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Screening of differential miRNAs in serum exosomes of early pancreatic cancer patients and the diagnostic value of hsa-let-7f-5p

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Journal Title:
Chinese Journal of Pancreatology
Key Word:
胰腺肿瘤;外泌体;微小RNA;糖类抗原19-9;胰腺炎,慢性;Pancreatic neoplasms;Exosomes;microRNA;Carbohydrate antigen 19-9;Pancreatitis, chronic

Abstract´╝Ü Objective:To screen the serum exosomal microRNAs differentially expressed in early pancreatic cancer patients and evaluate the diagnostic value of exosomal hsa-let-7f-5p.Methods:From January 2019 to January 2020, 19 patients with early pancreatic cancer (early pancreatic cancer group) and 16 patients with chronic mass-forming pancreatitis (pancreatitis group) were selected from Affiliated Hospital of Nanjing University of Chinese Medicine who underwent surgery and were confirmed by pathology. Serum samples of the two groups of patients were collected. At the same time, serum samples of 19 healthy volunteers were selected as the normal control group. The exoEasy Maxi Kit was used to isolate serum exosomes. The structural characteristics of exosomes were observed by transmission electron microscopy (TEM). The particle size of exosomes was observd by nanoparticle tracking analysis. CD 63 and CD 81, the specific protein marker on the surface of exosomes, were identified by western blotting. The total RNA of exosomes was extracted by the miRNeasy Serum/Plasma Kit, and a small RNA library was constructed after quality inspection. With reference to the small RNA database, the differentially expressed exosomal microRNAs in early pancreatic cancer group, pancreatitis group and normal control group were filtered out. The miRNA candidates were validated by quantitative polymerase chain reaction (qPCR) and different expressions of them were analyzed. The role of target genes and metabolic pathways of candidate miRNAs in the occurrence and development of early pancreatic cancer were analyzed by gene ontology (GO) and Kyoto Encyclopeda of Genes and Genomes(KEGG) enrichment pathway. Results:The isolated serum exosomes can be seen to have cup-like vesicle with the double lipid layer by TEM. The main peak of the particle size of target exosomes was about 150 nm. The expression of exosome specific protein markers CD 63 and CD 81 was positive. Comparing the expression of miRNAs among early pancreatic cancer group, pancreatitis group and normal control group, the specific tumor marker exosomal hsa-let-7f-5p was screened out in this study, and its expression in early pancreatic cancer group was significantly higher than that in pancreatitis group and normal control group (both P values <0.05). Receiver operating characteristic curve analysis (ROC) showed that the area under curve (AUC) of exosomal hsa-let-7f-5p to distinguish pancreatic cancer from pancreatitis was 0.843 (95% CI 0.640-1.000). The sensitivity and specificity were 100% and 81.82% respectively. The AUC for distinguishing pancreatic cancer from normal controls was 1.000 (95% CI 1.000-1.000), and both sensitivity and specificity were 100%. The diagnostic efficiency of exosomal hsa-let-7f-5p was equivalent to that of CA19-9 ( P>0.05). The GO analysis results showed that target genes of exosomal hsa-let-7f-5p were mainly involved in complement activation lectin pathway in biological processes, and the proteins expressed by target genes were mainly distributed in cilium, and molecules mostly functioned by combining with nitric-oxide synthase. The KEGG pathway enrichment analysis showed that the target genes were closely related to MAPK signaling pathway. Conclusions:Serum exosomal hsa-let-7f-5p has the potential to be a diagnostic biomarker for early pancreatic cancer.

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