Label-free Quantitative Proteomic Screening Of Candidate Plasma Biomarkers For The Prognosis Of Breast Cancer With Different Lymph Node Statuses

Object: Lymph node status is an important predictor of overall survival of invasive breast cancer.However,lymph node involvement was detected only in about half of HER2-positive patients.Since the prognosis of patients with lymph node involvement is poor and the risk of recurrence is high,we intend to explore the protein markers associated with lymph node metastasis in HER2-positive invasive breast cancer by proteomics.Experiment: A Label-free quantitative proteome method was used to construct a plasma proteome of 10 patients with small size HER2-positive breast cancer(5 patients lymph node metastasis versus 5 patients without lymph node metastasis).Proteins were identified by unique peptides by peptide fragmentation of proteins from different samples.The human plasma deep protein database and FDA-certified proteins were compared,and significant differential protein interaction network analysis,GO,and KEGG analysis were performed.Finally,Western blotting was performed to verify the random differential protein.Results: A total of 388 differentially expressed proteins were identified,and 33 differentially expressed proteins were screened according to the log2 fold change >1.5,p<0.05.Protein interaction analysis showed that 23 proteins were involved,15 of which were elevated in lymph node metastasis.Increased expression in the sample,decreased expression of 8 proteins,and these proteins are associated with immune regulation,complement activation,breast disease,cancer pathway,and lymph node morphology,lymph node metastasis.GO analysis and KEGG analysis showed that significant differential proteins were mainly related to complement activation,immune activation,enzyme activity,and functions in intracellular and extracellular exchange.Western blotting showed that the expression of FBLN5 and RARB in the lymph node metastasis group was higher than that in the non-metastasis group,and the WB results were consistent with the proteome experiments.Conclusion: The results of proteomics experiments were verified to be credible by Western blotting.Lymph node metastasis of small HER2-positive breast cancer is correlated with immune regulation and complement pathway activation.FBLN5 affects the invasion,proliferation and metastasis of breast cancer cells.PLG/PLA system is complex.The role of PLG in lymph node metastasis of HER2-positive small breast cancer needs further study in PLG/PLA system.RARB is correlated with lymph node morphology,tumor pathway,immune regulation and breast diseases.These proteins should be combined with existing detection methods to further verify their feasibility as predictors of lymph node metastasis by large-scale,multi-center experiments.