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Unveiling the microRNA landscape in pancreatic ductal adenocarcinoma patients and cancer cell models

BMC Cancer volume 24, Article number: 1308 (2024) Cite this article

Abstract

Background

Pancreatic ductal adenocarcinoma (PDAC) poses a significant challenge due to late-stage diagnoses resulting from nonspecific early symptoms and the absence of early diagnostic biomarkers. MicroRNAs (miRNAs) play a crucial role in regulating diverse biological processes, and their abnormal expression is observed in various diseases, including cancer. Cancer stem cells (CSCs) are thought to act as a driving force in PDAC spread and recurrence. In pursuing the goal of unravelling the complexities of PDAC and its underlying molecular mechanisms, our study aimed to identify PDAC-associated miRNAs and relate them to disease progression, focusing on their involvement in various PDAC stages in patients and in reliable in vitro models, including pancreatic CSC (PaCSC) models.

Methods

The miRNA profiling datasets of serum and solid biopsies of PDAC patients deposited in GEO DataSets were analyzed by REML-based meta-analysis. The panel was then investigated by Real Time PCR in serum and solid biopsies of 37 PDAC patients enrolled in the study, as well as on BxPC-3 and AsPC-1 PDAC cell lines. We extended our focus towards a possible role of PDAC-associated miRNAs in the CSC phenotype, by inducing CSC-enriched pancreatospheres from BxPC-3 and AsPC-1 PDAC cell lines and performed differential miRNA expression analysis between PaCSCs and monolayer-grown PDAC cell lines.

Results

Meta-analysis showed differentially expressed miRNAs in blood samples and cancerous tissues of PDAC patients, allowing the identification of a panel of 9 PDAC-associated miRNAs. The results emerging from our patients fully confirmed the meta-analysis for the majority of miRNAs under investigation. In vitro tasks confirmed the aberrant expression of the panel of PDAC-associated miRNAs, with a dramatic dysregulation in PaCSC models. Notably, PaCSCs have shown significant overexpression of miR-4486, miR-216a-5p, and miR-216b-5p compared to PDAC cell lines, suggesting the recruitment of such miRNAs in stemness-related molecular mechanisms. Globally, our results showed a dual behaviour of miR-216a-5p and miR-216b-5p in PDAC while miR-4486, miR-361-3p, miR-125a-5p, miR-320d expression changes during the disease suggest they could promote PDAC initiation and progression.

Conclusions

This study contributed to an enhanced comprehension of the role of miRNAs in the development and progression of PDAC, shedding new light on the miRNA landscape in PDAC and its intricate interplay with CSCs, and providing specific insights useful in the development of miRNA-based diagnostic biomarkers and therapeutic targets.

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Background

Pancreatic cancer (PC) is one of the most aggressive human tumors and is classified as the 3rd leading cause of cancer-related death in the United States [1]. It represents a global critical burden for its aggressiveness and poor prognosis [2]. Moreover, in 2024, it is estimated that there will be 66,440 PC cases and 51,750 PC-related deaths in the United States. With a 5-year overall survival rate of approximately 10%, largely attributed to late-stage diagnosis, early detection is crucial for enhancing survival rates and therapeutic outcomes [3, 4].

Pancreatic ductal adenocarcinoma (PDAC) accounts for over 90% of PC cases and manifests symptoms that include anorexia, fatigue, weight loss, malabsorption, jaundice, dyspepsia, nausea, and abdominal or back pain [5,6,7,8]. Diagnosis relies on histopathological analysis of solid biopsies, complemented by imaging tests such as computed tomography, positron emission tomography, magnetic resonance imaging and endoscopic ultrasound [8]. Despite Food and Drug Administration (FDA) approval of carbohydrate antigen 19–9, early PDAC diagnosis is hindered by the absence of reliable serum biomarkers [9, 10]. Surgical resection, the sole effective PDAC treatment, is often hampered by late-stage diagnosis. Patients who undergo surgery have a median survival of 20–23 months, and adjuvant chemotherapy is typically recommended afterwards. Traditionally, adjuvant chemotherapy has involved single therapy with gemcitabine, but recent advancements have led to the combination of cytotoxic compounds, such as nab-paclitaxel with gemcitabine or the FOLFIRINOX regime [11,12,13]. Advances in Next-Generation Sequencing and bioinformatics have enabled a deeper understanding of the altered pathways in the PDAC scenario holding promise for the development of novel targeted therapies [14].

According to the hierarchical carcinogenesis model, a small subpopulation of cells with stem-like properties, known as cancer stem cells (CSCs), is responsible for tumor aggressiveness, therapy resistance, and cancer recurrence [12, 15]. CSCs may recapitulate the cellular heterogeneity present within the tumor, thereby driving cancer initiation, progression and metastasis [5, 15, 16]. Pancreatic CSCs (PaCSCs) express several markers, including CD44, CD24, CD133, EpCAM, CxCR4, and ALDH1 [12, 16].

MicroRNAs (miRNAs) are small non-coding RNAs with a length of 19–24 nucleotides that act as negative post-transcriptional regulators of gene expression [17, 18]. The role of miRNAs is crucial in numerous cellular functions and their aberrant expression has been observed in a wide range of malignancies [17]. In cancer, they can act as oncogenic miRNAs (oncomiRs) or tumor suppressor miRNAs (anti-oncomiRs), depending on the role of their target mRNA in the tumor-initiating process. Due to their involvement in cancer initiation and progression, as well as their high stability in biological samples, they could serve as first-line biomarkers in early non-invasive diagnostic procedures [19]. To date, a series of observational and experimental studies have highlighted the contribution of certain miRNAs in PDAC [20,21,22]. However, in some cases, the data regarding certain miRNAs in the literature appear conflicting and inconsistent across different matrices, with the result that ultimately, no miRNA has yet been accepted and shifted into clinical diagnostic procedures.

