| Objective:To provide new monitoring indicators for the clinical diagnosis and prognosis assessment of breast cancer by studing the expression differences of Casitas Blineage lymphoma C(CBLC)in different breast cancer cell lines and tissues and exploring the biological role and mechanism of CBLC and its interacting proteins.Methods: The tissue CBLC gene expression profile matrix(FPKM)with complete related information was downloaded from the TCGA data set in the USCS Xena database to analyze the correlation between the temporal and spatial expression of CBLC and different molecular types,pathological types,TNM stage and prognosis in each tumor stage.The expression levels of CBLC in breast cancer and adjacent tissues were observed by immunohistochemical staining.qPCR and WB were used to verify the expression level of CBLC in 5 breast cancer cells,and the breast cancer cells with the lowest/highest expression level were used for CBLC overexpression/knockdown;Scratch test,transwell and CCK-8 experiments was used to assess the effect of CBLC on breast cancer cell biology behavior from several aspects including cell proliferation,migration and invasion.IP identification combined with LC/MS-MS,ppi,silver staining,immunoprecipitation and fluorescence confocal methods were used to screen and identify the CBLC interaction proteins,and then the effects of CBLC on the mRNA and protein levels of the interaction proteins were analyzed by qPCR and WB experiments,and the potential mechanisms were analyzed by stability experiments and ubiquitination experiments.CBLC and its interaction protein overexpressed cells were used in wound healing,Transwell and cck-8 experiments to explore the influence of both two on the biological behavior of breast cancer cells.Results: Among all molecular typing,mRNA expression of CBLC in Luminal A subtype was the highest,and the mean of FPKM was 3.55,which was significantly different from the other 3 subtypes(P<0.05).Among the pathological tissue types,the mRNA expression level of CBLC in mucinous carcinoma was the highest,and the FPKM was 4.01,which was significantly different from the other 3 types(P<0.05).Among all breast cancer cell types,the protein expression level of CBLC was the highest in MCF7 breast cancer cells and the lowest in MDA-MB-231.The ratio between the two groups was 3.49 times(P<0.01),and the expression level of CBLC in stage II breast cancer was lower than that in stage I(P<0.05).Median survival time of breast cancer patients in the group with high CBLC expression was 2.03 times that of the group with low CBLC expression(P<0.01),and the area under the ROC curve of the survival time of breast cancer patients in the group with high expression of CBLC exceeded the total median survival time was 0.81.The overexpression of CBLC can inhibit the proliferation,migration and invasion of breast cancer cells;conversely,reducing the expression of CBLC can promote them.CBLC protein and CTTN protein have a binding effect on each other and both of them colocate in cytoplasm,and can enhance the ubiquitination degree of CTTN at the protein level and promote its degradation.Overexpression of CTTN decreased the inhibition of CBLC on proliferation,migration and invasion of breast cancer cells.Conclusion:CBLC is highly expressed in breast cancer of low malignant molecular typing,highly differentiated histophysiological typing and low invasive cell type,which inhibits the proliferation,migration and invasion of breast cancer,and shows a downward trend in the process of breast cancer.Patients with high expression have a better prognosis situation.By increasing the ubiquitination of CTTN,CBLC promotes the degradation of CTTN protein,thus inhibiting the proliferation,migration and invasion of breast cancer. |
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