Application Research Of Aptamers In Tumor Recognition And Treatment

Tumors are a serious threat to human health and have become a major public health problem faced by the world.Early screening and subsequent effective treatment are the key to tumor control.Therefore,the development of new tumor identification probes and efficient tumor drug delivery strategies is of great significance for the diagnosis and treatment of tumors.Aptamers are a type of single-stranded DNA or RNA isolated via Systematic Evolution of Ligands by Exponential Enrichment(SELEX),which can specifically bind to targets with high affinity and hold the advantages of wide target range,high specificity,easy modification and synthesis,excellent thermal stability,and low immunogenicity.Aptamers have great application potential in biomedical fields such as molecular imaging,biosensing,early diagnosis of diseases,and drug targeted therapy.Based on the above research background,we focuses on the discovery of new tumor-targeting aptamers and aptamer-based targeted therapy in this study.The detailed contents are as follows:Renal cell carcinoma(RCC)is the most common malignant tumor of the urinary system,with a high frequency of local invasion and distant metastasis.In Chapter 2,an aptamer named SW-4 against RCC 786-O cells was identified from a known sequence pool named as SWAN library.The identified aptamer exhibited high binding affinity for target cells with dissociation constants in the nanomolar range.Binding analysis revealed that SW-4 only bound to RCC 786-O cells,but not human embryonic kidney 293 T cells or human proximal tubular HK-2 cells,indicating that SW-4 has excellent binding selectivity.By sequence optimization,the 26-nucleotide truncated SW-4b demonstrated improved binding affinity and was internalized into target cells via caveolae-mediated endocytosis in a temperature-dependent manner.Furthermore,fluorescence imaging confirmed that SW-4b accumulated at tumor sites in 786-O xenograft nude mice models and specifically recognized clinical RCC tissues.Meanwhile,SW-4b inhibited proliferation of 786-O cells by arresting cell cycle progression at the S phase.Taken together,these results indicate that SW-4b is a potential candidate for development into a novel tool for diagnosis and targeted therapy of RCC.Uveal melanoma(UM)is the most common primary intraocular malignancy.In Chapter 3,we found that CD71 was overexpressed in UM cell lines and tissues derived from UM.As a result,XQ-2d,which is a DNA aptamer that recognizes CD71,could distinguish UM cells from normal human uveal melanocyte.Furthermore,we conjugated the cytotoxic drug monomethyl auristatin E(MMAE)with XQ-2d.The corresponding conjugate(XQ-2d-MMAE)selectively inhibited the proliferation of UM cells in vitro and in vivo,indicating that XQ-2d-MMAE is a promising novel antitumor agent for targeted treatment of UM.Proteolysis targeting chimera(PROTAC)is a new technology of targeted protein degradation.With the development of PROTAC based on peptide and small molecular,we first proposed the nucleic acid-based PROTAC.In Chapter 4,we coupled the aptamer XQ-2d of targeting CD71 protein with the small molecule ligand Pomalidomide of E3 ubiquitin ligase to construct the Aptamer-based PROTAC.We found that the conjugate can internalize into cells by means of the targeting function of aptamer,and downregulated CD71 expression,which inhibited tumor cell proliferation.Aptamer-based PROTAC has shown its unique advantages as an anti-tumor drug,and is expected to achieve new breakthroughs in the research of targeted drugs.Chemotherapy is currently an effective method for clinical treatment of ovarian cancer,but the occurrence of drug resistance in patients seriously affects the effect of chemotherapy.In Chapter 5,we adopted the cell-SELEX strategy using the ovarian cancer paclitaxel-resistant cell line A2780-PTX as the target cell and the wild-type ovarian cancer cell line A2780 as the control cell.After 15 rounds of selection,an aptamer termed ZH-A2 was successfully selected against target cells.The aptamer ZH-A2 specifically bound to A2780-PTX with high affinity,and the Kd value was determined to be about 48 n M.Furthermore,we characterized the aptamer ZH-A2 and preliminary identified the potential target molecules of ZH-A2 by mass spectrometry.ZH-A2 is expected to provide the powerful molecular probe for ovarian cancer drug resistance research,and the identification of its target molecules is expected to reveal new drug resistance targets.