Study On The Selection And Application Of The Aptamers For Breast Invasive Ductal Carcinoma Based On Ser-SELEX Technology

Breast cancer is one of the common malignant tumors in women.In recent years,the incidence of breast cancer has been increasing rapidly,ranking first among all the tumors in women.The most common is the invasive ductal cancer(IDC),accounting for about50%~80%.The etiology of breast cancer is complex,so early diagnosis and early comprehensive treatment are the most effective means to prevent and treat breast cancer.Aptamers are short oligonucleotide molecules(ssDNA or RNA),which are selected from oligonucleotide libraries synthesized in vitro by Systematic Evolution of Ligands by Exponential Enrichment(SELEX).It can bind to the target molecules with high affinity and specificity by folding into space structure.Compared with antibodies,aptamers are widely used in cancer detection and treatment due to their advantages of high affinity,high specificity,low cost,easy synthesis and low immunogenicity.Therefore,as the most promising molecular recognition probe,aptamers can provide new ideas and tools for the diagnosis and treatment of invasive ductal cancer.In this study,magnetic bead-based Serum-SELEX(Ser-SELEX)technology was used to select specific aptamers for serum of invasive ductal cancer.In order to obtain high-specific aptamers for the serum of invasive ductal carcinoma,normal serum,lung cancer serum and gastric cancer serum were used as negative targets.After 12 rounds of target subtraction-replacement,SELEX was completed and high-throughput sequencing was carried out.By analyzing and comparing,the sequences were divided into four families,and six candidate aptamers(IDC11,IDC12,IDC2,IDC31,IDC32,IDC4)were selected for characteristic analysis.Sequence analysis of aptamers revealed that thymine(T)and guanine(G)were the two most abundant bases,occupying 55~75% of the 40 random sequence domains in six aptamers.By predicting the secondary structure of the aptamer,it is found that the dominant domains of aptamers are stem-loop and bulge-loop structures.QGRS Mapper analysis showed that the aptamer IDC11,IDC12,IDC2 and IDC4 had a tendency to form G-quadruplex structure.The affinity of the aptamer to target molecules was determined by q-PCR,and the affinity constants Kd were in the nanomolar range,indicating that theaptamer has a high affinity to target molecules.q-PCR was used to identify the specificity of aptamer and target.It was found that aptamer IDC11,IDC12,IDC31 and IDC4 had higher specificity,and the positive detection was more than 70%.ELASA(Enzyme Linked Aptamer-sorbent Assay)further showed the specificity of aptamer IDC31.In this study,a specific aptamer for the serum of invasive ductal cancer was obtained,especially the aptamer IDC31,which had a good affinity(16.05 ± 2.19 nM)and specificity(P<0.05)with the target,and the positive detection rate was more than 80%,indicating that the aptamer had great potential in the diagnosis of breast invasive ductal carcinoma.