the examined group.

MicroRNAs isolation

Blood was collected in EDTA (ethylenediaminetetraacetic acid) tubes and then centrifuged (2000 × g, for 10 min.) to obtain plasma. Plasma was stored at –80°C until miRNA isolation.

Isolation of total RNA with miRNAs fraction from plasma was performed using miRNeasy Serum/Plasma Kit (Qiagen, Germany). The amount and purity of RNA were assessed using an Eppendorf BioPhotometer (Eppendorf, Germany). RNA was stored at –80°C until the reverse transcription reaction was performed.

Reverse transcription reaction

The TaqMan™ Advanced miRNA cDNA Synthesis Kit (Applied Biosystems, USA) was used to transcribe the miRNAs into complementary DNA (cDNA) according to the manufacturer’s instructions. RT (Reverse transcription) was performed in a TPersonal Biometra thermocycler (Analytik-Jena Company, Germany). cDNA was stored at –20°C until quantitative polymerase chain reaction (qPCR) was performed.

Quantitative polymerase chain reaction

The expression of six microRNAs, which are attributed to properties of oncogenes or tumor suppressors (miRNA-17-5p, miRNA-93-5p, miRNA-320a-5p, miRNA-519d-3p, miRNA-526b-3p, miRNA-5590-3p), was assessed. Expression was examined by the qPCR method on the Illumina Eco (Illumina Inc, USA) device. The 20 microliter PCR mix for assessing miRNAs expression consisted of 10 µL TaqMan Fast Advanced Master Mix (Applied Biosystems, USA), 1 µL TaqMan Advanced miRNA Assay (separate reaction for each miRNA), 4 µL RNase-free water and 5 µL cDNA. Quantitative polymerase chain reaction was carried out under the following conditions: 95°C for 30 sec. and then 40 cycles: 95°C for 3 sec. and 62°C for 30 sec. miRNA-191-5p and cel-miR-39-3p were used as an internal control and spike-in control, respectively. Commercial sets of TaqMan primers and probes were used for each of the microRNAs and the internal control (Applied Biosystems, USA)

The 2-∆Ct method was used for the calculation of the expression.

Statistical analysis

Statistical analysis was performed using Statistica 13 software (Tibco Software, USA). The Mann-Whitney U-test was used to assess the differences in expression of particular miRNAs between individual groups. Receiver operating characteristic (ROC) curves with area under the curve (AUC) analyzes were used to assess the diagnostic utility of miRNAs in distinguishing patients from healthy participants, as well as CP and PTAC patients. A p-value below 0.05 was considered significant.

Results

Comparison of microRNA expression in patients and healthy donors

MiRNA-17 expression was higher in patients with colorectal cancer compared to healthy participants (p = 0.05). Moreover, significantly higher expression of miRNA-93 was observed in patients with pancreatic cancer and colorectal cancer compared to healthy individuals (p = 0.02 and p = 0.005, respectively). Further, significantly lower expression of microRNA-519 was found in chronic pancreatitis and pancreatic cancer compared to healthy subjects (p = 0.01 and p = 0.02, respectively) (Fig. 1).

Figure 1. Comparison of the expression of selected miRNAs in patients with chronic pancreatitis (CP), pancreatic ductal adenocarcinoma (PDAC), and colorectal cancer (CRC), and healthy volunteers

MiRNAs expression in patients

Significantly higher expression of miRNA-93 was observed in patients with CP who had a higher concentration of CA-19-9 compared to patients with a low level or unknown status of this marker (p = 0.03). Significantly lower expression of miRNA-519 was found in patients with chronic pancreatitis who were diagnosed with diabetes compared to patients without this disease (p = 0.02). Moreover, significantly higher expression of miRNA-320 was noticed in female in comparison to male patients with chronic pancreatitis (p = 0.004) (Fig. 2). No differences were found in the relative expression of the examined miRNAs between CP and PDAC patients.

Figure 2. Expression of selected microRNAs in patients with chronic pancreatitis (CP) depending on CA 19-9 concentration, sex, and diabetes coexistence

The diagnostic value of miRNAs expression assessment

We found three molecules that differentiated CP, PC, and CRC from healthy subjects.

