Page 99 - Demo

Plasma microRNAs in oesophageal and gastric cancer975to vitamin supplementation was observed (median expression 0.8, range 0.2-1.2; n=3 vs 10.0, range 2.4-28; n=4; p=0.06). Differences in miR-141-3p and miR-218-5p expression in GC samples could not be analyzed due to the small sample size. DiscussionThe measurement of miRNAs in heparinized plasma samples is challenging. Heparin interferes with the detection of miRNAs by RT-qPCR [34]. Measurement of miRNA expression with ddPCR was managed despite storage in heparin over a longer period by using heparinase treatment to antagonize the effect of heparin. The optimal reagent contained 12 U heparinase with 30 %u00b5l RNA input. According to recent literature an endogenous normalization method was considered not necessary for ddPCR[40]. Cel-miR-39-3p was used as a control for technical variability. Adequate detection of cel-miR-39-3p was observed after a relatively high number of quantification cycles compared to fresh blood samples of the healthy donors (during the optimization experiment) and other publications[33]. The reason for this observation is not clear. A significant relation was found between a high plasma miR-375 expression and a longer OS in patients with EAC treated with first-line palliative chemotherapy with gemcitabine and cisplatin. To our knowledge the prognostic value of circulating miR-375 in patients with EAC treated with palliative chemotherapy has not been shown before. High plasma miR-375 expression could indicate that tumors from these patients overexpress miR-375 although the plasma level may not reflect the level of microRNA in the tumor. Previous studies reported similar findings in tissue samples and cell lines, low levels of miR-375 were associated with a worse prognosis in tissue of Barrett associated EAC[41,42]. Upregulation of miR-375 in tissue and cell lines of pancreatic adenocarcinoma also inhibited cell growth and induced apoptosis, while the downregulation of miR-375 had the opposite effect. miR-375 may suppress the malignant behavior of pancreatic adenocarcinoma through decreased activation of the AKT signaling pathway[43]. An increased AKT pathway has been associated with resistance to several drugs, including cisplatin and gemcitabine. These drugs activate the AKT pathway which may act as a survival pathway[44]. Activating mutations of the AKT signaling pathway are also found in EAC[45]. The relation of miR-375 and gemcitabine and cisplatin is not