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. 2018 Sep 13;10(9):328.
doi: 10.3390/cancers10090328.

Clinical Impact of Epithelial-to-Mesenchymal Transition Regulating MicroRNAs in Pancreatic Ductal Adenocarcinoma

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Free PMC article

Clinical Impact of Epithelial-to-Mesenchymal Transition Regulating MicroRNAs in Pancreatic Ductal Adenocarcinoma

Sameer Abdallah Dhayat et al. Cancers (Basel). .
Free PMC article

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive carcinoma entities worldwide with early and rapid dissemination. Recently, we discussed the role of microRNAs as epigenetic regulators of Epithelial-to-Mesenchymal Transition (EMT) in PDAC. In this study, we investigated their value as diagnostic and prognostic markers in tissue and blood samples of 185 patients including PDAC, non-malignant pancreatic disorders, and age-matched healthy controls. Expression of the microRNA-200-family (microRNAs -141, -200a, -200b, -200c, -429) and microRNA-148a was significantly downregulated in tissue of PDAC Union internationale contre le cancer (UICC) Stage II. Correspondingly, stromal PDAC tissue showed strong expression of Fibronectin, Vimentin, and ZEB-1 (Zinc finger E-box-binding homeobox) versus low expression of E-cadherin. Transient transfection of microRNA-200b and microRNA-200c mimics resulted in the downregulation of their key target ZEB-1. Inversely, blood serum analyses of patients with PDAC UICC Stages II, III, and IV showed a significant over-expression of microRNA-200-family members, microRNA-148a, microRNA-10b, and microRNA-34a. Correspondingly, Enzyme-linked Immunosorbent Assay (ELISA) analyses revealed a significant over-expression of soluble E-cadherin in serum samples of PDAC patients versus healthy controls. The best diagnostic accuracy to distinguish between PDAC and non-PDAC in this patient collective could be achieved in tissue by microRNA-148a with an area under the receiver-operating-characteristic (ROC) curve (AUC) of 0.885 and in blood serum by a panel of microRNA-141, -200b, -200c, and CA.19-9 with an AUC of 0.890. Both diagnostic tools outreach the diagnostic performance of the currently most common diagnostic biomarker CA.19-9 (AUC of 0.834). Kaplan Meier survival analysis of this patient collective revealed an improved overall survival in PDAC patients with high expression of tissue-related microRNA-34a, -141, -200b, -200c, and -429. In conclusion, EMT-regulating microRNAs have great potential as liquid and solid biopsy markers in PDAC patients. Their prognostic and therapeutic benefits remain important tasks for future studies.

Keywords: biomarker; epigenetics; epithelial-to-mesenchymal transition; microRNA; pancreatic ductal adenocarcinoma.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Expression of EMT-regulating microRNAs and EMT-proteins in-vitro. (A) EMT-regulating microRNA expression by qRT-PCR in mesenchymal human pancreatic ductal adenocarcinoma (PDAC) cell line Mia-PaCa-2 and epithelial BxPC-3. Fold change values interpreted with the ΔΔCt-method and data expressed as mean ± Standard error of the mean (SEM) (n = 3). * indicates significance (p ≤ 0.05) against Mia-PaCa-2. (B) Immunohistochemistry (IHC) staining of E-Cadherin, Vimentin, Fibronectin and ZEB-1 (Zinc finger E-box-binding homeobox) in human PDAC cell lines Mia-PaCa-2 and BxPC-3. Scale bar: 50 μm. (C) EMT-marker protein expression of ZEB-1 by Western blot in mesenchymal wild type (WT) Mia-PaCa-2 after transient transfection with microRNA-200b and -200c mimics.
Figure 2
Figure 2
EMT-regulating microRNAs in human pancreatic tissue and blood serum samples. Fold change values for tissue and blood serum samples analyzed with qRT-PCR and interpreted with the ΔΔCt-method. Data expressed as the mean ± SEM. * indicates significance (p ≤ 0.05) against healthy controls; # against chronic pancreatitis and § against intraductal papillary mucinous neoplasm (IPMN).
Figure 3
Figure 3
EMT-marker protein expression in human pancreatic tissue and blood serum samples. (A) IHC staining of the epithelial marker E-cadherin, mesenchymal markers vimentin, fibronectin and the transcription factor ZEB-1 in human PDAC and healthy pancreatic tissue. Scale bar: 100μm. (B) Protein expression of ZEB1 in macrodissected tissue specimens analyzed with Western blot. (C) EMT-marker protein analyzed in blood serum samples by Enzyme-linked Immunosorbent Assay (ELISA). * indicates significance (p ≤ 0.05) against healthy controls.
Figure 4
Figure 4
Diagnostic potential of EMT-microRNAs by receiver-operating-characteristic (ROC) analysis in tissue and blood serum samples of PDAC Union internationale contre le cancer (UICC) Stages II-IV.
Figure 5
Figure 5
Prognostic impact of EMT-microRNAs by Kaplan-Meier analysis of overall survival in tissue of PDAC UICC Stage II.
Figure 6
Figure 6
Scheme of the main findings of this study.

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