Examination of miRNA-519 expression distinguished patients with chronic pancreatitis from healthy volunteers with sensitivity and specificity of the diagnostic test at 60% and 80%, respectively [AUC = 0.68; 95% confidence interval (CI) 0.55–0.80; p = 0.006] (Fig. 3A).

Examination of miRNA-93 and miRNA-519 expression distinguished pancreatic cancer patients from healthy participants. The sensitivity and specificity of the diagnostic test for assessment of miRNA-93 expression were 60% and 77%, respectively (AUC = 0.69; 95% CI 0.53–0.85; p = 0.002). While the sensitivity and specificity of the diagnostic test for miRNA-519 were 59% and 79% (AUC = 0.69; 95% CI 0.53–0.85; p = 0.002) (Fig. 3B).

Examination of miRNA-17 and miRNA-93 expression distinguished patients with colorectal cancer from healthy participants. The sensitivity and specificity of the diagnostic test for examination of miRNA-17 expression were 78% and 50% (AUC = 0.65; 95% CI 0.49–0.80; p = 0.05). Test for miRNA-93 expression had 78% sensitivity and 80% specificity (AUC = 0.71; 95% CI 0.57–0.85; p = 0.004) (Fig. 3C).

Figure 3. Sensitivity of the tests assessing the expression of selected miRNAs in distinguishing patients with chronic pancreatitis (CP), pancreatic ductal adenocarcinoma (PDAC), and colorectal cancer (CRC) from healthy participants; AUC — area under the curve; CI — confidence interval

Discussion

Despite advancement of research into early detection of malignant neoplasms, many cancers are still detected too late, especially pancreatic cancer, which is characterized by an aggressive course and a poor prognosis. Another problem is that the image on CT (computed tomography) or MRI (magnetic resonance imaging) is similar in pancreatic cancer and CP patients, so there is an urgent need to find markers allowing for differentiation of these two diseases. Nowadays, CA 19-9 is believed to be a blood marker in the early detection of PDAC, but on the other hand, it has a reduced diagnostic value because of false positive and false negative results [26]. This means that CA 19-9 level is elevated not only in PDAC but also in the biliary tract, stomach, colorectal, lung, or thyroid tumors and also in non-malignant pathologies, including pancreatitis, diabetes mellitus, as well as other pulmonary, thyroidal, and gynecologic diseases [27]. Moreover, another limitation of CA 19-9 examination is the fact that as a sialylated Lewis blood group antigen, also CA 19-9 is not detected in people who lack the expression of fucosyltransferase, an enzyme required for the production of both CA 19-9 and Lewis antigen [28]. The above problems do not allow the detection of PDAC in early stages and prevent effective surgical treatment [28].

We have much greater possibilities in the early detection of colorectal cancer thanks to the wide availability of screening tests, including colonoscopy. However, due to the reluctance (aversion) of patients to this examination, the need for tiring preparation and high costs, markers are necessary to facilitate identification of the best candidates for this study. The CEA tumor marker has fallen short of expectations and is not recommended for screening. Also in our study, the majority of CRC patients had normal or slightly raised CEA level at the moment of diagnosis.

Aberrant miRNA expression profiles have been studied in many types of cancers, including PDAC and colorectal cancer [29]. MiRNAs may be interesting blood-based biomarkers in clinical practice because they are stable in circulation, not degraded by endogenous RNases, non-invasive, and simple to collect [30]. Chronic inflammation regulates carcinogenesis on different levels, starting from tumor initiation, through proliferation and progression, ending up in metastasis; miRNAs are involved in this process [31]. Depending on the type of tumor and immune cells involved in this process, mediators produced by inflammatory cells increase mutagenesis and activate epigenetic machinery, including histone modifications, long non-coding RNA and miRNAs that modulate gene expression and promotor gene methylation [32]. Accumulating evidence indicates that miRNAs are frequently dysregulated in human cancers, and alterations of miRNAs expression in CRC have been well documented [33].

