Materials and methods
Patients and tissue samples
Specimens from 74 patients with PDAC (37 males, 37 females) and 18 patients with CP (12 males, 6 females) who had undergone resection from August 2001 through April 2012 at the Department of Surgery (Faculty Hospital Brno, Czech Republic) were used. Moreover, control pancreatic tissue samples without signs of inflammation or dysplastic changes from 9 patients were included. All subjects were of the same ethnicity (European descent). The ages of patients ranged between 30 and 79 years with a median of 60.5 years. Written informed consent was obtained from all patients and the study has been approved by the local Ethical Board.
Isolation of total RNA enriched for small RNAs was performed using formalin-fixed paraffin-embedded samples with more than 90% of cancerous, inflammatory or normal tissue. All samples were deparaffinized, treated with DNAse I, proteinase K and RNA extraction was undertaken using mirVana miRNA Isolation Kit (Ambion Inc, Austin, TX, USA) according to the manufacturer's instructions. Concentration and purity of RNA were determined spectrophotometrically by measuring its optical density (A260/280 > 2.0, A260/230 > 1.8) using Nanodrop ND-1000 (Thermo Fisher Scientific, Waltham, MA, USA).
Real-time quantification of miRNAs
Complementary DNA was synthesized from total RNA according to the TaqMan MicroRNA Assay protocol (Applied Biosystems, Foster City, CA, USA) using T100™ Thermal Cycler (Bio-Rad, Hercules, CA, USA). Real-Time PCR was performed according to the standard protocol using the TaqMan MicroRNA Assay kit and the Applied Biosystems 7500 Sequence Detection System (both Applied Biosystems, Foster City, CA, USA).
Data normalization and statistical analysis
The threshold cycle data were calculated by SDS 2.0.1 software (Applied Biosystems, Foster City, CA, USA). All real-time PCR reactions were run in triplicates. The average expression levels of all measured miRNAs were normalized using miR-1233 (Assay No. 002768; Applied Biosystems, Foster City, CA, USA) and subsequently analyzed by the 2-ΔCt method. Statistical differences between the levels of analyzed miRNAs were evaluated by non-parametric Mann-Whitney U-test and Kruskal-Wallis test. Survival analyses were carried out using the log-rank test and Kaplan-Meier plots approach. All calculations were performed using GraphPad Prism version 5.00 (GraphPad Software, San Diego, CA, USA). P-values of less than 0.05 were considered statistically significant.
Discussion
Successful management and treatment of PDAC patients remains one of the key challenges in clinical oncology. Although the early stages of the disease can be treated surgically, most patients are diagnosed at advanced stages, when surgical resection is not possible. Moreover, differential diagnosis of pancreatic lesions is challenging [
10]. Therefore, there is an urgent need for novel diagnostic biomarkers that would enable precise differential diagnosis of pancreatic lesions. In addition, molecular biomarkers that could serve as prognostic factors would be very valuable.
MiRNAs have been described to be deregulated in a variety of solid cancers, including PDAC [
11-
13]. In this study, the utility of miR-21, miR-34a, miR-198 and miR-217 as novel diagnostic and prognostic biomarkers of PDAC was evaluated. Consistently with the previous data, significantly increased levels of miR-21 and miR-198 [
7,
14,
15] and decreased levels of miR-217 [
7,
15,
16] have been observed in PDAC tissue. Despite the fact that miR-34a is generally described as an important tumor suppressor [
12,
17], the expression of this miRNA has been significantly higher in our PDAC samples compared to healthy tissue. Therefore, it seems that this miRNA may have dual functioning as both oncogene and tumor suppressor, depending on the cellular and tumor microenvironment [
18].
Importantly, all analyzed miRNAs had a high potential to differentiate CP from PDAC tissue, therefore, they might be involved in early events of pancreatic carcinogenesis. Habbe
et al. [
19] showed that miR-21 is highly expressed in early non-invasive intraductal papillary mucinous neoplasms. Further, using
in situ hybridization increased miR-21 expression was found in 79% of PaCs; however, only 8% of benign pancreas and 27% of CP expressed this miRNA suggesting its important role in the development of PaC [
20]. MiR-217 was described to play a crucial role in regulation of acinar-to-ductal metaplasia [
21], in addition, this miRNA is deregulated not only in PDAC but also in its precursor lesions, compared to non-neoplastic pancreatic tissues [
15]. The function of miR-34a and miR-198 in early development of PaC has not been described till now, nevertheless, it seems that miR-21, miR-34a, miR-198 and miR-217 could be used as tumor markers to distinguish PDAC and its precursors from a benign lesions.
Given the dismal prognosis of PaC, second aim of this study was to identify miRNAs with the potential to differentiate between patients with good (DFS ≥ 12 months, OS ≥ 18 months) and poor (DFS < 12 months, OS < 18 months) prognosis. We proved that high levels of miR-21 and/or miR-198 significantly correlate with poor prognosis. Concerning miR-21, several studies have been previously published demonstrating prognostic function of this miRNA in PaC [
12,
20]. Moreover, high levels of miR-21 were associated with a poor response to gemcitabine and its levels were increased after the exposure to this drug [
22,
23]. Concerning miR-198, there are two contradictory reports analyzing the prognostic function of this miRNA in PDAC. Whereas Marin-Müller
et al. [
24] described high levels of this miRNA to be associated with good prognosis, Schultz
et al. [
7] observed correlation between over-expression of miR-198 and poor prognosis. Taken together, our data indicate that miR-21 and miR-198 could be used as potential prognostic biomarkers in PDAC patients. Importantly, the value of clinical utility of these miRNAs could be enhanced by measurement prior to resection in PDAC tissue obtained by endoscopic ultrasound-guided fine needle aspirates [
25] with the aim to improve the clinical management of borderline resectable cases and identification the patients who will benefit most from the surgical resection.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.
The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
PVF, IK, SK, VP, JHl, JM, JHa and PK performed the research; OS, IK, ZK and RV conducted the project; JHa, JM, LK and MH performed the histopathological analysis of tumour samples; PVF, SK and OS performed statistical analysis; PVF, IK and OS drafted the manuscript; ZK and MH edited and revised the manuscript. All authors read and approved the final version of the manuscript.