Preliminary Study Of MicroRNA Abnormal Expressed In Pancreatic Cancer

BackgroundThe prevalence of pancreatic cancer has shown an increased tendencies over the past decades. Pancreatic cancer is usually diagnosed at an advanced stage due to no specific clinical presentation and the lack of reliable early diagnostic markers, the effectiveness of treatment is poor, the 5-year survival rate is under 5%. Therefore, there is an obvious need to discover unique early detection marker(s) and molecular targets to combat pancreatic cancer. This paper discusses abnormal expression of microRNA in pancreatic cancer.ObjectivesTo describe miRNA profile in cell line ASPC-1, pancreatic cancer and normal pancreatic tissues, and to screen abnormal miRNA in pancreatic carcinoma cell line and tissue. Choose two of the differentially expressed miRNAs: miR-21 and miR-10b, they were confirmed by real time RT-PCR assay.Unique patterns of deregulated miRNA expression in pancreatic cancer provides valuable information that may serve as molecular biomarkers for tumor diagnosis, identifying low and high risk populations of patients, disease prognosis , prevention of cancer, and prediction of therapeutic responses.Methods1. miRNA Microarray was used to determine differential expressed miRNA in pancreatic cancer cell line ASPC-1 and malignant pancreatic tissues compared with normal pancreatic tissues. The total RNA were extracted and isolated by TRIzol(?)Reagent. Hybridizations were made by applying the miRNAs to Exiqon miRNA microarray. Scanning is performed with the Axon GenePix 4000B microarray scanner. GenePix pro V6.0 is used to read the raw intensity of the image. Date analysis was proceeded by Microsoft Excel.2. According to the measuring results of miRNA profile. We choose two of the differential expressed miRNA . Detected miR-10b , miR-21 expression in pancreatic cancer tissues and normal pancreatic tissues by real time RT-PCR assay(SYBR GREEN). To confirm the results of miRNA Array. the method of RT-PCR in accordance with the operation procedure of Invitrogen-NCode(?) EXPRESS SYBR(?) GreenER(?) miRNA qRT-PCR Kit Universal, we use U6 RNA as internal standard to carry out normalization, data analysis was proceeded by LightCycler480 1.5.0 software.Results1. Contains more than 1700 miRNAs tested by miRNA Microarray(covering all microRNAs annotated in miRBase 14.0, as well as all viral microRNAs, related to these species), 306 miRNAs were found to be down-regulated and 354 up-regulated in pancreatic cancer tissues and cell lines.2. The melting curves of miRNA RT-PCR is simple spike, it illustrate the specificity of PCR amplification is well. Compared with normal pancreas tissues, the values of relative quantification(RQ)of miR-10b in human pancreatic cancer tissues and normal pancreas tissues are (0.0743±0.0222 )and(0.0287±0.0129),there is a significant difference between human pancreatic cancer tissues and normal pancreatic tissues (p<0.05). miR-21 expression in pancreatic cancer tissues and normal pancreatic tissues are (0.3062±0.1117) and (0.0240±0.0137), there is a significant difference between them (p<0.05).Conclusion1. There exists a lot of abnormal expressed miRNAs molecules in Pancreatic cancer tissues and cell lines.2. RT—PCR results were accordant to the miRNA microarrays results and indicated a positive correlation between the quantities of transcripts measured by both microarray and RT-PCR assay. miR-10b, miR-21 are high expression in the pancreatic cancer tissues, which suggested that miR-10b, miR-21 may play an oncogene role in the occurrence and development of cancer.