As miRNAs are mediators in carcinogenesis and inflammation, we have selected a group of miRNAs potentially involved in CRC, PDAC, and CP pathophysiology. We chose also some miRNAs known for having a potential diagnostic and prognostic role in patients with other cancers, especially lung cancer. In the next part of the discussion, we will look at those miRNAs that were associated with CP, PDAC, or CRC in our study: miRNA-17-5p, miRNA-93-5p, and miRNA-519d-3p.

The examination of the miRNA-93-5p molecule appears to have a diagnostic, predictive, and therapeutic potential. The test based on the evaluation of the level of this molecule in the serum or plasma was very promising; however, some limitations of this microRNA should be pointed out. In our study, high expression of miRNA-93-5p occurred in colorectal-cancer and pancreatic-cancer patients, with no significant difference between these cancers. That is, this molecule is not tumor-specific and, arguably, cannot be used as a stand-alone diagnostic or prognostic/predictive factor. However, it can be a valuable ancillary parameter for cancer screening, early cancer diagnosis, or the likelihood of resistance to treatment. Shao et al. indicated that a test based on a combined analysis of the miR-93-5p and miR-18a expression in serum (miR-93-5p+miR- -18a marker) has a better value potential for diagnosis and prognosis in non-small cell lung cancer (NSCLC) patients than the examination of single markers [34]. Likewise, Vila-Navarro et al. indicated that multiple miRNAs assessed simultaneously in one test provided much better information (in terms of sensitivity and specificity) in the identification of PDAC. Moreover, they showed that CA19.9 increased the diagnostic potential of test-examined miRNAs signatures. The test combining miRNAs and CA19.9 (miR-33a-3p+miR- -320a+CA19.9) achieved an AUC of 0.95 (93% sensitivity and 85% specificity) [35]. In our study, we observed significantly higher expression of the miR-93 molecule in the group of patients suffering from chronic pancreatitis, with a concentration of this marker above 37 U/mL. Nevertheless, we did not observe such a relationship in pancreatic cancer patients, and we found no differences in the expression of this miRNA between CP and PDAC. We think it is worthwhile to expand our study to an enlarged group of CP and PDAC patients and include protein tumor markers and other microRNAs that have the possibility of differentiating these two diseases.

The miR-93-5p molecule is considered to be oncogenic, whereas in our study we also observed a significant decrease in expression of the tumor suppressor miR-519d-3p in patients with both chronic pancreatitis and pancreatic cancer. Furthermore, we found that the expression of this molecule is significantly reduced in patients with chronic pancreatitis who were also diagnosed with diabetes. We tentatively suggest that this microRNA could be disease-tissue-specific. However, there is limited research on pancreatic diseases involving this molecule. Table 3 [20–25, 34–42] contains information on the miRNA-17-5p, miRNA-93, and miRNA-519d-3p molecules and their role in cancer in relation to the results obtained in this study.

There is no evidence of the impact of circulating miRNA-519d-3p as a biomarker in chronic pancreatitis. We showed that examination of miRNA-519 expression distinguished patients with chronic pancreatitis from healthy volunteers with sensitivity and specificity of 60% and 80%, respectively, while the sensitivity and specificity of this test in distinguishing pancreatic cancer patients from healthy participants were 59% and 79%, respectively. However, this finding must be confirmed and validated in an independent enlarged study group.

Our studies have some limitations, such as small study groups or lack of data on the stage or localization of possible metastases. However, there may be strong indications to extend the research to an enlarged group of patients and to conduct it using biological tests that would indicate the target transcripts for the studied miRNA molecules.

Conclusions

Our study indicated that microRNA-93 has diagnostic potential in colorectal and pancreatic cancers, but literature data indicated that it cannot be a stand-alone diagnostic/predictive factor. In the case of miRNA-519d-3p, due to limited literature data on serum/plasma studies in pancreatic cancer or chronic pancreatitis, we could draw conclusions based on our own studies, which suggested that this molecule probably has a suppressor function and its expression can be a supportive factor for the diagnosis of PDAC or CP. However, it does not distinguish between these two diseases.

Data availability statement

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflict of interest

Authors declare no conflict of interest.

References