Aptamer Selection For Human PDAC Cell Line PL45 And Identification Of The Target

Pancreatic adenocarcinoma (PAC) is one of the most lethal cancers at present. It is a kind of digestive tract highly malignant tumor which malignant degree is high and is very difficult to diagnose and cure. The pathologic types include Ductal adenocarcinomas, Special type ductal carcinoma origin, Acinar cell carcinoma, Small gland carcinoma, large eosinophilic granular cell carcinoma, and etc. Among them, pancreatic ductal adenocarcinomas (PDAC), as the main class of PAC, often found in the terminal stages, it is account for around 95% of the incidence of PAC. At present, surgery remove is the most effective treatment for PAC, but research shows the five years of survival rate is less than 6%, so it is very important to find new molecular probes and new tumor markers to detect pancreatic ductal adenocarcinoma.In order to obtain the specific identification of pancreatic ductal adenocarcinoma aptamer, we synthesized ssDNA library and conducted the cell-SELEX screening with the pancreatic ductal adenocarcinoma PL45 cell line as target cells, and with hTERT-HPNE cell line as control cells. After 15 rounds of screening, we found the ssDNA library maximum enrichment. Using high-throughput sequencing technologies, the sequence are divided into six groups on the basis of the sequence of homogeneity. We chose the first sequences of each group for further identification, and find the XQ-2 specificity can bind to the human pancreatic ductal adenocarcinoma PL45 cell line with no binding to hTERT-HPNE control cell line. Studies show the dissociation constant of combination with the human pancreatic ductal adenocarcinoma PL45 cell line is about 82.5 nM.By reducing the end of sequences gradually, we analyzed and optimized the sequenced aptamer XQ-2. The result of flow cytometry shows that the aptamer reduced XQ-2 has stronger ability to combine with target cell PL45. Then we did the chemical modification to the aptamer XQ-2 sequence which has been reduced for improving the stability of aptamer in complex environment, it is found that the methoxy-modificated aptamer not only can keep the ability to combine with target cells efficiently but also can significantly improved stability, which is beneficial to its application in biomedicine.To test the aptamer application in complex biological environment, we tested whether the aptamer combined with clinical pathological paraffin tissue section and the vivo tumor in mice. Section results shown that the aptamer recognition efficiency to cancer tissue is as high as 82.5%. It is much higher than the recognition to normal tissues. In mouse xenograft model, we detected the signal indicating the enrichment in tumor location 5 minutes after tail vein injection, and the signal became strongest 60 minutes after injection, while it still lasted 180 minutes after injection. Therefore, the aptamer in complex environment can still keep good recognition ability, and maintain a high level of tissue specificity.At present, there are few biomarkers for pancreatic ductal adenocarcinoma, which are of no specificity and are not widely used, it is necessary to find a new biomarker for pancreatic ductal adenocarcinoma. In this paper, using mass spectrometry, siRNA interference technology, flow cytometry, SDS-PAGE, western blot and other techniques, we successfully identified the specific aptamer XQ-2 and validated its potential biological target protein transferrin receptor 1. Above all, this protein is expected to be a new biomarker for pancreatic ductal adenocarcinoma, and it provides a new method and thought for the treatment to pancreatic ductal adenocarcinoma.