The experimental workflow of our study included GEO DataSets-based (https://www.ncbi.nlm.nih.gov/gds) meta-analysis of miRNA levels in serum and solid biopsies of PDAC patients, followed by validation in 37 PDAC patients enrolled for the study [23]. The panel was also explored in PDAC in vitro models, including BxPC-3 and AsPC-1 cell lines, and their respective PaCSCs-enriched pancreatospheres. The identified PDAC-associated miRNAs enhances our understanding of miRNA-based molecular mechanisms in PDAC, inspiring new potential diagnostic, prognostic, and therapeutic avenues in the fight against this challenging cancer.

Methods

Selection of public datasets for meta-analysis

To select miRNAs whose expression could significantly vary between PDAC cases and controls, we performed a meta-analysis of non-coding RNA profiling data available on the GEODatasets Platform as of October 2022. The search strings used were: "microRNA in PDAC", "microRNA in pancreatic ductal adenocarcinoma", "microRNA in pancreatic cancer", "miRNA in PDAC", "miRNA in pancreatic ductal adenocarcinoma" and "miRNA in pancreatic cancer". All the eligible studies met the following inclusion criteria: 1) Organism: we selected only studies on Homo sapiens; 2) Biological matrices: we choose only studies where the miRNA evaluation was performed on serum and solid biopsies; 3) Type of analysis: we included datasets of non-coding RNA profiling by means of high-throughput methods. The search strings allowed us to identify a total of 9 studies, 4 on serum and 5 on solid biopsies. All the eligible datasets were obtained from studies of non coding RNA profiling by arrays, as the studies with other methods did not meet the first two eligibility criteria. Eligible GEO DataSets of serum GSE59856 [24], GSE85589 [25], GSE124158 [26], and GSE140196 [27] were obtained from similar platforms with a comparable number of tested miRNAs. Data from solid biopsies GEO DataSets GSE140719 [28], GSE24279 [29], GSE41369 [30], GSE60978 [31], GSE71533 [32], were heterogeneous as they were provided from different platforms, with a highly variable number of miRNAs analyzed. Based on the above, we conducted a meta-analysis exclusively on the blood datasets. Instead, tissue datasets were analyzed independently, and significant miRNAs were identified as those showing a statistically significant difference between cases and controls in at least 3 out of the 5 datasets. Series characteristics are illustrated in Table 1. Some studies have been conducted on different tumors, not only on pancreatic cancer. For all of them, we used data related to PDAC.

Table 1 GEO DataSets selected for meta-analysis
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Statistical analysis

For each dataset, we were able to conduct a miRNA-transcriptome-wide association study by investigating the relationship between each miRNA and disease status using linear regression models. In these models, miRNA expression served as the dependent variable, and case/control status was the independent variable. Age and sex were included as covariates when available. For blood datasets, miRNA expression data were standardized, so each miRNA had a mean of 0 and a standard deviation of 1. This standardization allowed us to express results in terms of standard deviation differences rather than absolute differences, thereby making results from different datasets comparable and avoiding bias in the meta-analysis. The meta-analysis was performed using the Random Effect Maximum Likelihood (REML) method, implemented in the R package metafor [33]. The results were reported as standardized miRNA expression differences between cases and controls. To control for false positives, a False Discovery Rate (FDR) correction for multiple testing was applied.

Patient selection

A total of 37 patients diagnosed with PDAC, who had never received chemotherapy or radiotherapy were provided by the Surgical Clinic of the University Hospital of Sassari. The use of human samples was approved and supervised by the Ethics Committee of Cagliari (Prot. PG/2021/8575). The collection of solid biopsies was not possible when patients were inoperable, which was the case for 14 patients, or due to certain conditions adverse to the collection by the Surgical Clinic, such as nodules that were too small and therefore only intended for histopathological analysis. For the patients who underwent surgical resection, we collected pathological biopsies and their healthy counterparts. Biopsies were classified according to the AJCC/TNM Cancer Staging Manual [34]. 20 healthy controls were enrolled for the study. Patients and controls characteristics are provided in Tables 2 and 3.

Table 2 Characteristics of patients enrolled in the study. Age was dichotomized to 65 years as it was the mean age of the recruited patients
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Table 3 Characteristics of the controls enrolled in the study. Age was dichotomized to 65 years as it was the mean age of the control group
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Sample collection

A total of 37 preoperative blood samples were collected in S-Monovette 9 mL Serum Gel with Clotting Activator tubes (Sarstedt, Nümbrecht, Germany). The tubes were left at room temperature (RT: 15 °C – 25 °C) for 10 min to 1 h to allow the complete clotting. The serum was separated through two consecutive centrifugations at 4 °C, with the first at 1900 rcf and the second at 16,000 rcf. After the separation of serum, it was stored at -80 °C. 19 tumoral biopsies and 18 healthy counterparts were collected from patients who underwent surgical resection. To stabilize and protect cellular RNA, solid biopsies were immediately transferred in RNAlater-filled 15 ml tubes (Invitrogen, Thermo Fisher Scientific, Waltham, USA) for 24 h. Subsequently, solid biopsies have been removed from the stabilization solution, dried, and stored at -80 °C.

