Selection Of Aptamers Against Human Breast Cancer Cell SK-BR-3 And Their Applications For Molecular Classification Of Breast Cancer

Breast cancer is a highly heterogeneous tumor with four major molecular subtypes.Different breast cancer subtypes show distinct response to therapy and prognosis.Most currently,the commonly used methods for breast cancer molecular classification is immunohistochemistry(IHC),which is recommended as the gold standard for the pathogenic detection of breast cancer.However,IHC is always fussy,time-consuming and also very expensive,and cannot meet the requirement of fast detection in clinics.Therefore,it is in urgent need to develop effective,fast and more sensitive methods for breast cancer molecular classification,which is of great significance for decreasing the mortality and improving the therapeutic effect of breast cancer.Aptamer is a short RNA or ssDNA that is derived from SELEX(Systematic Evolution of Ligands by Exponential enrichment)method and can specifically bind with varieties of targets.Compared with antibodies,aptamer is more specific with high binding affinity,good stability,easy modification,low cost and a wide arrange of targets,which is known as "chemical antibodies".Recently,the biomedical application of aptamers for biological detection,molecular diagnosis,biological imaging,tumor molecular classification and drug delivery has attracted the most attention.Moreover,the Cell-SELEX,which is evolved from general SELEX,can help select aptamers against various tumor cells without knowing the targets.What is more,aptamers evolved from Cell-SELEX can sensitively bind with the targets with native conformation,surmounting the drawback of aptamers selected by purified protein based SELEX that they are not sensitive enough to native targets.In this study,six specific aptamers of human breast cancer cell SK-BR-3 were selected by Cell-SELEX in vitro.Then,the characteristics and optimization of the aptamers were analyzed systematically.Finally,the optimized aptamer sk6Ea was identified,which shows great potential in breast cancer classification.The main points of this study are as follows:1.Selection of aptamers against human breast cancer cell SK-BR-3In this detailed Cell-SELEX procedure,human breast cancer cell SK-BR-3 was set as the target cell,human breast cancer cell MCF-7,MDA-MB-231 and human normal breast epithelial cell MCF-10A were set as the negative cells.After successive selection of 21 cycles,the library was cloned and 30 randomly picked clones were sequenced.Finally,six aptamers were identified and the secondary structures of the aptamers were simulated.All the aptamers had typical stem-loop structures.2.Study of the characteristics and optimization of aptamersThe characteristics of the six aptamers were systematically studied,all the aptamers showed high specificity and affinity toward breast cancer cell SK-BR-3,among which sequence 6 owned both the optimal specificity and affinity.Then,the length of sequence 6 was optimized and a new aptamer sk6Ea of 53 nt was identified.Finally,the specificity,binding affinity,the influence of different temperatures and enzymatic dissociations for the binding ability of sk6Ea were tested.The results indicated that the aptamer sk6Ea had high specificity and its Kd value is 49.32±14.53 nM.Aptamer sk6Ea could bind with SK-BR-3 cells very well at any temperature of 4 ?,25? and 37 ? without significant difference.When SK-BR-3 cells were dissociated by trypsin and proteinase K,aptamer sk6Ea showed no binding with the target cell SK-BR-3,indicating the target of sk6Ea was likely to be a protein.3.Application of aptamers for breast cancer classificationAptamer sk6Ea was utilized for breast cancer classification including breast cancer cells classification,breast cancer tissue classification and in vivo imaging.The results showed that the aptamer sk6Ea could distinguish breast cancer cell SK-BR-3 from MCF-7,MDA-MB-231 and normal breast epithelial cell MCF-10A.As for breast cancer tissue fluorescence staining,aptamer sk6Ea could distinguish HER2 overexpressed breast cancer tissue from Luminal A,Luminal B,triple negative breast cancer tissue and normal breast tissue.For in vivo imaging,aptamer sk6Ea could distinguish SK-BR-3 xenografted tumor from MCF-7 xenografted tumor.The biodistribution of aptamer sk6Ea in vivo was also examined at 1 h post-injection.The results showed that the tumor site had a stronger fluorescence singal than kinder,liver,lung,spleen and heart.All the results indicated that aptamer sk6Ea was very promising for breast cancer classification and in vivo applications.