| BackgroundBreast invasive carcinoma(BC)is one of the most prevalent malignancies and represents the first leading cause of death among women.The mechanism of the development and progression of BC is still uncleara.Cell proliferation and apoptosis inhibition are difficult to treat.At the same time,chemotherapy resistance and refractory diseases caused by drug resistance are also special challenges to comprehensive treatment.Gene p5 3(tumor suppressor protein p5 3)is the most relevant tumor suppressor gene in human,which can induce the expression of genes such as p21,CDKN1A,BTG2 and BAX,inhibit cell cycle progression by inhibiting cell cycle dependent kinases,and promote cell apoptosis by mitochondrial pathway.If p53 is inactivated or mutated,cell proliferation progresses out of control,and the execution of cell apoptosis is greatly reduced.In addition,the p53-mediated pathway can be activated by genotoxic compounds,such as the chemotherapy drug cisplatin,resulting in tumor cell cycle arrest and apoptosis.Therefore,the activation of p5 3 function is regarded as the therapeutic target of cancer,and the maintenance of its protein stability is of great significance for the normal development of its antitumor function.Wild-type p53 is present in about 70%of BC cases.In these cases,the functional activity of p53 plays an important role in guiding treatment and prognosis.At present,in addition to point mutations and gene deletions,abnormal post-translational protein modification has been proved to be an important mechanism of p5 3 inactivation.Among these post-translational modifications,the UPS(Ubiquitin proteasome system)is the most important protein degradation pathway in vivo.Amazingly,deubiquitylases(DUBs)could mediate substrates,such as p53 protein,ubiquitin and stability of the protein level.More and more abnormally expressed DUBs have been found in BC,including tumor-promoting DUBs and tumor inhibiting DUBs.However,only two types of DUBs have been shown to deubiquitinate and stabilize p53 in breast cancer.Therefore,the relevant mechanism of regulating p5 3 deubiquitination is in urgent need of further study.OTU deubiquitinase 3(OTUD3),which is located in human chromosome 1 p3 6.1 3,is a little studied gene.Animal experiments confirmed that OTUD3 may specifically promote the development of lung cancer and inhibit the development of breast cancer.However,the role of OTUD3 in the occurrence and development of malignant tumors deserves further study,and its relationship with the occurrence and development of breast cancer and whether it can be used as a therapeutic target for breast cancer also deserves further exploration.Our previous study found that OTUD3 could deubiquitinate and stabilize PTEN.However,compared with PTEN,high expression levels of OTUD3 and p 5 3 were more indicative of better prognosis of BC.This topic not only detect the OTUD3 expression in breast cancer and cancer adjacent tissues,and tested the correlation between OTUD3 and p53.Since the breast cancer cell proliferation and apoptosis in the process of OTUD3 with p53 play a role of regulation is not reported,we perform cell proliferation experiments,apoptosis detects,ubiquitin experiment and Co-IP assay in the presence of WT p5 3 in breast cancer cell lines,further verify the OTUD3 positive regulatory role of p5 3 and ubiquitin enzyme activity in breast cancer cells.In conclusion,the expression of OTUD3 in breast cancer and its related biological functions and mechanisms were preliminarily explored in this paper,so as to provide new ideas and directions for the treatment of breast cancer.ObjectiveTo determine whether the deubiquitination enzyme OTUD3 is an important tumor suppressor in the development of breast cancer.To clarify the biological function of the deubiquitination enzyme activity of OTUD3 on wild-type p5 3 breast cancer cells.To study the molecular mechanism of OTUD3 deubiquitination to modify p53 and regulate the stability of p53 protein.Method(1)The expression of OTUD3 mRNA and protein in human breast cancer tissuesthe were analyzed online.The relationship between OTUD3,p53 and PTEN and patient prognosis were also analyzed online using TCGA data set.(2)80 pairs of Qilu hospital of Shandong University(Qingdao)breast cancer tumor tissue specimens and the corresponding tissue adj acent to carcinoma specimens were collected.OTUD3 protein expression was detected and analyzed using immuno-histochemical method.The relationship between the clinical pathological features of Qilu queue verify OTUD3 expression were analyzed.