Cell lines

We purchased the hTERT-HPNE E6/E7 (ATCC CRL-4036), BxPC-3 (ATCC CRL-1687), and AsPC-1 (ATCC CRL-1682) cell lines from ATCC and cultured them following the manufacturer's instructions. The hTERT-HPNE E6/E7 cell line, representing the normal pancreatic ductal epithelium, was cultured in a complete medium composed as follows: 75% DMEM without glucose (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) with an additional 2 mM L-glutamine and 1.5 g/L sodium bicarbonate (Gibco™, Thermo Fisher Scientific, Waltham, USA), 25% Medium M3 Base (Incell Corp. San Antonio, USA) and fetal bovine serum 5% (Gibco™, Thermo Fisher Scientific, Waltham, USA), 10 ng/ml human recombinant EGF (Cedarlane Lab, Burlington, Canada), 5.5 mM D-glucose (1 g/L) (Gibco™, Thermo Fisher Scientific, Waltham, USA), 750 ng/ml puromycin (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). As for BxPC-3 and AsPC-1, representing primary and metastatic pancreatic tumors, respectively, they were cultured in RPMI 1640 Medium with ATCC modification (Gibco™, Thermo Fisher Scientific, Waltham, USA) supplemented with 10% of FBS. The cells were maintained in the incubator under standard conditions at 37 °C with 5% CO2. We chose the hTERT-HPNE E6/E7 cell line as a representative of the normal pancreatic ductal epithelium while employing the BxPC-3 (WT for KRAS) and AsPC-1 (mutated KRAS) PDAC cell lines as models for primary and metastatic PDAC stages, respectively. This experimental asset allowed us to investigate miRNA recruitment in a quantitative and qualitative manner during the transition from normal pancreatic ductal epithelial to primary tumoral and metastatic PDAC phenotypes.

CSC enrichment

To obtain an enriched subpopulation of PaCSCs, the BxPC-3 and AsPC-1 cell lines were cultured in a specific sphere-forming medium according to the protocol WO2016020572A1 [35,36,37]. The cells were cultured in Ultra-Low Attachment Six-Well Plates (Corning® Costar®, USA) with DMEM F/12 without FBS and supplemented with the following components: 4 ng/mL heparin (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), 1 mg/mL hydrocortisone (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), 10 µL/mL p15, 10 µg/mL insulin (Insulin–Transferrin–Selenium, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), 1X B27 (B-27™ Supplement [50 ×], Vitamin A; Invitrogen, Carlsbad, CA, USA). Before cells seeding, the CSCs medium was supplemented with 10 ng/mL of Epidermal Growth Factor (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), 10 ng/mL of Interleukin-6 (Miltenyi, Bergisch Gladbach, Germany), 10 ng/ml Hepatocellular Growth Factor (Miltenyi) and 10 ng/mL of Fibroblast Growth Factor (Sigma-Aldrich). After 72 h, primary spheres were separated using trypsine or a syringe. Following disaggregation, the cells were re-seeded in low attachment multi-well plates in spheres conditioned medium for an additional 72 h, before further use.

RNA extraction and reverse transcription

Unless otherwise stated, the reagents and instruments used for RNA extraction and retrotranscription were purchased from Qiagen (Hilden, Germany). For the RNA extraction from serum, miRneasy Serum/Plasma kit was used, following the manufacturer's instructions. Before RNA extraction from solid biopsies, tissue homogenization was carried out using 700µL of Qiazol Lysis Reagent and Stainless-Steel Beads with 5 mm of diameter. The tissue disruption was performed using the Tissue Lyser LT. Then, RNA was extracted from homogenized solid biopsies and cell lines using the miRneasy Mini kit according to the manufacturer’s instructions. After extraction, the quality of the extracted RNA was evaluated by measuring the 260/280 ratio using the NanoDrop™ One/OneC Microvolume UV–Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and RNA concentration was quantified. Before proceeding with reverse transcription with miRCURY LNA RT Kit, all samples were adjusted to a concentration of 5 ng/µL. The cycling conditions for RT were set to 42 °C for 60 min, followed by 95 °C for 5 min and carried out in the BioRad MJ Mini 48-well Personal Thermal Cycler (BioRad, Hercules, California, USA).

Real time PCR

Unless otherwise stated, the reagents used for Real Time PCR were purchased from Qiagen (Hilden, Germany). To quantify the expression of miR-4486, miR-4741, miR-20b-5p, miR-361-3p, miR-324-5p, miR-125a-5p, miR-320d, miR-216a-5p, and miR-216b-5p in cDNA from serum samples miRCURY LNA miRNA Custom PCR Panel has been used. In serum samples, where RNA and miRNA concentrations are typically low, 96-well pre-spotted plates were used and supplied ready-to-use, each well containing a specific primer assay. UniSP6 and UniSP3 were used as interplate calibrators, while miR-24-3p was used as a serum reference miRNA. For Real Time PCR on solid biopsies and cell lines miRCURY LNA miRNA PCR Assays with U6 as a reference gene have been used. Real Time PCR reactions were run using the StepOne Plus thermocycler (Applied Biosystems, Waltham, Massachusetts, USA) with the following cycling conditions: 95 °C for 2 min (PCR initial heat activation), 95 °C for 10 s (denaturation), 56 °C for 60 s (Combined annealing/extension) for 40 cycles. Data were analyzed using the 2−∆∆Ct method and evaluated statistically using a two-sided non-paired Student’s T test, considering significant results with a p-value < 0.05 [38].

