Pancreatic Juice-Derived microRNA-4516 and microRNA-4674 as Novel Biomarkers for Pancreatic Ductal Adenocarcinoma

Abstract

Background and aims: Precise diagnostic biomarkers are urgently required for pancreatic ductal adenocarcinoma (PDAC). Therefore, the aim of this study was to identify PDAC-specific exosomal microRNAs (Ex-miRs) from pancreatic juice (PJ) and evaluate their diagnostic potential.

Methods: Exosomes in PJ and serum were extracted using ultracentrifugation and confirmed morphologically and biochemically. PDAC-specific Ex-miRs were identified using our original miR arrays, in which "Ex-miRs derived from the PJ of patients with chronic pancreatitis (CP)" were subtracted from Ex-miRs commonly expressed in both "human PDAC cell lines" and "the PJ of patients with PDAC." We verified the expression of these miRs using quantitative real-time reverse transcription polymerase chain reaction. Changes in serum Ex-miR levels were assessed in 2 patients with PDAC who underwent curative resection. In situ hybridization was performed to directly visualize PDAC-specific miR expression in cancer cells.

Results: We identified novel Ex-miR-4516 and Ex-miR-4674 from the PJ of patients with PDAC, and they showed 80.0% and 81.8% sensitivity, 80.8% and 73.3% specificity, and 90.9% and 80.8% accuracy, respectively. The sensitivity, specificity, and accuracy of a triple assay of Ex-miR-4516/4674/PJ cytology increased to 93.3%, 81.8%, and 88.5%, respectively. In serum samples (n = 88), the sensitivity, specificity, and accuracy of Ex-miR-4516 were 97.5%, 34.3%, and 68%, respectively. Presurgical levels of serum-derived Ex-miR-4516 in 2 patients with relatively early disease stages declined after curative resection. In situ hybridization demonstrated that Ex-miR-4516 expression exclusively occurred in cancer cells.

Conclusion: Liquid assays using the in situ-proven Ex-miR-4516 may have a high potential for detecting relatively early-stage PDAC and monitoring its clinical course.

Keywords: Cancer Early Detection; Exosomes; Extracellular Vesicles; Liquid Biopsy.

Graphical abstract

Figure 1

Characterization of PJ-derived exosomes and overview of the PDAC-specific Ex-miR identification process. (A) TEM showing the ultrastructural appearance of PJ-derived exosomes from patient CP-3. Arrowheads indicate exosomes. (B) NTA showing the size distribution of PJ-derived exosomes from patient CP-3. The black line represents the mean of 5 experiments, and the red line indicates the standard deviation. (C) Exosomes isolated from PJ were analyzed by western blotting using antibodies against exosomal markers, including CD9 (28 kDa), CD63 (53 kDa), CD81 (26 kDa), and HSP70 (53–70 kDa). A representative image of western blot analysis using samples obtained from patients PDAC-2 and CP-3 is shown. (D) Venn diagram representing a compartment of the identified PDAC-specific Ex-miRs. (E) Heat map of the miR array analysis depicts the top 15 most upregulated Ex-miRs. The samples are cell culture supernatants from 3 PDAC cell lines and PJ from 4 patients with PDAC and 3 patients with CP. (F) qRT-PCR analysis reveals the Ex-miR-4484, Ex-miR-6800-5p, Ex-miR-4516, Ex-miR-3940-5p, Ex-miR-3621, Ex-miR-149-3p, Ex-miR-4674, and Ex-miR-3656 expression in the supernatants of PDAC cell lines (n = 3) and PJ of PDAC (n = 4) and CP cases (n = 2). Relative values normalized to those for the control miR-6858-5P are expressed as fold-changes compared with those in patient CP-1. The results are presented as mean ± SD. CP, chronic pancreatitis; Ex, exosome; miR, microRNA; NTA, nanoparticle tracking analysis; PDAC, pancreatic ductal adenocarcinoma; PJ, pancreatic juice; TEM, transmission electron microscopy.

Figure 2

Diagnostic potential of PJ-Ex-miRs-4516 and -4674 for PDAC. PJ-Ex-miR-4516 (A) and -4674 (B) expression in patients with PDAC (n = 15) and CP (n = 11). Fold changes in the expression of the 2 PJ-Ex-miRs are compared with those in the patient CP-1. ROC curves for the sensitivity and specificity of PJ-Ex-miR-4516 (C) and -4674 (D), coupled with their AUC and P values are shown. The results are presented as mean ± SD. AUC, area under the curve; CP, chronic pancreatitis; Ex, exosome; miR, microRNA; PDAC, pancreatic ductal adenocarcinoma; PJ, pancreatic juice; ROC, receiver operating characteristic.

Figure 3

Detection of S-Ex-miRs-4516 and -4674 in the sera of patients with PDAC and diagnostic potential of S-Ex-miR-4516 for PDAC. (A and B) S-Ex-miR-4516 (A) and -4674 (B) expression in patients with PDAC (n = 4) and CP (n = 5), respectively. Relative fold changes compared with the expression in patient CP-3 are shown. (C and D) Expression levels of S-Ex-miR-4516 at the time of preresection and postresection in patients PDAC-4 and PDAC-8. (E) Expression levels of S-Ex-miR-4516 in the PDAC (n = 40) and non-PDAC groups (n = 35; healthy donors = 8, chronic pancreatitis = 9, intraductal papillary mucinous neoplasm = 4, hepatocellular carcinoma = 8, and colorectal cancer = 6). Relative fold-change compared with the expression level in patient CP-3 is shown. (F) ROC curve for the sensitivity and specificity of S-Ex-miR-4516 and its AUC and P values are presented. (G) The focus is on the differential diagnosis of PDAC from other pancreatic diseases, including CP and intraductal papillary mucinous neoplasm (IPMN). S-Ex-miR-4516 expression in patients with PDAC (n = 40) is significantly higher than that in CP (n = 9) and IPMN (n = 4). Relative fold-change compared with the expression level in patient CP-3 is shown. (H) ROC curve for pancreatic diseases is shown. The values are presented as mean ± SD. CP, chronic pancreatitis; Ex, exosome; miR, microRNA; PDAC, pancreatic ductal adenocarcinoma; S, serum.

Figure 4

In situ hybridization (ISH) of miR-4516 (A) and miR-4674 (B) in PDAC tissues Representative ISH images in patient PDAC-4 using the LNA-miR-4516 probe, LNA-miR-4674 probe, and scrambled-miR control probe are shown. Scale bars, 100 μm. LNA, locked nucleic acids; miR, microRNA; PDAC, pancreatic ductal adenocarcinoma.

Figure 5

Functional analysis of miR-4516 in PDAC cells. (A) Expression of miR-4516 in PANC-1 cells and its knocked-down counterparts is demonstrated. Fold-change in expression is shown for miR-4516 relative to its levels in PANC-1 cells. Relative fold changes compared with the expression in PANC-1 cells are shown. The values are presented as mean ± SD. (B) MTT proliferation assay shows a significant inhibitory effect of miR-4516 knockdown on cellular proliferation in PANC-1 cells. ∗∗P < .01, ∗∗∗P < .001. miR, microRNA; PDAC, pancreatic ductal adenocarcinoma.