Identification Of An Aptamer Targeting Early Clinical Colon Cancer Tissue Using Tissue-SELEX

Colon cancer is a malignant tumor originated from colon tissue,which is the second cancer-related death rate.In recent years,the incidence of colon cancer has an increasing trend.Although fecal occult blood test and colonoscopy can improve the diagnosis rate of colon cancer,non-invasive liquid biopsy is still the expected diagnosis strategy for early detection of colon cancer and reduction of colon cancer-related mortality.At present,most screening methods are not enough to replace the traditional diagnosis methods of colorectal cancer digital rectal examination,imaging examination,colonoscopy,pathological examination),and CEA,as a disease diagnostic marker,has little value in the diagnosis of early tumors.Therefore,it is of great significance for the early diagnosis of colon cancer to find a molecular probe that can specifically identify early colon cancer.As a new probe molecule,aptamer is widely used in biomedicine,biochemistry and other fields because of its small molecular weight,easy modification,wide range of targets,good stability,easy synthesis,high binding force,low immunogenicity and other characteristics.At present,more and more aptamers are obtained by protein SELEX and cell SELEX,but few aptamers can be used in clinical application.Tissue screening technology(tissue SELEX)is aimed at clinical tumor tissue,taking clinical cancer tissue as positive screening sample and clinical paracancerous tissue as negative screening sample to screen the aptamers that can specifically identify clinical tumor samples,and using tissue SELEX technology to screen out the aptamers with good clinical application prospects.Therefore,in this work,our research contents are as follows:(1)Aptamer screening of early clinical colon cancerBased on the classic cell SELEX and the previous experience of tissue SELEX,we optimized the screening process of tissue SELEX by combining the clinical tumor tissue and the aptamer microsphere library modified by affinity group,and set up the positive screen library sample,the positive screen library and the clinical colon cancer tissue incubation sample,the positive screen Library and the adjacent colon cancer tissue incubation sample,the negative screen library sample,the negative screen library and the clinical six experimental groups,including colon cancer tissue incubation samples,negative screening library and colon cancer adjacent tissue samples,sent the amplified products to the second-generation sequencing.Finally,through the quantitative and accurate analysis of flow cytometry and the qualitative and visual evaluation of laser confocal fluorescence microscopy,it is verified that MYH-1 can recognize the colon cancer cell SW480 and the colon cancer cell HCT 116,and has no recognition ability to the human intestinal epithelial cell FHS 74 INT at the same time.(2)the properties of MYH-1 aptamer in early clinical colon cancerWe further identified MYH-1,the aptamer with the largest migration by flow cytometry.Firstly,the dissociation equilibrium constants of MYH-1 in colon cancer cell SW480 and colon cancer cell HCT 116 cells and the stability of MYH-1 in serum were determined.In addition,the target of MYH-1 binding to colon cancer cell SW480 is membrane protein,and the target of MYH-1 binding to colon cancer cell HCT 116 is a protein with high resistance to enzyme action or glycosylation.Later,the internalization experiment found that MYH-1 showed the same binding ability with colon cancer cell SW480 and colon cancer cell HCT 116 at 4 ? and 37 ?,which indicated that MYH-1 could be used conveniently in vitro and in vivo.In addition,we found that MYH-1 may be localized in lysosomes after internalization into cells.The above results show that MYH-1 can target and recognize colon cancer cell SW480 and colon cancer cell HCT 116,so it is expected to be used as a new molecular probe in early clinical diagnosis of colon cancer.