Results

GEO DataSets meta-analysis results

We investigated differences between PDAC cases and healthy controls for 2,526 miRNAs through meta-analysis of GEO DataSets. In serum, we found significant association for miR-6729-5p, miR-6125, miR-4734, miR-4486, miR-6075, and miR-4741 that were upregulated (FDR adjusted p < 0.05) while miR-6799-5p and miR-20b-5p were downregulated (FDR adjusted p < 0.05). Solid biopsies bioinformatical analysis allowed us to identify a significant increase (FDR adjusted p < 0.05) in the expression of let-7i, miR-361-3p, miR-324-5p, miR-221, miR-125a-5p, miR-484, miR-320d. A significant reduction of the expression in solid biopsies of cases compared to controls was observed for miR-216b, miR-217, miR-216a, miR-375 (FDR adjusted p < 0.05). To evaluate the relative variation of miRNAs between serum and solid biopsies, their meta-analysis results were compared. Increased expression of miR-4486 and miR-4741 was found between cases and controls in both serum and solid biopsies and the result was statistically significant in both matrices. On the other hand, the miRNAs miR-361-3p, miR-324-5p, miR-125a-5p, miR-320d were overexpressed although not significantly in serum and solid biopsies from PDAC patients. A significant difference between serum and solid biopsies was observed for miR-20b-5p which was downregulated in serum (p < 0.001) and upregulated in solid biopsies (p < 0.05) of cases compared to controls. MiR-216a-5p and miR-216b-5p both showed a significant decrease (p < 0.001) in the expression on solid biopsies of cases compared to controls, while in serum they were upregulated without any statistical significance. The following miRNAs were then selected and evaluated in serum and solid biopsies of PDAC patients and in vitro in cellular models of PDAC: miR-4486, miR-4741, miR-361-3p, miR-324-5p, miR-125a-5p, miR-320d, miR-20b-5p, miR-216a-5p and miR-216b-5p (Figs. 1 and 2).

Fig. 1
figure 1

Forest plot of the standardized mean differences (SMD) between pancreatic cancer (PC) cases and controls of a miR-4486, b miR-4741, c miR-20b-5p expression from 4 independent studies based on serum. Estimate and 95% CI indicate individual study estimated SMD and its 95% confidence interval

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Fig. 2
figure 2

Forest plot of the standardized mean differences (SMD) between pancreatic cancer (PC) cases and controls of a miR-361-3p expression from 3 independent studies, b miR-324-5p expression from 5 independent studies, c miR-125a-5p expression from 3 independent studies, d miR-320d from 3 independent studies, e miR-216a-5p from 5 independent studies, f miR-216b-5p from 3 independent studies. Studies were performed on solid biopsies. Estimate and 95% CI indicate individual study estimated SMD and its 95% confidence interval

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PDAC-associated miRNAs in serum of PDAC patients and controls

To validate the meta-analysis, Real Time PCR was performed on serum samples from 37 PDAC patients and 20 healthy controls; the latter were used for data normalization. We statistically analyzed the results by performing a Student’s T test for unpaired data. Patients were divided into four groups according to TNM grading, and the relative expression values of miRNAs were compared among the stage groups. Additionally, the population was stratified into low-grade PDAC (stages I-II) and high-grade PDAC (stages III–IV) and miRNA variations between the two groups were evaluated. The levels of miR-4486 (p < 0.01), miR-20b-5p (p < 0.001), miR-361-3p (p < 0.001), miR-320d (p < 0.001), and miR-216b-5p exhibited a significant increase in the serum of cases compared to controls. The expression of miR-4741 was not detected. Under the same conditions, we observed an upward trend in miR-125a-5p, miR-324-5p, and miR-216a-5p in the serum of cases, although the difference did not reach statistical significance (Fig. 3). No statistically significant differences were found in the comparison between stage I and stage II PDAC serum samples. MiR-125a-5p was upregulated in stages III (p < 0.05) and IV (p < 0.05) when compared to stage I, while miR-324-5p and miR-216b-5p were both significantly downregulated (p < 0.001) in stage III compared to stage I. In stage IV serum samples, emerged the downregulation of miR-216a-5p (p < 0.001) and miR-216b-5p (p < 0.001) compared with stage I serum samples (Fig. 4). Considering the stratification in low-grade and high-grade PDAC, miR-4486 (p < 0.01), miR-20b-5p (p < 0.001), miR-320d (p < 0.001), miR-216a-5p (p < 0.05), miR-216b-5p (p < 0.01), miR-324-5p and miR-125a-5p were upregulated while miR-361-3p was downregulated (p < 0.001) in low-grade PDAC when compared to controls. In high-grade PDAC patients, miR-4486 (p < 0.01), miR-20b-5p (p < 0.01), miR-361-3p (p < 0.001), miR-125a-5p (p < 0.001), miR-320d (p < 0.001), miR-324-5p, and miR-216b-5p were upregulated, while miR-216a-5p (p < 0.001) was downregulated compared to controls. The direct comparison between low-grade and high-grade PDAC serum samples resulted in the upregulation of miR-4486, miR-20b-5p (p < 0.05), miR-361-3p (p < 0.05), miR-324-5p, miR-125a-5p (p < 0.001) and miR-320d (p < 0.001) in high-grade PDACs and the downregulation of miR-216a-5p (p < 0.001) and miR-216b-5p (p < 0.05) in the same patients (Fig. 4).

