Investigate The Degradation Pathway Of MYC Protein In Breast Cancer Based On Molecular Dynamics Simulation

1.Background At present,tumor has become one of the difficult problems in current medical science,and breast cancer is the female morbidity and mortality ranked the first malignant tumor.Breast cancer patients often face a variety of physiological and psychological pressure.Long-term exposed to chronic stress,patients' sympathetic nervous system overexcites,which lead to homeostasis disorders,activation of the hypothalamic-pituitary-adrenal(HPA)axis and excessive secretion of adrenalin and then affect the progress of the tumor.Tumor stem cells(CSCS)are a small group of tumor cells in the tumor tissue that possess the potential of self-renewal,infinite proliferation and multidirectional differentiation and can initiate and reconstruct the phenotype of the tumor tissue.Tumor stemness markers are important targets to help study tumor stem cells.Among them,MYC is one of the earliest found tumor stem cell markers.The study of its expression,mechanism of action and degradation pathway in tumor cells is conducive to the in-depth study of tumor stem cells and related tumor cells.Molecular dynamics is an integrated technique that combines physics,mathematics,and chemistry.Molecular dynamics(MD)is a set of molecular simulation method.This method is mainly relyed on Newtonian mechanics to simulate the movement of molecular system,which extracted samples from the system composed by different states of molecular system so that to calculate configurational integral of the system and the thermodynamic quantities and other macroscopic properties of the system are calculated based on the results of configuration integral.The molecular dynamics simulation technology can help us to understand the relationship between the structure and function of biological macromolecules,explore the interaction between molecules,and understand various biological phenomena more accurately.2.Methods(1)(1)Real-time quantitative PCR assay was used to detect the changes of Myc in the level of m RNA in breast cancer cells(MDA-MB-231,MCF-7)after epinephrine treatment;(2)The expression of MYC protein in breast cancer cells(MDA-MB-231,MCF-7)treated with epinephrine was detected by Western blot.;(3)Changes of MYC protein in breast cancer cells(MDA-MB-231,MCF-7)after treatment with epinephrine and CHX were detected by Western blot;(4)Western blot assay was used to detect the changes of MYC protein in breast cancer cells(MDA-MB-231,MCF-7)after treatment with epinephrine and proteasome inhibitor,MG132;(5)Changes of MYC ubiquitination level in 293 T cells after treatment with epinephrine and proteasome inhibitor were detected by immunoprecipitation and Wwestern blot.(2)(1)The expression of MYC protein in breast cancer cells(MDA-MB-231)transfected with different plasmids and treated with epinephrine were detected by Western blot;(2)Western blot detected the expression of USP28 protein and MYC protein in 231 cells;(3)Western blot detected the expression of USP28 protein and MYC protein in 231 cells after knocking down USP28 protein and treatment with epinephrine;(4)Western blot assay detected the expression of USP28 protein and MYC protein in 231 cells after overexpression of USP28 and treatment of epinephrine;(5)Changes in MYC ubiquitination level in 293 T cells were detected by immunoprecipitation and Western blot assay after knocking down USP28 and treatment of epinephrine.(3)(1)Immunoprecipitation assay detected the binding of USP28 protein and MYC protein;(2)The binding sites of MYC protein and USP28 protein were detected by molecular dynamics simulation;(3)Changes in MYC ubiquitination levels after transfection of wild-type and mutated USP28 proteins treated with epinephrine in 293 T cells were detected by immunoprecipitation and Western blot.(4)(1)Western blot detected the expression of USP28 protein and MYC protein in normal and tumor tissues;(2)Immunohistochemical experiments were performed to detect the differences in the expressions of USP28 and MYC proteins in the two groups of patients with breast cancer;(3)Analyze the correlation between the expression levels of USP28 protein and MYC protein and patient survival.3.Results(1)(1)Real-time quantitative PCR results showed that epinephrine did not affect the level of MYC gene in RNA;(2)Western blot results showed that epinephrine promoted the expression of MYC protein in breast cancer cells;(3)Western blot results indicated that epinephrine significantly delayed the half-life of MYC protein;(4)Western blot results revealed that MG132 significantly enhanced the expression of MYC protein,while epinephrine could not affect the effect of MG132 on MYC protein;(5)The results of the ubiquitination experiment showed that MG132 significantly enhanced the ubiquitination level of MYC protein,while epinephrine could reverse the ubiquitination enhancement of MYC protein caused by MG132.(2)(1)Western blot results showed that the level of MYC protein stabilized by epinephrine was determined by the deubiquitination enzyme USP28;(2)Western blot results demonstrated that the knockdown of the deubiquitination enzyme USP28 could lead to the decreased expression of MYC protein;(3)Western blot results stated that the knocking down of the deubiquitination enzyme USP28 could reverse the promoting effect of epinephrine on the expression of MYC protein;(4)Western blot results showed that the overexpression of deubiquitination enzyme USP28 could simulate the promoting effect of epinephrine on the expression of MYC protein;(5)The results of the ubiquitination experiment showed that epinephrine could reduce the phenomenon of increased ubiquitination level caused by knock down deubiquitination enzyme.(3)(1)The immunoprecipitation experiment showed that the deubiquitination enzyme USP28 could bind to MYC protein;(2)The molecular dynamics simulation experiment stated that the catalytic center of deubiquitination enzyme USP28 could bind to MYC protein;(3)The results of ubiquitination experiments indicated that the overexpression of mutant USP28 could not simulate the deubiquitination of MYC protein promoted by the overexpression of wildtype USP28.(4)(1)Western blot results demonstrated that the expressions of USP28 protein and MYC protein in tumor tissues were significantly higher than those in normal tissues in breast cancer patients;(2)The results of immunohistochemical experiments indicated that the USP28 protein and MYC protein in the tissues of patients with high epinephrine were significantly higher than those with low epinephrine in breast cancer patients;(3)The results of the survival experiment showed that the expression levels of USP28 protein and MYC protein were negatively correlated with the survival time of breast cancer patients.4.Conclusions(1)Epinephrine stabilizes the expression of MYC protein by deubiquitination in breast cancer;(2)USP28 mediates the deubiquitination of MYC protein by epinephrine;(3)Molecular dynamics simulation revealed the binding site of USP28 to MYC protein;(4)Increased expressions of USP28 and MYC in breast cancer patients resulted in poor prognosis of patients.