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. 2021 Feb 26:14:1441-1451.
doi: 10.2147/OTT.S296816. eCollection 2021.

Serum Exosomal miRNA-1226 as Potential Biomarker of Pancreatic Ductal Adenocarcinoma

Cheng Wang #  1   2 , Jianhua Wang #  1 , Wenjing Cui #  1 , Yongkang Liu  1 , Hao Zhou  1 , Yajie Wang  1 , Xin Chen  1 , Xiao Chen  1 , Zhongqiu Wang  1
Affiliations

Serum Exosomal miRNA-1226 as Potential Biomarker of Pancreatic Ductal Adenocarcinoma

Cheng Wang et al. Onco Targets Ther. .

Abstract

Background: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related mortality and it is urgent to find biomarkers for early detection of PDAC. Exosomal miRNAs are useful biomarkers for cancer detection. The aims of this study were to investigate the potential role of serum exosomal miRNA in detection of PDAC and to analyze the correlation between the levels of exosome miRNA and the tumor biological behaviors.

Materials and methods: Thirteen serum samples were collected from five patients with PDACs, three healthy individuals (HIs) and five benign pancreatic lesions (BP) for a high throughput profiling analysis to identify an altered miRNA expression patterns in PDAC. Candidate exosomal miRNAs were filtered based on a second independent cohort that included 17 PDACs and 12 benign pancreatic lesions by quantitative real-time polymerase chain reaction (qRT-PCR). Four miRNAs were selected for miRNA validation as PDAC biomarkers in a subsequent set of samples. The association between candidate exosomal miRNA and tumor behavior (tumor invasion or metastases) was evaluated in 17 PDACs. In vitro studies were performed to evaluate the role of candidate exosomal miRNA on cell viability, apoptosis and cell migration in two PDAC cell lines.

Results: The expression of 11 miRNAs showed same trend between PDAC and BP, and between PDAC and HIs. Six of them were upregulated (miR-203b-5p, miR-342-5p, miR-337-5p, miR-149-5p, miR-877-5p, miR-203a-3p), and five were downregulated (miR-1226-3p, miR-3182, miR-625-3p, miR-624-5p, miR-664a-5p). miR-1226-3p was selected as the candidate exosomal biomarker for the PDAC detection. The expression of serum exosomal miRNA-1226-3p was downregulated in PDACs compared to the BPs (p = 0.025). miR-1226-3p had acceptable performance in predicting [area under the curve (AUC) = 0.74] PDAC. Exosomal miRNA-1226-3p level in PDAC with invasion or metastases was lower than that without invasion or metastases (p = 0.028). Transfection of miRNA-1226-3p significantly inhibited the proliferation of PANC-1 and BXP-3 cells, stimulated cell apoptosis and inhibited cell migration.

Conclusion: Serum exosomal miRNA-1226-3p is a potential biomarker in diagnosing and predicting the tumor invasion or metastases of PDAC.

Keywords: exosome; miRNA; miRNA-1226-3p; pancreatic ductal adenocarcinoma.

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Conflict of interest statement

There are no conflicts of interest to declare.

Figures

Figure 1
Figure 1
Identification of exosomes. Exosomes were purified from serum of human and morphological characterization was observed by scanning electron microscopy (A). Exosomes were nanometer-sized micro-vesicles. Exosome (red circle) derived from the serum were cup-like vesicle with the double lipid layer. Exosome protein markers (CD63) were identified by Western blot analysis (B). The small RNA libraries were qualified to sequence (C).
Figure 2
Figure 2
The results of next-generation sequencing analysis. (A) The volcano map of altered expressed miRNAs between 5 PDACs and 3 HIs. (B) The volcano map shown the different expression miRNAs between 5 PDACs and 5 BPs.
Figure 3
Figure 3
The expression level of serum exosomal miRNA-1226-3P from validation cohort (17PDACs VS 12 BPs) by qRT-PCR. The expression of miRNA-1226-3p was significantly higher in the patients with BPs compared to the PDACs (p = 0.025) (A). Receiver operating characteristic (ROC) curve analyses showed that the diagnostic value of miRNA-1226-3p for PDAC and the area under curve (AUC) was 0.74 (95% CI 0.55, 0.92) (B).
Figure 4
Figure 4
A 55-year-old man with PDAC. (A) Axial unenhanced CT image demonstrates an iso-hypoattenuation mass in the tail of pancreas. The tumor shows hypovascular enhancement in the arterial phase (B and C). The CT image demonstrates two metastases in the right posterior lobe of the liver (arrow) (D). The miRNA-1226 expression level is 0.14.
Figure 5
Figure 5
A 62-year-old man with PDAC. (A) Axial unenhanced CT image demonstrates an iso-hypoattenuation mass in the neck of pancreas. The tumor shows hypovascular enhancement in the arterial and venous phase (B–D). miRNA-1226 expression level is 2.84.
Figure 6
Figure 6
The effects of miRNA-1226 on proliferation, apoptosis and migration of pancreatic cell. (A) The expression of miRNA-1226. (B) Cell proliferation for PaNc-1 and BXPc-3 cells after the miRNA-1226 transfection. (C and D) Apoptosis assays for PaNc-1 and BXPc-3 cells after the miRNA-1226 transfection. (E and F) Migration of PaNc-1 and BXPc-3 cells after the miRNA-1226 transfection, evaluated by wound healing assay. (G and H) Migration of PaNc-1 and BXPc-3 cells after the miRNA-1226 transfection, evaluated by transwell assay. Control: BXPC-3-CON, BXPC-3 was transfected with the control plasmid.
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Grants and funding

This work was supported by the National Natural Science Foundation of China (Grant No. 81771899, 81773460), the Key Program of Research and Development of Jiangsu Province (Grant No. BE2017772), Jiangsu Provincial Hospital of Chinese Medicine Peak Academic Talent Project (NO: y2018rc04) and Jiangsu Provincial Traditional Chinese Medicine Science and Technology Development Plan(NO: ZD201907).

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