Fig. 3
figure 3

Relative expression of serum between PDAC cases and controls. Data were obtained also from low-grade and high-grade patients; *p < 0.05; **p < 0.01; ***p < 0.001

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Fig. 4
figure 4

Relative expression of serum microRNAs in disease progression, from PDAC patients of stage I to stage IV; *p < 0.05; **p < 0.01; ***p < 0.001

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PDAC-associated miRNAs in tumoral solid biopsies and healthy counterparts

All nine miRNAs were assessed in solid pathological biopsies and their corresponding normal counterpart. Because a great number of patients were inoperable, fewer solid biopsies were collected than blood specimens. Consequently, it was not possible to do comparisons between stages, as done in the serum samples. Upon comparing the relative expression data of the miRNAs between pathological biopsies and their healthy counterpart, it was observed that miR-4486 was significantly downregulated (p < 0.001) in tumoral tissues while no miR-4741 expression was observed. Not statistically significant differences were detected in miR-20b-5p, miR-361-3p, miR-125a-5p, miR-320d, miR-216a-5p and miR-216b-5p, which were upregulated, while miR-324-5p was downregulated (Fig. 5).

Fig. 5
figure 5

Relative expression of miRNAs in pathological solid biopsies of PDAC patients and their healthy counterparts; *p < 0.05; **p < 0.01; ***p < 0.001

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PDAC-associated miRNAs in PDAC in vitro models

Building upon the results of the bioinformatic analysis, we evaluated the expression levels of candidate miRNAs in hTERT-HPNE E6/E7, BxPC-3, and AsPC-1 cell lines to understand how relative expression varies during the transition from a normal pancreatic ductal epithelial cell to a tumoral and metastatic one. The relative miRNA expressions of primary pancreatic tumor cell line BxPC-3 were compared with the relative expression in hTERT-HPNE E6/E7 cells, a normal pancreatic epithelial cell line. MiR-4741 wasn’t detected in any in vitro model considered. MiR-4486, miR-361-3p, miR-324-5p, miR-125a-5p (p < 0.01), miR-320d, miR-216a-5p, and miR-216b-5p were upregulated in BxPC-3 but, apart from miR-125a-5p, the differences were not statistically significant. MiR-20b-5p was not expressed in hTERT-HPNE E6/E7 cell lines but only in BxPC-3. However, as we progressed from the hTERT-HPNE E6/E7 cell line to the metastatic phenotype, represented by the AsPC-1 cell line, the relative expression of miR-361-3p (p < 0.001), miR-324-5p (p < 0.05), miR-125a-5p (p < 0.05), miR-320d (p < 0.05), miR-216a-5p (p < 0.05) increased significantly in AsPC-1 cells. MiR-4486 was downregulated in AsPC-1, while miR-216b-5p was upregulated, but the difference was not statistically significant. Considering the transition between BxPC-3 and AsPC-1, miR-4486, miR-324-5p (p < 0.001), miR-125a-5p (p < 0.001), miR-320d (p < 0.01), miR-216a-5p (p < 0.001) and miR-216b-5p were downregulated, while miR-361-3p showed increased expression (p < 0.01) (Fig. 6).

Fig. 6
figure 6

hTERT-HPNE E6/E7 vs BxPC-3, AsPC-1 and Tumoral cell lines. Relative expression of miRNAs in normal ductal epithelial cell line (hTERT-HPNE), primary tumor cell line (BxPC-3), and metastatic cell line (AsPC-1). Tumoral cell lines include BxPC-3 and AsPC-1 mean values for miRNAs expression; *p < 0.05; **p < 0.01; ***p < 0.001

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PDAC-associated miRNAs in PaCSCs-like models

To investigate the potential role of candidate miRNAs in CSC phenotype we evaluated their expression levels on CSCs-enriched pancreatospheres obtained from BxPC-3 and AsPC-1 cell lines. Compared to monolayers, miR-4486, miR-216a-5p, and miR-216b-5p were upregulated in BxPC-3 CSC, even if the differences were not statistically significant. MiR-20b-5p, miR-361-3p (p < 0.01), and miR-125a-5p (p < 0.001) were downregulated in the same model. MiR-4741, miR-324-5p, and miR-320d were not detected in BxPC-3 CSC-like. In AsPC-1 pancreatospheres, miR-361-3p and miR-320d were significantly downregulated (p < 0.001) while miR-4486 (p < 0.05), miR-216a-5p, and miR-216b-5p were upregulated. MiR-4741, miR-20b-5p, miR-324-5p, miR-125a-5p were not detected on AsPC-1 CSC-like models (Fig. 7). Relative expression values are listed in Table 4.

Fig. 7
figure 7

Relative expression of miRNAs between cells cultured in monolayers and CSC-like models (BxPC-3 spheres and AsPC-1-spheres). Avg. spheres refers to mean value of the two CSC-like models; *p < 0.05; **p < 0.01; ***p < 0.001

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Table 4 Relative expression values of miRNAs between cells cultured in monolayers and CSC-like models (BxPC-3 spheres and AsPC-1-spheres). Avg. spheres refers to mean value of the two CSC-like models; ND means not detected. Relative expression values were obtained from qPCR
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Discussion

This study is aimed at the identification of a panel of miRNA aberrantly expressed in PDAC and with a role in its pathogenesis, to improve the knowledge about miRNA regulation in PDAC and introduce new potential diagnostic and prognostic biomarkers.