(3)2 6 pairs of fresh samples from breast cancer and para-cancer normal tissues were collected.WB was used to detect the protein levels of OTUD3,p53 and p21 in cancer and para-cancer,to confirm the relationship between OTUD3 expression and tumor cell histological grading,and to analyze the correlation between OTUD3 expression and p53/p21 expression.(4)The proliferation rate of MCF7 and DU4475 cells transfected with OTUD3 and OTUD3C76A and negative control cell lines was detected by MTS proliferation test.MCF7 and DU4475 cell stable strains with OTUD3 knockdown were constructed,and the cell proliferation rate was detected by MTS proliferation test kit and enzyme marker.The cell proliferation rate was detected after the OTUD3 and the enzyme activity mutant OTUD3C76A were respectively reverted and overexpressed without interference of shRNA.(5)The growth rates of MCF7 and DU4475 cells transfected with OTUD3 and OTUD3C76A and negative control cell lines were detected by clone formation experiments.MCF7 and DU4475 stable cell strains with OTUD3 knockdown were constructed,and the growth rate of cells was detected by cloning and formation experiments.After the overexpression of OTUD3 and enzyme activity mutant OTUD3C76A without interference from shRNA were respectively reverted,the growth rate of cells was detected by cloning and formation experiments.(6)MCF7 and DU4475 cells of OTUD3 and OTUD3C76A were transfected with the chemotherapy drug cisplatin and the negative control cell lines,respectively.MCF7 and DU4475 cell stable strains with OTUD3 knockdown were constructed,and the sensitivity of cells to cisplatin induced apoptosis was detected by using apoptosis kit.The changes of apoptosis rate were detected after the overexpression of OTUD3 without shRNA interference and the mutant OTUD3C76A with enzyme activity were detected respectively.(7)Wild-type p53 breast cancer cells MCF-7 and DU4475 were transfected with Flag-OTUD3 respectively.Flag empty vector was used as a negative control,and WB was used to detect the protein levels of p53,p21 and BAX.(8)Wild-type p53 breast cancer cells,MCF-7 and DU4475,were transfected with OTUD3 shRNA or Control shRNA lentivirus,and after the down-regulation of OTUD3,the protein levels of p53,p21 and BAX were detected by WB.After treating the cells with the proteasome inhibitor MG132,WB was used to detect the protein levels of p53,p21 and BAX.(9)After OTUD3 was down-regulated by OTUD3 transfected cells with shRNA,the cells were treated with the protein synthesis inhibitor actinomycone(CHX).Compared with the control cells and the rotatory overexpression of OTUD3,the p53 protein level was detected by WB at the corresponding time point.(10)Myc-MDM2 overexpression of MDM2 was transfected into MCF7 cells and DU447 5 cells,respectively.My c empty vector was used as the negative control,and the endogenous p53 level was detected by WB.Meanwhile,Flag-otud3 overexpression of OTUD3 was transfected,and p53 protein level was detected by WB.(11)The interaction between OTUD3 and p5 3 was determined by immunoprecipitation.(12)The OTUD3 truncated body and the corresponding eukaryotic expression vectors(Myc-OTUD 3,Myc-D1,My c-D2)and the p 5 3 truncated body and the corresponding eukaryotic expression vectors(My c-p5 3,Myc-T1,Myc-T2,Myc-T3,and My c-T4)were constructed,and the immunoco-precipitation experiment was conducted to verify the interaction region between the two.(13)The prokaryotic expression vector of p53 with GST tag and the prokaryotic expression vector of OTUD3 with GST tag were constructed,respectively.Direct interaction between OTUD3 and p5 3 was detected by GST pulldown experiment,and the interaction region was verified again.(14)MCF7 cells and DU4475 cells were transfected with OTUD3 shRNA,respectively,and Control shRNA was used as the negative Control.The proteasome inhibitor MG 132(20ul)was added 48 hours after transfection to inhibit the degradation of the protein.Flag-OTUD3,Flag-OTUD3C76A and Myc-MDM2 and MG 13 2 were transfected into the treated cells to detect the ubiquitination level of p53.Results1.The expression of OTUD3 in breast cancer was analyzed online:Online analysis of U ALCAN gene expression data platform of TCGA database of human BC showed that the level of OTUD3 mRNA in BC tissues was significantly lower than that in normal breast tissues(p<1 e-12).The mRN A level was independent of the clinical staging of BC.However,TNBC was slightly higher than Her-2 positive type(p=0.0337),but there was no difference in expression between Luminal type and Her-2 positive type(p=0.0714),nor between Luminal type and TNBC(p=0.10 5).The expression level of OTUD3 protein in BC tissues was significantly lower than that in normal breast tissues(p=0.0023).In the early stage 1(p=0.1 743),there