| Objective:TRIM45(Tripartite motif-containing protein 45),a member of TRIM family,is an E3 ubiquitin ligase containing RING(Really Interesting New Gene)domain,which plays an inhibitory role in cancer.As a tumor prognostic biomarker,SQSTM1/p62 can interact with key molecules in apoptosis,autophagy,oxidative stress and other processes to affect tumor progression.This study aims to explore the role and mechanism of TRIM45 in regulating Nrf2/Keap1 signaling pathway by targeting p62,thus inhibiting the malignant behavior of breast cancer cells.Method:1.Correlation prediction of TRIM45 gene and breast cancer occurrence was conducted through bioinformatics.Female breast cancer gene sets were screened from GEO database,and the expression of TRIM45 gene in breast cancer and adjacent normal tissues in GSE5364 and GSE3744 databases were used to draw boxplot,and the difference of TRIM45 expression in clinical breast cancer tissues was evaluated.The clinical survival data of female breast cancer were screened in the TCGA database by UCSC website to explore the effect of TRIM45 gene on the survival and prognosis of breast cancer patients.And the correlation between TRIM45 and clinicopathology.2.Breast cancer cell lines of TRIM45 overexpression group and no-load control group were established using TRIM45 plasmid and no-load plasmid.The effect of TRIM45 overexpression on the proliferation of breast cancer cells was investigated by CCK-8 and Ed U experiments.Transwell and Wound healing experiments investigated the effect of TRIM45 on transverse and longitudinal migration of breast cancer cells.The effects of TRIM45 on the expression of N-cadherin,E-cadherin,Vimentin,MMP2,MMP9,VEGF and IGF-1 were investigated by Western Blot.3.IP experiment was used to explore whether TRIM45 was bound to p62 in the cleavage fluid of breast cancer cells in the overexpression group of TRIM45.CHX chase test and proteasome inhibitor(MG132)were used to explore the effect of TRIM45 on p62 and its mechanism.4.The effect of TRIM45 on the expression of p62,Keap1 and Nrf2 proteins in breast cancer cells was explored through Western Blot experiment,and the mechanism of TRIM45’s inhibitory effect on breast cancer cells was further explored.5.Breast cancer cell lysates of TRIM45 overexpression group and no-load control group were extracted to detect the contents of glutathione peroxidase(GSH-PX),glutathione(GSH)and malondialdehyde(MDA),so as to explore the effect of TRIM45 gene on oxidative stress of breast cancer cells.Results:1.Through bioinformatics,it was found that the expression of TRIM45 in female breast cancer tissues was lower than that in adjacent normal tissues.Clinical survival data of women with breast cancer screened by the database showed that high expression of TRIM45 could significantly improve the survival rate of breast cancer patients,while patients with low expression of TRIM45 had a poor prognosis.The expression level of TRIM45 was correlated with the clinicopathology of breast cancer.2.CCK-8 test results showed that the number of proliferating cells in MCF-7 and BT549 cells in the overexpression group of TRIM45 was less than that in the no-load control group.EdU results showed that the amount and intensity of fluorescence in the overexpression group of TRIM45 was significantly lower than that in the no-load control group,and the expression of proliferation-related gene IGF-1 and its receptor IGF-1R were down-regulated,confirming that TRIM45 could inhibit the proliferation of breast cancer cells.The results of Transwell and Wound healing experiments showed that the migration ability of cells in the overexpression group of TRIM45 was decreased.3.TRIM45 inhibited the expression of Epithelial mesenchymal transformation(EMT)and migration-related proteins in breast cancer.In addition,the expression levels of important extracellular matrix degradation enzymes and angiogenesis related factors--MMP2,MMP9 and VEGF--were also down-regulated in the TRIM45 overexpression group compared with the no-load control group.4.The IP experiment showed that TRIM45 could bind to p62,the expression of p62 in the overexpression group of TRIM45 was significantly down-regulated,while MG132 could significantly increase the expression of p62 in the overexpression group of TRIM45.The half-life of p62 was shortened in MCF-7 and BT549 cells overexpressed by TRIM45 compared with the no-load group.5.Differential genes were analyzed in breast cancer samples associated with TRIM45 expression.GSEA enrichment analysis showed that differential genes were enriched in oxidative stress and Nrf2 signaling pathway.According to the expression level of TRIM45 in the TCGA breast cancer database,correlation analysis was conducted between p62,Keap1 and Nrf2 pairs,and there were statistical differences in the expression levels of p62,Keap1 and Nrf2.Further detection of the expression of Nrf2 and Keap1 interacting with p62 showed that overexpression of TRIM45 would decrease the expression of Nrf2 and increase the expression of Keap1.6.Compared with no-load control group,GSH-PX activity decreased,MDA content increased and GSH content decreased in TRIM45 overexpression group.These results suggest that TRIM45 disrupts the antioxidant stress mechanism of breast cancer cells and can induce cancer cell death by activating oxidative stress.Conclusion:1.Compared with normal tissues,the expression of TRIM45 in breast cancer is lower than that in normal tissues,and is positively correlated with the survival of clinical patients.2.Phenotypically,TRIM45 can inhibit the proliferation and migration of breast cancer,and inhibit the expression of EMT-related proteins and migration-related proteins.3.In terms of mechanism,it was confirmed that TRIM45 could interact with p62 to reduce the expression of p62,thus destroying the reciprocal checks and balances of Keap1/ Nrf2,thus destroying its protective effect on anti-oxidative stress of cancer cells,and thus inhibiting the development of breast cancer. |
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