The research began with a meta-analysis of non-coding RNA profiling data, which allowed the selection of miR-4486, miR-4741, miR-20b-5p, miR-361-3p, miR-125a-5p, miR-320d, miR-216a-5p, and miR-216b-5p as PDAC-associated miRNAs. Their expression levels were then assessed by Real Time PCR on serum and solid biopsies of 37 patients enrolled for the study: a perfect match between the meta-analysis data and both biological matrices was obtained for miR-125a-5p, miR-320d and miR-361-3p, which were always overexpressed in PDAC patients. A partial alignment to the meta-analysis data, consistent with only one of the two matrices, was reported for miR-4486, miR-324-5p, miR-216a-5p and miR-216b-5p. MiR-4741 was not detected on any model adopted. In vitro, when switching from the normal to the primary tumor phenotype, all miRNAs were overexpressed. The same trend was observed in the metastatic tumor cell line, apart from miR-4486. In these models, miR-20b-5p was not detected.

By using the CSC-enriched pancreatospheres we have ascertained that miRNAs vary even more than previously observed, and this difference is not always consistent with the dysregulations found in the other models. This is the case for miR-361-3p or miR-324-5p, whose expression, increased on tumor conditions, appears in pancreatospheres strongly reduced and completely absent respectively. On the contrary, concerning miR-216a-5p and miR-216b-5p, a consistent overexpression behaviour was observed between tumor and CSC-like models. Previous research efforts have focused on examining the relationship between miRNA and PDAC, but conflicting results have been found.

Several studies reported miR-216a-5p downregulation in PDAC cases and suggested different mechanisms to explain its aberrant expression and its potential oncosuppressive role [39,40,41,42]. In our study, by assessing its expression in biological matrices of PDAC patients, miR-216a-5p was upregulated in cases compared with controls. In serum, it was significantly upregulated in the early stages while its expression gradually decreased along the evolution of the disease: it was upregulated in stages II and III compared to stage I while in stage IV its expression was dramatically reduced (p < 0.001). Consistent with this data, miR-216a-5p expression increased in low-grade patients (p < 0.05) and decreased in high-grade patients (p < 0.001). This observation is coherent with Zhang et al. who reported that miR-216a-5p expression in solid biopsies was negatively associated with advanced TNM stage and poor prognosis [42]. During the progression from the primary tumor cell line (BxPC-3) to the metastatic one (AsPC-1), miR-216a-5p expression decreased (p < 0.001) but it showed an increasing trend in CSCs models. This supports our idea that miR-216a-5p could act as oncomiR in the early phase of PDAC while later it could exert an oncosuppressive function and its differential expression may be affected by other regulators.

Our study revealed a dichotomy in miR-216b-5p dysregulation. The microRNA was upregulated in both serum (p < 0.05) and solid biopsies of PDAC cases. The same trend was detected in vitro, where it showed a non-significant upregulation in PDAC cell lines and CSC models. Nevertheless, serum comparisons provided us with an interesting scenario: miR-216b-5p expression gradually decreased with disease progression, reaching a significant downregulation in stages III and IV compared to stage I (p < 0.001). Likewise, switching from low-grade serum samples to high-grade serum samples miR-216b-5p expression decreased (p < 0.01), even if in the two groups it was higher than in controls. Various studies reported miR-216b-5p decreased expression. You et al. related its downregulation to tumor size and advanced TNM stage, suggesting a target for its oncosuppressive function [43, 44]. We propose that the molecule could exert a dual role during the progression of the disease.

The observation of miR-216a/b-5p expression changes during PDAC agrees with Petrovich et al. who discussed how miRNA levels could vary during disease progression, following a time profile [45]. It has been reported that miR-216a/b-5p is abundant in the pancreas and localized in acinar cells. We believe that the dichotomy in miR-216a/b-5p behaviour could result from acinar cell degeneration and necrosis that occurs with PDAC: miR-216 serum levels increase in the early stages of the disease, following the disruption of the acinar cell membrane and the release of the miRNA into the bloodstream [46, 47]. In advanced disease, when acinar cells that contain miR-216a-5p are replaced by cancerous cells, miR-216a-5p levels are dramatically reduced. The increased serum levels of these molecules in the early stages of the disease suggest their potential as biomarkers for early diagnosis. However, further studies are needed.

The role of miR-125a-5p in cancer has been extensively debated, yielding contradictory findings across various tumor types. While it has been described as anti-oncoMir in hepatocellular carcinoma, lung cancer and glioblastoma, our results indicate a potential oncogenic role in PDAC, especially in the advanced stages, highlighting the tissue-specific nature of miR-125a-5p. We found that it was upregulated in the biological matrices of patients enrolled, perfectly matching with meta-analysis. In serum, as the disease progressed, miR-125a-5p expression increased, particularly in stages III and IV compared to stage I (p < 0.05), and in high-grade patients compared to controls (p < 0.001). In vitro, it was significantly upregulated in tumoral cell lines compared to the normal ductal cell line, but the expression decreased (p < 0.001) passing from the primary tumoral cell line to the metastatic one. Interestingly, in the CSC model, miR-125a-5p expression was lower in BxPC-3 CSC than in adherent BxPC-3, while it was not detected on AsPC-1 CSC-enriched spheres. This specific CSC pattern suggests a unique role of miRNAs in this subpopulation, which is distinct from the broader cancerous cells. However, miR-125a-5p remains poorly addressed in the context of PDAC [48]. Chen et al. suggested that it promotes PDAC cell proliferation, migration and invasion by the activation, mediated by miR-216a-5p, of the ERK/EMT pathway [49].

