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

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.

Keywords: Biomarkers; CSC; Meta-analysis; MiRNA; PDAC; Serum; Solid biopsies.

Fig. 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

Fig. 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

Fig. 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

Fig. 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

Fig. 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

Fig. 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

Fig. 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