Expression Of A20 In Breast Cancer And The Effect Of A20 And P53 On Human Breast Cancer Cell Line MCF-7

Objective:A20,a ubiquitin-editing enzyme,which has the ability to modulate inflammatory signalling cascades and down regulate NF-?B signalling induced by TNF-?and other cytokines.There is a strong correlation between A20 and many kinds of tumors.However,A20 exerts different effect on different tumors,which manifests as the effect of tumor suppression or carcinogenesis.As there is still not report about the expression in breast cancer and the effect of A20 and p53 on human breast cancer cell line MCF-7,our study focuses on the discussion of these problems.Methods:1.The expression of A20 protein in 145 breast cancer samples and 46 para-carcinoma samples which have complete survival datas was detected with a two-step immunohistochemical staining.2.We collected 26 breast cancer specimens and 14 benign breast tumor specimens from the hospital's operating room.By using qRT-PCR and Western blot,we investigate the expression of A20 between the two groups at mRNA level and protein level.3.Firstly,the MCF-7 cell was divided into six groups: 1)A20 overexpression vector group.2)A20 empty vector group.3)Breast cancer cell blank control group.4)Pifithrin-?(p53 inhibitor)group.5)A20 overexpression vector+ Pifithrin-?(p53 inhibitor)group.6)A20 empty vector group+ Pifithrin-?(p53 inhibitor)group.Then,the human breast cancer MCF-7 cell line was transfected with a A20 overexpression plasmid.We detected the biological behavior of the grouped cells by using CCK-8 cell proliferation test,cell cycle experiment,wound healing assay and cell invasion experiment.After adding the p53 inhibitor,we also detect the effect of the treatment factor on A20 overexpression cancer cells,whose p53 function was inhibited.Results:1.A20 mainly expresses in cytoplasm and the over-expression rate in cancerous tissue is higher than in para-carcinoma(71.4% VS 34.3%)a significant difference is founded in the expression rate of A20 between 35 breast cancer samples and 35 para-carcinoma samples(*p<0.05).During 145 breast cancer samples,71 samples show a high expression state while 74 samles show a low expression state,and the survival rate of high expression group is 59.2%(42/71)while the survival rate of low expression group is 81.1%(60/74).There is a significant difference in survival rate between high expression group and low expression group(*p<0.05).Univariate and multivariate analyses of the factors correlated with overall survival of breast cancer show that the expression state of A20 is a independent prognostic indicator.2.The experimental results of Real-time fluorescent quantitative PCR and Western blot show that the expression quantity of A20 in human breast cancer tissure was significantly higher than that in human breast benign tumor tissure both at mRNA level and at protein level(*p<0.05).3.Compare with Con group,the ability of cell proliferation of Pifi+NC group and Pifi group was stronger than than that of Con group,which had statistical significance.By contrast,the ability of cell proliferation of A20 group and Pifi+A20 group had a significant decline(*p<0.05).Compare with Con group,the cell number in G1 phase of Pifi group and Pifi+NC group were significantly higher than that of Con group.By contrast,the cell number in S phase of A20 group and Pifi+A20 group had a significant decline(*p<0.05).There was no significant difference in cell apoptosis rate between NC group,Pifi group,Pifi+NC group and Con group.After comparing A20 group and Pifi+A20 group with Con group,we found that the cell apoptosis rate of A20 over-expressed cells increased.Compare with Con group,the cell migration speed of Pifi group and Pifi+NC was significantly higher than that of Con group.The cell migration speed of A20 group significantly declined.The ability of cell invasion of Pifi group was stronger than that of Con group and the ability of cell invasion of Pifi+NC group was stronger than that of NC group which both had statistical significance.Nevertheless,the ability of cell invasion of A20 group and Pifi+A20 group had a significant decline(*p<0.05).Compare with Con group,the expression quantity of A20 and p53 in Pifi group and Pifi+NC group significantly declined,while the expression quantity of A20 and p53 in A20 group significantly increased both at mRNA level and protein level(*p<0.05).Conclusion:1.The expression quantity of A20 in breast cancer tissue microarray was significantly higher than that in para-carcinoma tissue microarray at protein level.And the survival rate of A20 high expression group was significantly lower than that of A20 low expression group in breast cancer tissue microarray at protein level.The expression state of A20 is a independent prognostic indicator for breast cancer patients.2.The expression quantity of A20 in human breast cancer specimens was significantly higher than that in human breast benign tumor specimens both at mRNA and at protein level.3.Compare with control group,the expression quantity of A20 and p53 increased significantly in A20 over-expressed group whatever at mRNA level or at protein level.And the ability of proliferation,migration and invasion of A20 over-expressed MCF-7 cells was significantly inhibited and the cell apoptosis rate significantly increased.It indicates that A20 may play a role in tumor suppression in breast cancer.