Hu et al. demonstrated that miR-361-3p regulate ERK/EMT pathway by targeting DUSP2, promoting metastasis in PC. They found increased miR-361-3p expression levels and associated them with advanced disease and poor prognosis [50]. Consistent with this evidence, our results showed elevated miR-361-3p expression in both serum (p < 0.001) and solid biopsies of cases, in line with our meta-analysis. In our cohort, the miRNA exhibited an increase along the disease progression. This trend was mirrored in cell models, being upregulated in BxPC-3 and AsPC-1 cell lines (p < 0.001) compared to the normal cell line. However, we found a significant decrement in CSC models. Combining our data with existing literature, we propose that miR-361-3p may play a promoting role in PDAC initiation and progression. Its oncogenic role in PDAC and the molecular mechanisms associated have been demonstrated by Huang et al. who highlighted the intricate regulatory network influencing miR-361-3p in PC [51].

Our results revealed miR-4486 upregulation in the serum of PDAC cases (p < 0.01) and its downregulation in pathological solid biopsies (p < 0.001). MiR-4486 serum levels followed an increasing trend during the progression of the disease. Such behaviour was recapitulated in vitro, where the expression of miR-4486 increased shifting from the normal pancreatic ductal epithelial cell line to the BxPC-3 and AsPC-1 cell lines and CSC models. Importantly, miR-4486 was significantly higher in CSC models in relation to BxPC-3 and AsPC-1 cell lines, paving the way for future functional studies that may indicate a role of miR-4486 in stemness and cancer spread, Its aberrant expression, significantly increased in the early-stage disease, suggests a potential promoting role in tumorigenesis. Lee et al. also reported miR-4486 dysregulation, proposing it, along with other miRNAs, as potential biomarkers to improve PDAC detection rate [25]. While our results hint at its potential as an early diagnostic biomarker for PDAC, further investigations are needed.

There are numerous discrepancies regarding the expression of miR-324-5p in diverse tumor types. While some studies have found its expression to be extremely low and characterized it as a tumor suppressor, others have reported differing results. Wan et al. reported miR-324-5p upregulation in PDAC solid biopsies and cell models, highlighting its crucial role in PDAC progression by targeting KLF3 and, thereby, regulating the proliferation and apoptosis of PC cells [52, 53]. According to our findings, miR-324-5p expression was increased in the serum of enrolled cases, confirming meta-analysis results, and decreased in pathological solid biopsies. In the analysis of serum samples for cancer staging and grading, profound dysregulation was observed, such that it cannot be linked to the progression of the disease. In vitro, miR-324-5p was upregulated in primary tumor and metastatic PDAC cells, mirroring our serum findings. Its expression decreased from primary to metastatic cells (p < 0.001) until it was no longer detectable in CSC models.

The observed behaviour on the in vitro task suggests a potential promoting role in the early PDAC phases. However, in advanced stages and PaCSCs, its role may be subject to inhibitory regulations.

While the role of miR-320d in PDAC has not yet been discussed, its aberrant expression has been found in several human malignancies [54,55,56]. In colorectal cancer (CRC), certain studies have suggested that it may have tumor-suppressive properties, while others have indicated that its levels are elevated in exosomes from patients with metastatic CRC, suggesting its potential use as a blood-based biomarker [57, 58]. From our analysis, miR-320d consistent upregulation occurred in both serum (p < 0.001) and solid biopsies of PDAC cases, aligning with meta-analysis results. Notably, when considering the PDAC stage, it showed an increasing trend in stages III and IV compared to stage I. Moreover, elevated miR-320d levels were observed in both low and high-grade groups compared to controls (p < 0.001), with a more pronounced increase in high-grade samples versus their low-grade counterparts (p < 0.001). These findings were also reflected in certain conditions of our in vitro models, reinforcing our hypothesis regarding miR-320d potential oncogenic role.

Numerous studies have reported elevated expression of miR-20b-5p across various cancers, indicating its potential role as oncomiR. Tavano et al. explored the utility of circulating miR-20b-5p in identifying new-onset diabetes in PDAC, though not necessarily reflective of early cancer stages [59, 60]. Our analysis revealed its elevated expression in both serum (p < 0.001) and solid biopsies of PDAC cases. Notably, in serum samples, it displayed upregulation in low-grade (p < 0.001) and high-grade (p < 0.05) groups compared to controls, as well as in stages II and III compared with stage I. However, its expression significantly declined in stage I compared to controls and in stage IV, compared to stage I. In vitro, its contribution was limited. The observed upregulation in PDAC cases implies a potential regulatory role in disease progression, underscoring the need for further investigation.

Conclusions

In conclusion, the study is based on a comprehensive approach that merges meta-analysis, patients' biological matrices and in vitro models of PDAC. The integration of different methods allowed us to highlight the thick dysregulation that the molecules undergo depending on the disease stage, the tissue considered, the model and the phenotype.

