In the past few years, three protein molecules-USP53, NPY2R, and DCTN1-AS1-have garnered significant attention in scientific research due to their potential implications in tumor development. Mass spectrometry and proteomics techniques were used to analyze the three-dimensional structure of these protein molecules and predict their active sites and functional domains. The effects of USP53, NPY2R and DCTN1-AS1 on biological behavior of tumor cells were studied by constructing gene knockout and overexpression cell models. The data showed that when USP53 was overexpressed, there was a notable enhancement in both the proliferation and invasion capabilities of tumor cells, indicating its potential role in promoting cancer aggressiveness. Conversely, the knockout of USP53 resulted in a significant reduction of these capabilities, highlighting its importance in tumor cell behavior. Similarly, the overexpression of NPY2R correlated with an increased apoptosis rate among tumor cells, suggesting that this protein may play a role in regulating cell survival. On the other hand, the knockout of DCTN1-AS1 markedly diminished the metastatic ability of the tumor cells, emphasizing its potential involvement in cancer spread. Furthermore, the analysis of clinical data provided compelling evidence that high levels of USP53 and NPY2R expression are closely linked to a poor prognosis for patients, suggesting that these proteins could serve as negative prognostic indicators. In contrast, low levels of DCTN1-AS1 expression were found to predict a poor response to treatment, further underscoring its importance in the context of therapy outcomes.