| Background and Objective:Breast cancer is the most common malignant tumor in women,and its mortality rate is always the highest.Therefore,if breast cancer patients want to achieve good treatment effect and prognosis survival,early screening and diagnosis are essential.Among the basic detection methods for breast cancer,X-ray photography is relatively important.However,due to its low sensitivity of less than 60% in the detection of breast cancer,it often cannot assist clinicians in diagnosis,resulting in fuzzy diagnosis results with high false negative and false positive rates.The development of new therapeutic strategies,especially targeted therapies,has greatly reduced the mortality rate of breast cancer.At the same time,there is an increasing demand for personalized and precise treatment in the treatment of cancer.Therefore,we urgently need to identify biological targets that play a key role and can be used for clinical diagnosis and prognosis.Bioinformatics analysis is one of the newest fields of biological research,which uses mathematical statistical methods to process and analyze biological data.In the past few years,the rapid growth of informatics technology in the era of "genomics" and "omics" has shown great advantages.The combination of bioinformatics analysis and traditional experimental methods has promoted the development of biological research and accelerated people’s understanding of biological information.At the same time,the analysis and interpretation of biological data helps people better understand and study the progression of diseases and select the optimal treatment.In combination with computer technology,the study of life is moving from a confined laboratory environment to a "virtual laboratory".Therefore,bioinformatics analysis can help us to find molecular biomarkers that can be used for the diagnosis and treatment of breast cancer.Breast-conserving surgery is the most important treatment for early-stage breast cancer.However,despite the continuous improvement of surgical methods,postoperative pathologic evaluation shows that 20% to 60% of patients who undergo breast-conserving surgery leave residual tumor tissue.Therefore,efforts are being made to develop new methods for tumor edge assessment during surgery.Activable fluorescent probes can identify cancer biomarkers rapidly,sensitively and accurately.Recently,intraoperative fluorescence imaging navigation technology has gradually entered everyone’s field of vision.With the discovery of new fluorescent probes,surgeons can more accurately distinguish the boundaries of tumors,identify residual tumors and completely remove the lesions during surgery.These technologies have revolutionized cancer surgery by maximizing the efficiency of tumor resection and improving patient survival.In addition,with the continuous development of molecular design theory and nanotechnology,more novel multimodal and multifunctional fluorescent probes will be designed and developed,showing great potential for clinical transformation.Therefore,this study mainly explored appropriate molecular markers of breast cancer through bioinformatics analysis methods.Then a specific fluorescent molecular probe was constructed according to the structural characteristics of the molecular target,and its physical and chemical properties and application were further explored.Methods:1.The correlation between CES2 and breast cancer was analyzed based on bioinformatics methodsWe extracted 1222 samples from the TCGA database,there were 113 normal breast samples and 1109 invasive breast cancer samples.Download and standardize the relevant data information of all samples.We first analyzed the expression of CES2 in 30 human cancers,1109 invasive breast cancer tissues,113 normal breast tissues,and 113 matched breast cancer tissues.Then the survival package and p ROC package were used to analyze the prognosis and diagnostic value of CES2 in breast cancer patients.Then we built PPI network in string database.Then we constructed the protein-protein interaction network of CES2 through string tool,and carried out go and KEGG enrichment analysis through clusterprofiler package to explore the biological function of CES2.Then we analyzed the relationship between CES2 and breast cancer infiltrating cells through GSVA package.Finally,we verified the expression of CES2 m RNA level and protein level from the cell level in breast cancer cell lines through Western Blot and Real-time PCR experiments.2.Explore the biological application of CES2-targeted fluorescent probe DDAB in breast cancerFirstly,CES2-targeted fluorescent probe DDAB was constructed in our laboratory,and its biological toxicity was explored through CCK-8 experiment,and the safe biological dose of DDAB probe in breast cancer cells was detected.Then,the cell fluorescence imaging experiments were conducted to study the imaging performance of DDAB probes in various types of breast cancer cell lines.At the same time,flow cytometry fluorescence assay showed that the DDAB probe had the ability to label large amounts of free normal breast and breast cancer cells.Results:1.The correlation between CES2 and breast cancer was analyzed based on bioinformatics methodsWe extracted the data of 1222 samples through TCGA database,and obtained that the expression difference of CES2 in 24 of 30 cancer types was significant.In non paired and matched samples of invasive breast cancer,the expression level of CES2 in normal breast tissues was significantly higher than that in breast cancer tissues.The low expression level of CES2 in stage T4 of breast cancer is associated with poor prognosis.ROC analysis showed that the diagnostic value of CES2 was significant in all stages of TNM,IDC and ILC.The co-expressed genes with CES2 mainly include ESD,CES1,UGT1A8,UGT1A1,UGT1A6,GUSB,CYP3A4,LRAT,RBP4 and CDA.CES2 is mainly enriched in the metabolic pathway.The results also suggest that CES2 may be involved in the immune infiltration of breast cancer.The expression of CES2 was significantly higher in normal breast cells than in other breast cancer cells at both protein and RNA levels.2.Explore the biological application of CES2-targeted fluorescent probe DDAB in breast cancerIn the early stage of our laboratory,we successfully constructed a probe molecule called DDAB,which is a new near-infrared fluorescence probe for detecting CES2 activity in breast cancer cells and tissues.CCK-8 results showed that the biological toxicity of DDAB probe was relatively low,and had little effect on cell proliferation.The cells with high DDAB expression in CES2 showed good imaging performance,and the fluorescence intensity of each breast cancer cell line was significantly lower than that of normal cells.DDAB probe also has good labeling and sorting ability in a large number of breast cells,and its ability to recognize normal breast tissue is much greater than that of various types of cancer cells.Conclusion:1.Bioinformatics analysis showed that CES2 was expressed in many cancer tissues and normal tissues,and the difference was significant.The expression level in breast cancer was significantly lower than that in normal breast tissues.At the same time,it is concluded that CES2 may be related to metabolism and immunity.We also validated this view from the breast cancer cell level.Similarly,the expression of CES2 on protein and RNA levels was significantly higher than that of other types of breast cancer cells.Therefore,we can predict that CES2 may be a biomarker of breast cancer and may be a potential target for breast cancer treatment.2.We have learned from experiments that CES2 targeted NIR fluorescent probe DDAB has low toxicity and specificity in breast cancer cells,and has strong ability to screen and identify CES2 cells with high expression.This shows that DDAB fluorescent probe molecule will have a wide application prospect in tumor surgery It is worth our indepth exploration and provides theoretical and experimental basis for early clinical transformation of fluorescent probes for surgical navigation. |
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