| Cancer is a major global public health issue and the second leading cause of death worldwide after cardiovascular disease.With the growth of the global population and the expansion of the sub-healthy population,the development of cancer is expected to continue to worsen.Cancer cells often rapidly develop due to their vigorous proliferation ability and apoptosis resistance,making it difficult to cure them completely through their transfer and invasion.Therefore,early and accurate diagnosis of cancer is crucial for improving its clinical treatment outcomes and reducing post-treatment complications.Recent research has shown that during the development of cancer,epigenetic changes occur in cells,resulting in the underexpression or overexpression of certain proteins.These overexpressed proteins,as cancer-specific markers,are important targets for diagnosis and treatment.Therefore,the development of appropriate molecular probes for these cancer markers is advantageous for early cancer screening and the development of new treatments.Aptamers are molecular recognition probes composed of 20-120 nucleotide bases(ss DNA or RNA),which have high binding affinity and specificity for a variety of targets,such as metal ions,small molecules,and proteins.Compared to traditional protein antibodies,aptamers have natural advantages such as low production cost,ease of synthesis,easy modification,flexible design,and low immun ogenicity.Due to their unique characteristics,they have become excellent molecular probes for cancer diagnosis and treatment in recent years.According to the above research background,this paper carried out the following work:(1)Currently,the clinical treatment of bladder cancer mainly involves surgical resection,with chemotherapy such as bladder infusion as a supplementary approach.However,for patients in advanced stages or with m etastasis,there is still a need for effective systemic targeted therapy.In Chapter 2,we successfully obtained an aptamer BC-3 that specifically recognizes bladder cancer cell line 5637 using X-Aptamer-based rapid screening technology.We conducted studi es on BC-3’s targeting recognition ability and found that not only does it specifically bind to 5637,T24,and other bladder cancer cell lines in vitro,but it also maintains its recognition performance in vivo and in complex environments such as clinical pathological tissues,demonstrating good biocompatibility and stability.Subsequently,through a series of experiments including proteomics mass spectrometry,we discovered and validated the target protein of BC-3as RPS7.Importantly,we found that BC-3 targets RPS7 protein and is internalized through the meshwork protein-lysosomal pathway mediated by its target protein,ultimately locating to the endoplasmic reticulum.These findings are significant for the development of effective systemic targeted therapy for bladder cancer,particularly for late-stage patients or those with metastasis.(2)In the development and progression of urological system cancers,some cancer cells are shed and excreted in urine.Therefore,a highly sensitive,specific,easy to operate,rapid,and low-cost method for detecting shed cells is crucial for cancer diagnosis and treatment.In Chapter 3,we designed and constructed a stimuli-responsive aptamer probe,Aptamer-Complex,based on different nucleotide fragments of the bladder cancer-specific aptamer BC-3 and using the hybridization chain reaction(HCR)nucleic acid signal amplification technol ogy.We first confirmed the feasibility of the probe and signal amplification strateg y by complementary base closure of the core binding region of the aptamer BC-3 and a series of experiments,such as flow cytometry and gel electrophoresis,and optimized i ts reaction time and reactant ratio.This stimuli-responsive probe can be used to specifically enhance the signal for bladder cancer cells,effectively improving the sensitivity and selectivity of bladder cancer cell detection.(3)Cancer is a complex disease,and its pathogenesis and invasion process are influenced by various factors in the body,such as the vascular microenvironment and cells surrounding tumor cells.In Chapter 4,we developed an optimized Tissue-SELEX method based on the X-Aptamer library.We used clinical bladder cancer tissue as the positive screening sample and bladder cancer adjacent tissue as the negative screening sample.After a single round of screening,we obtained a n aptamer TB-5 that specifically binds to clinical bladder cancer tissue and characterized its clinical tissue recognition and targeting capabilities.We then used a series of experiments including aptamer pull-down,Coomassie blue staining,proteomics mass spectrometry,and si RNA interference to further verify the target molecule of TB-5 as NCL protein.We also explored the correlation between NCL protein expression levels and bladder cancer through bioinformatics and immunohistochemistry experiments,which is of great significance for the early diagnosis of bladder cancer.(4)In Chapter 5,we examined the biological effects of aptamer TB-5 on bladder cancer cells.Firstly,we discovered through a CCK-8 experiment that TB-5 can specifically kill migratory bladder cancer cell line,253J-BV,with high expression of NCL.Additionally,utilizing an autophagy fluorescence transfection experiment,we found that TB-5 induces autophagy in 253J-BV cell line,which effectively kills them.Furthermore,in a scratch experiment,we determined that TB-5 inhibits the migration ability of 253J-BV cells and in a Transwell invasion experiment,we verified that TB-5can also inhibit the invasive ability of migratory bladder cancer cell line,253J-BV.Finally,through transcriptome sequencing and WB experiments,we explained and validated the intrinsic molecular mechanisms of TB-5’s anti-cancer biological function.(5)In the previous study of our research group,we used the human fibrosarcoma cell line HT1080 as the positive screening cell and the h uman normal embryonic lung fibroblast cell line MRC-5 as the negative screening cell line,and utilized the non-SELEX strategy to screen for the aptamer S11 e,which has anti-cancer function.Building on this foundation,in Chapter 6,we further investigated the intrinsic anti-cancer mechanism of S11 e by identifying its functional target protein Diablo/SMAC through aptamer pull-down experiments.Moreover,we explored the intrinsic molecular mechanisms underlying the biological function of S11 e through the expression levels of its related proteins. |
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