Research Of The Application Of Plasma Exosome Mirna In The Diagnosis Of Pancreatic Cancer

Background and purposeThe pathogenesis of pancreatic duct adenocarcinoma(PDAC)is concealed,and the mortality rate is high.The testing and examination methods are difficult to meet the needs of clinical diagnosis.The most commonly used diagnostic marker for PDAC is CA19-9,which has a low diagnostic rate for early patients and is susceptible to jaundice.Therefore,we urgently need new tumor biomarkers to assist the diagnosis of pancreatic cancer.Exosomes secreted by tumor cells can contain nucleic acids and proteins specifically expressed by tumors,so it is expected that we can diagnosis pancreatic cancer by detecting exosome components in peripheral blood of pancreatic cancer patients.miRNAs are a kind of small non-coding RNA,it can target mRNAs,inhibit its translation and degradation.Some studies have reported that miRNA is involved in the proliferation,migration,invasion,metastasis and chemotherapy tolerance of pancreatic cancer.MiRNAs have the function of both oncogenic factor and tumor suppressor.Previous studies have also found that miRNAs expression in tumor tissues are different from that in normal tissues.Therefore,we believe that there may be differences in exosome miRNA in peripheral blood between pancreatic cancer patients and healthy people,and these differences are expected to be used in the diagnosis of pancreatic cancer.MethodsFrom June 2017 to June 2018,we collected 141 samples of patients with pancrease disease.After excluding patients whose pathological examination does not meet the requirements and samples of hemolysis,a total of 90 patients were included in the training cohort and the validation cohort.The training cohort consists of 27 patients with PDAC(11 metastatic PDAC involved),15 chronic pancreatitis.The validation cohort included 30 patients with PDAC(12 metastatic PDAC involved)and 18 chronic pancreatitis.Plasma was separated and exosomes were purified by centrifugation.Then miRNA was extracted from exosomes and sequenced.The miRNA expression of all samples was standardized.Bayesian networks were established based on the standardized miRNA expression levels of all patients to identify plasma exosome miRNAs potentially associated with the diagnosis and prognosis of PDAC.The receiver operating characteristic curve(ROC-curve)was used to analyze the diagnostic value of individual miRNAs and paired miRNAs for PDAC in the training group,and miRNAs with high diagnostic value were veryfied in the validation cohort.Meanwhile,prognostic data of patients were collected to find miRNAs related to the prognosis of patients.ResultTo characterize the exosomes obtained in this study,Western blotting(WB)and scanning electron microscopy were performed.The concentration of miRNA also met our research needs.According to the expressions of miRNA in different samples,we plotted the bayesian network.The bayesian network consisted of 337 nodes and 713 edges.In the bayesian network,miR-95-3p existed downstream of pancreatic duct adenocarcinoma and miR-26-5p existed upstream of chronic pancreatitis.The diagnostic sensitivity and specificity of miR-95-3p/miR-26-5p for PDAC in the training group were 81.5% and 93.3% respectively.The sensitivity and specificity of miR-95-3p/miR-335-5p in the validation group were 86.7% and 100%,respectively.MiR-335-5p/miR-340-5p was associated with the diagnosis of metastatic PDAC.The AUC of the training group was 0.798,and the AUC of the validation group was 0.798.Combined with patients’ survival data,the survival time of patients with the ratio of miR-335-5p/miR-340-5p lower than 0.15 was significantly longer than that of patients with the ratio higher than 0.15(412 days vs.207 days,P=0.0200).ConclusionIn this study,the bayesian network was formed by the expression of plasma exosome miRNA,from which miRNA biomarkers could be identified for the diagnosis and prognosis of PDAC.The ratio of miR-95-3p/miR-26-5p could be used to assist the clinical diagnosis of PDAC.The ratio of miR-335-5p/miR-340-5p can assist in the diagnosis of metastatic PDAC and in the prediction of patients’ prognosis.