Screening And Identification Of Aptamers Related To EVs Of Triple Negative Breast Cancer

Triple negative breast cancer has a high degree of malignancy,a high recurrence rate,and a very low five-year prognostic survival rate.The lack of effective early diagnosis markers is one of the main reasons that hinder the early diagnosis and screening of the cancer.Therefore,it is of great significance to screen the specific tumor markers of triple negative breast cancer.Extracellular vesicles(EVs)are hot research topic in tumor liquid biopsy,and their membrane surface proteins are very promising tumor markers.In this work,EVs secreted by the triple-negative breast cancer MDA-MB-231 cell line were directly used as targets,and the aptamers that can specifically bind to EVs were screened by Systematic Evolution of Ligands by an Exponential Enrichment(SELEX).The main research contents are as follows:1. Screening of aptamers specific for triple negative breast cancer EVsEVs from cell supernatant were extracted by particle size selection method,and plasma EVs were extracted by differential centrifugation method.Transmission electron microscopy(TEM),nanoparticle tracking analyzer(NTA)and Western blot(WB)were used to characterize the EVs.TEM images showed clear vesicle structure and exosomes were saucer-like shape.The average particle size of MDA-MB-231,MCF-10A and plasma EVs are 211.8 nm,204.4 nm,and 203.5 nm,and the 1.03×10~9+/-5.56×10~7 particles/m L and 1.44×10~9+/-2.15×10~7particles/m L.Then,the EVs from triple negative breast cancer cell line MDA-MB-231 and breast epithelial cell line MCF-10A were separately used as positive and negative screening targets for SELEX experiment.After 5 rounds of positive screening,the negative screening was added.In the screening process,the screening pressure was gradually increased by shortening the positive screening incubation time,increasing the negative screening incubation time,increasing the number of washings,and performing negative screening with plasma EVs.During the screening process,secondary libraries were prepared by PCR amplification and automatic magnetic separation for the next round of screening.During the optimization of PCR conditions,when the template was 30 ng and the annealing temperature was 58.2℃,the amplification efficiency was the best.Each round of screening needs to be optimized for the number of cycles.After 18rounds of screening,the final screening products were amplified and purified and sequenced by the High-throughput sequencing,finally 7 sequences with high frequency were selected through the analysis of this data.Then,using DNAMAN software to predict the secondary structure of these 7 sequences,the results showed that these sequences have obvious neck Ring structure.2. Identification of aptamers specificity for triple negative breast cancerThe 7 sequences obtained by screening were incubated with MDA-MB-231 EVs,enriched by particle size selection method and used as a template,and detected by fluorescence quantitative PCR.According to the Ct value,Seq1,Seq5,Seq6 Seq7 has the best combination efficiency with EVs from MDA-MB-231 breast cancer cell line.Then,the 4 labeled fluorescent sequences were incubated with EVs derived from breast cancer cell line MDA-MB-231,breast epithelial cell MCF-10A,liver cancer cell He PG2,and lung adenocarcinoma cell A549.After enrichment,they were detected by nanoflow cytometry.It can be seen from the flow histogram that the fluorescence intensity of Seq1 and Seq4 is greater than that of the control group,indicating that Seq1 and Seq4 have the best specificity.3. Sequence optimization of triple negative breast cancer EVs-specific AptamerWithout breaking the stem-loop structure of Seq1,it was truncated for binding specificity experiments without destroying its structure.After incubating the truncated sequences S1a and S1b with the target separately,the complex of EVs and aptamers were enriched,the fluorescence intensity of the combination of S1b and the target was about 20 times higher than that of the control group by nanoflow cytometry,detection,while the combination of S1a and the target were only 2 times higher than that of the control group.this result indicates that S1b has better specificity for triple negative breast cancer cells MDA-MB-231 EVs.This paper successfully screened S1b that can specifically bind to triple-negative breast cancer EVs.The results of this work laid a solid foundation for the subsequent enrichment and identification of new markers for the membrane surface proteins of EVs.