Globally, our results suggest a PDAC-promoting role for miR-4486, miR-361-3p, miR-324-5p, and miR-125a-5p. Occasionally, the miRNA behaviour suggested a dual role in different tumoral stages. This is the case of miR-216a-5p and miR-216b-5p, whose levels are increased in the early PDAC and reduced in the advanced disease. Serum dysregulation of miR-216a/b-5p in early PDAC may also occur as a result of acinar cell degeneration and represents an important observation in the future perspective of developing non-invasive biomarkers for the early detection of PDAC. The consistent dysregulation that occurred in PaCSCs models suggests that miRNAs could exert a prominent role in the acquisition or regression of stemness traits, closely associated with carcinogenesis and tumour aggressiveness. Further studies are needed to functionally characterize this potential role.

The integrated approach allowed the discovery of connections between data obtained from the multiple models considered. However, the intricate regulation to which microRNAs are subjected can lead to conflicting data under certain conditions. Another noteworthy limitation is the relatively small number of enrolled patients and challenges in obtaining solid biopsies, resulting in discrepancies between serum and solid biopsies. Finally, we believe that the results of our work could be implemented and ameliorated through the employment of PDAC organoids in future research. Our findings have provided valuable insights into the role of miRNAs in PDAC, both in patients and in vitro models. These observations have significantly enhanced our comprehension of the complex regulatory mechanisms of miRNAs in cancer. Given the challenging clinical context of PDAC, characterized by a late-stage diagnosis and a dismal prognosis, our serum results strongly support the potential of circulating miRNAs as non-invasive diagnostic tools for the early detection of PDAC. Furthermore, exploring the involvement of miRNAs in the pathways that contribute to the CSC phenotype may pave the way for the development of novel therapeutic strategies that target CSCs and related processes, such as metastasis and tumor recurrence.

Availability of data and materials

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

Abbreviations

PDAC:

Pancreatic ductal adenocarcinoma

MiRNA:

MicroRNA

CSC:

Cancer stem cell

GEO:

Gene Expression Omnibus

PC:

Pancreatic cancer

FDA:

Food and Drug Administration

PaCSC:

Pancreatic cancer stem cell

oncomiR:

Oncogenic microRNA

anti-oncomiR:

Oncosuppressive microRNA

REML:

Random Effect Maximum Likelihood

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Acknowledgements

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Funding

This research received no external funding.

Author information

Author notes

  1. Grazia Fenu and Carmen Griñán-Lisón contributed equally to this work.

Authors and Affiliations

  1. Department of Biomedical Science, University of Sassari, Sassari, 07100, Italy

    Grazia Fenu, Andrea Pisano, Cristiano Farace, Federica Etzi, Angela Sabalic, Maria Giuliana Solinas & Roberto Madeddu

  2. Biopathology and Regenerative Medicine Institute (IBIMER), Centre for Biomedical Research (CIBM), University of Granada, Granada, 18016, Spain

    Carmen Griñán-Lisón, Aitor González-Titos & Juan Antonio Marchal

  3. Instituto de Investigación Biosanitaria Ibs.GRANADA, University of Granada, Granada, 18071, Spain

    Carmen Griñán-Lisón, Aitor González-Titos, Belén Toledo & Juan Antonio Marchal

  4. Excellence Research Unit “Modeling Nature” (MNat), University of Granada, Granada, 18016, Spain

    Carmen Griñán-Lisón & Juan Antonio Marchal

  5. Department of Biochemistry and Molecular Biology II, Faculty of Pharmacy, University of Granada, Granada, 18071, Spain

    Carmen Griñán-Lisón

  6. National Institute of Biostructures and Biosystems, Rome, 00136, Italy

    Cristiano Farace & Roberto Madeddu

  7. Clinical Bioinformatics Unit, IRCSS Istituto Giannina Gaslini, Genoa, 16147, Italy

    Giovanni Fiorito

  8. Department of Medicine, Surgery and Pharmacy – Unit of General Surgery, University of Sassari, Sassari, 07100, Italy

    Teresa Perra & Alberto Porcu

  9. Department of Health Sciences, University of Jaén, Jaén, 23071, Spain

    Belén Toledo & Macarena Perán

  10. Department of Human Anatomy and Embryology, Faculty of Medicine, University of Granada, Granada, 18016, Spain

    Juan Antonio Marchal

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  1. Grazia Fenu
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Contributions

Conceptualization, C.F., J.A.M. and R.M.; methodology, G.Fe, C.G.L., A.Pi, A.G.T., C.F., F.E., T.P., A.S., B.T. and A.Po.; software, G.Fi, M.G.S.; formal analysis, G.Fe, G.Fi, M.G.S.; data curation, G.Fe, C.G.L., A.Pi, F.E and G.Fi; writing—original draft preparation, G.Fe, A.Pi and F.E.; writing—review and editing, C.F., J.A.M. and R.M. All authors have read and agreed to the published version of the manuscript.

Corresponding authors

Correspondence to Cristiano Farace or Juan Antonio Marchal.

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Ethics approval and consent to participate

The study was conducted in accordance with the Declaration of Helsinki and approved by the Regional Ethics Committee of Sardinia (Prot. PG/2021/8575) for the use of human samples for research purpose. Informed consent to participate was obtained from all of the participants by surgeons of the Unit of General Surgery of the University of Sassari.

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Not applicable.

Competing interests

The authors declare no competing interests.

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Fenu, G., Griñán-Lisón, C., Pisano, A. et al. Unveiling the microRNA landscape in pancreatic ductal adenocarcinoma patients and cancer cell models. BMC Cancer 24, 1308 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12885-024-13007-w

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12885-024-13007-w

Keywords

BMC Cancer

ISSN: 1471-2407

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