| BACKGROUND: Pancreatic cancer(Pa C) is found in patients at late stage with hidden incidence. Its high degree of malignancy always leads to a poor Pa C prognosis and only 5% survival rates of 5-years. Therefore, searching for innovative Pa C biomarkers has become extremely urgent and important tasks in the pancreatic research field. Back to early 20 th century, there has been a close relationship between Pa C and diabetes mellitus(DM) or hyperglycaemia. Many recent studies relating to large-scale clinical epidemiological investigations and relevant fundamental experimental researches have shown that patients carrying 1-3 years diabetes having a high risk of being diagnosed with pancreatic cancer, that has a significant clinical impact on Pa C-associated DM(Pa C-DM) studies.The pathogenesis of Pa C-DM is currently not clear since this Pa C related glucose metabolism disorder is obviously not caused by tumor invasion or chronic obstructive pancreatitis. For patients with early Pa C, who tend to have high frequency DM and glucose stimulated insulin secretion(GSIS). The physiological status favors humoral process rather than local tumor effect. Currently, many in vitro and human studies indicate that insulin resistance and β-cell damage found in Pa C may be caused by secretion of low level proteins or polypeptides from the tumors. For other insulin resistance cases such as obesity, and polycystic ovary syndrome, lacking of insulin related β-cell damage would not cause diabetes. The damage of β-cell is normally caused by humoral circulatory factors. Insulin tend to increase its resistance in Pa C status and causes β-cellmetabolic disorder and even diabetes. Therefore, we speculate the β-cell is the main target for Pa C-DM.The discovery of circulatory mi RNA in serum has provided a great opportunity for early diagnosis of Pa C. These stable existing mi RNAs have been potentially used for early diagnosis of cancer and other diseases, individual treatment, prognosis and many other aspects, which would provide an innovative non-invasive methodology for clinical value, as well as social economic benefits. Instead of free radical forms, most of circulatory mi RNAs are captured by microvesicles(MV). MVs can freely enter into target cells so as to change the biological activity of the target cells. Its free-passing ability provides a new idea for the investigation and progression of the molecular study of Pa C-DM.AIMS: We aimed to construct of serum mi RNA expression profile of Pa C-DM patients and evaluation of relavent diagnostic value, predict and verify of significant differential expression of particular mi RNA target genes and investigation of the effect of glucose metabolism via subcutaneously Pa C tumor transplantation and islet β-cell function.METHODS: Sera of newly enrolled Pa C-DM, type II DM patients and normal controls were collected. We screen of Pa C-DM specific serum mi RNA by using Taq Man Low density array and quantitative PCR techniques to construct mi RNA expression profile. Validation of the microarray results uses stem-loop quantitative PCR. In vitro, isolation of MV from cell supernatant uses differential centrifugation and density gradient centrifugation method. Identification of mi RNAs in MV and quantification of the significant expressed mi RNAs. Identification of mi RNAs’ biological effect on islet β-cells via manipulating either mi RNAs enrichment or depeltion mi RNA target gene prediction based on several authorized websites. Verification of target genes via dual luciferase reporter assay, quantitative the PCR and Western blot. SW1990 cell subcutaneous xenograft mouse model construction and identification of the metabolic effect caused by subcutaneous tumor transplantation using glucose tolerance test(IPGTT) and insulin tolerance test(ITT) detection. The specific mi RNAs in serum and target gene expression was detected by using quantitative PCR and Western blot.RESULTS: Serum samples of Pa C-DM(70 cases), DM(75 cases) and normal controls(70 cases) were tested using low-density chip technology and q RT-PCR method. Six mi RNAs showed increased expression level in Pa C-DM patients: mi R-483-5p, mi R-19 a, mi R-29 a, mi R-20 a, mi R-24, mi R-25. And in type II diabetes and normal controls distinct expressional changes indentified and were statistically significant(with a fold change of 2.45 to 3.88, p <0.0001). 6 mi RNAs diagnosed from Pa C-DM showed the area under the ROC curve AUC=0.841, which was higher than tumor marker CA199(AUC = 0.741). Mi R-19 a in Pa C-DM patients increased significantly, and MV extracted from Pa C-DM serum and cultured Pa C cells speficically captured mi R-19 a. Three kinds of Pa C cells Bxpc3, PANC-1 and SW1990 secreted mi R-19 a could enter into insulin βcells, which in turn downstream regulating insulin transcriptional Neuro D1 level. MV secreted by islet β-cells can increase the apoptotic effect of pancreatic islet ? cells and primary islet. In vivo, SW1990 pancreatic subcutaneous Pa C transplantation induced an increase of the serum mi R-19 a level and deregulation of Neuro D1.CONCLUSION:(1) Six mi RNAs(mi R-483-5p, mi R-19 a, mi R-29 a, mi R-20 a, mi R-24, mi R-25 6 micro RNAs) found to have potential for the early diagnosis of diabetes induced Pa C, especially for diagnosing patients with Pa C who suffered from new-onset type II diabetes less than 3-years. As important serum markers,(2) The serum and the supernatant of Pa C cell culture of Pa C-DM patients identified with MVs that carries particular mi R-19 a. In vivo and in vitro experiments showed that three kinds of Pa C cells Bxpc3, PANC-1 and SW1990 secreted mi R-19 a could enter into insulin β cells, which in turn downstream regulating Neuro D1 level and affect insulin secretion and lead to Pa C-DM. MV secreted by insulin cells can trigger an increase of apoptosis of pancreatic islet ? cells and primary islet, which participates Pa C-DM occurance. |
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