The Study For Functions And Mechanism Of ClpP In Breast Cancer

Background : Breast cancer has the highest mortality rate of the female cancers in developed and developing countries,and its incidence is steadily increasing.Mitochondria,as well known organelles,are widely involved in the physiological and pathological processes of human body.Mammalian cell mitochondria contain ClpXP,an evolutionarily conserved atp-dependent unfoldable peptidase protein complex that mediates proteolysis to remove misfolded proteins.There is evidence that the pathway in which the protein is involved may regulate the mitochondrial unfolded protein response(UPR)and be involved in the pathogenesis of human disease.The ClpXP protease complex consists of two proteins:AAA+ ATPase(ClpX)and casein hydrolyzed protease P(ClpP).ClpP is a serine protease with a typical catalytic triplet domain.In mammals,ClpP is encoded by nuclear genes,translated and transported to mitochondria,where it degrades mitochondrial proteins damaged by oxidative stress in the mitochondrial matrix,and plays a central role in the quality control of mitochondrial proteins.In the field of oncology,the role of ClpP shows some tissue differences.Studies have shown that the expression of ClpP is lower in gastric adenocarcinoma than in normal adjacent tissues.In acute myeloid leukemia(AML)cells,the expression of ClpP is higher than that of normal hematopoietic cells.However,the expression and biological function of ClpP in breast cancer have not been studied.Objective: In this study,the expression level of ClpP in breast cancer tissues and cells was detected,and the correlation between ClpP andclinicopathological features and prognosis of patients was analyzed,so as to further explore the biological functions and related mechanisms of ClpP.Methods: 1.The Cancer Genome Atlas(TCGA)and kaplan-meier plotter databases were used to analyze the expression level of ClpP in breast Cancer tissues,further explore the relationship between the high and low expression level of ClpP and clinicopathological characteristics as well as the correlation analysis between ClpP expression and the prognosis of breast cancer patients.2.mRNA was extracted from 18 pairs of breast cancer and paracancer tissues,and the difference of ClpP mRNA expression was detected by real-time fluorescence quantitative PCR(RTqPCR).Immunohistochemical staining(IHC)was performed on 29 cases of breast cancer and 24 cases of para-cancer tissues to detect the expression of ClpP protein.3.The specific small interfering RNAs(siRNAs)of ClpP were transfected into breast cancer cell lines MDA-MB-231 and ZR-75-1with high expression of ClpP,and the knockdown efficiency was verified by RT-qPCR and Western blot after 48 h,and the proliferation,migration,invasion and apoptosis of the cells in the experimental group and the control group were compared by the clone formation,transwell and flow cytometry experiments.4.Western blot was used to detect the expression of Src,PI3 K,Akt and other related proteins in the experimental group and the control group.Results: 1.Through the analysis of TCGA database data,it was found that the expression of ClpP in breast cancer tissues was significantly upregulated compared with normal tissues,and the difference was statistically significant(p < 0.001).The expression level was significantly correlated with T stage(p = 0.0154)and ER expression(p = 0.0164).In the Kaplan Meier-plotter database,high ClpP expression was associated with shorter RFS(p = 0.00071).2.The results of RT-qPCR and IHC showed that theexpression of ClpP mRNA and protein in breast cancer tissues was higher than that in para-cancer normal tissues,and the difference was statistically significant(p < 0.01).3.According to the clone formation experiment,transwell experiment and flow cytometry experiment,compared with the control group,the proliferation,migration and invasion of the experimental group were inhibited,while the apoptosis was increased.The above results were statistically significant(p < 0.01).4.Compared with the control group,the phosphorylation of Src,PI3 K and Akt in the experimental group was inhibited by Western blot.Conclusion: 1.Through the analysis of TCGA data,it was found that the expression of ClpP increased significantly in breast cancer tissues,and the high expression of ClpP was correlated with clinical characteristics.These results indicate that ClpP has a high diagnostic potential.Further,through the Kaplan Meier-plotter database,it was found that high ClpP expression was significantly correlated with shorter RFS.It indicates that ClpP has a potential important role in the development of breast cancer.2.The transcriptional level and translation level of ClpP expression in human breast cancer and paracancer tissue samples were analyzed by RT-qPCR and IHC experiments,which verified the conclusion of high expression of ClpP in breast cancer obtained from data analysis of TCGA.3.Relevant functional experiments in breast cancer cell lines with knockdown ClpP expression showed that si-ClpP could inhibit the proliferation,migration and invasion of breast cancer cells and promote apoptosis.These results further confirmed the pro-cancer effect of ClpP.4.Western blot confirmed that ClpP knockdown can inhibit the Src/PI3K/Akt signaling pathway,and the activation of this pathway can promote the proliferation,invasion and migration of tumor cells.The results are consistent with the results of the functional test,which further indicates that the role of ClpP in promotingbreast cancer is carried out by regulating the Src/PI3K/Akt signaling pathway.