was no difference in the expression in BC tissues and paracancer tissues,but the expression in stage 2(p=0.003 0)and stage 3(p=0.0099)breast cancer tissues was lower than that in normal tissues,and the expression in stage 2 BC tissues was higher than that in stage 3(p<le-12).However,there was no difference in the expression level of OTUD3 among the molecular types.2.The relationship between tumor suppressor molecules and breast cancer prognosis was analyzed online:Kaplan-meier survival was used to analyze the relationship between the expression of OTUD3,p53 and PTEN and the survival of BC patients online.The results indicated that the RFS rate of the high OTUD3 expression group(n=1986)was better than that of the low OTUD3 expression group(n=1965)(p=0.00034).Similarly,the RFS rate of the high p5 3 expression group(n=1977)was better than that of the low OTUD3 expression group(n=1974)(p=0.00054).However,the level of PTEN expression was not an independent prognostic factor in BC patients(p=0.62).The results suggested that compared with PTEN,the expression of OTUD3 and p53 was related to the prognosis of BC patients,and the BC patients with high expression of OTUD3 or p53 had better prognosis.3.Relationship between OTUD3 expression and clinicopathological features in BC tissues of Qilu cohort:Use the center data to verify the online analysis data.Immunohistochemical results showed that the protein level of OTUD3 in the BC tissues of the qilu cohort was significantly lower than that of the normal tissues surrounding breast cancer,and was not related to molecular typing or clinical staging,but suggested that it was related to the histological grading of tumor cells:3.1 The relationship between the expression of OTUD3 in breast cancer tissues and the histological grade of breast cancer:the expression of OTUD3 protein was related to the histological grade of tumor cells(?2=7.424),and the difference was statistically significant(p=0.024).3.2 The relationship between the expression of OTUD3 in breast cancer tissues and the tumor size:the expression of OTUD3 protein was not correlated with the primary tumor size of breast cancer(?2=0.120),and the difference was not statistically significant(p=0.942).3.3 The relationship between the expression of OTUD3 in breast cancer tissues and axillary lymphatic metastasis:The expression of OTUD3 protein was not correlated with the number of lymph node metastasis(?2=3.963),and the difference was not statistically significant(p=0.265).3.4 The relationship between the expression of OTUD3 in breast cancer tissues and clinical staging:The expression of OTUD3 protein was not correlated with clinical stage(?2=6.070),and the difference was not statistically significant(p=0.194).3.5 The relationship between the expression of OTUD3 and ER,PR,Her-2 and Ki67 in breast cancer tissues:The expression ofOTUD3 was independent of ER,PR,Her-2 and Ki67(?2=0.13 1/3.41 8/4.940/0.491),and the difference was not statisticallysignificant(p=0.717/0.064/0.026/0.483).3.6 Relationship between expression of OTUD3 and molecular typing in breast cancer tissues:The expression of OTUD3 protein was not correlated with the molecular typing(?2=7.292),and the difference was not statistically significant(p=0.121).3.7 Relationship between expression of OTUD3 and age in breast cancer tissues:There was no significant difference between OTUD3 protein expression and age(?2=1.054)(p=0.305).4.OTUD3 inhibited the proliferation of wild-type p53 breast cancer cells by deubiquitination enzyme activity:After overexpression of OTUD3,the proliferation of MCF7 and DU4475 cells slowed down,while overexpression of OTUD3C76A could lead to accelerated cell proliferation.MCF-7 and DU447 5 cells had stable OTUD3 knockdown,and the cell proliferation rate was significantly higher than that of the negative control.However,after overexpression of shRNA-free OTUD3,DU4475 cells proliferated at a significantly slower rate.There was no change in cell proliferation rate after OTUD3C76A.5.OTUD3 inhibits the cloning of wild-type p53 breast cancer cells by deubiquitination enzyme activity:After transfection with OTUD 3 in MCF-7 and DU4475 cells respectively,the ability of cell cloning formation was weakened;however,the ability of cell cloning formation was enhanced by transfection with the mutant OTUD3C76A with enzyme activity.After stable knocking down of OTUD3 in MCF-7 and DU4475 cells,the ability of cell clone formation was significantly enhanced.However,after overexpression of OTUD 3 without shRNA interference,the cloning ability of MCF-7 and DU4475 cells was significantly reduced.After OTUD3C76A,the ability of cell cloning was significantly enhanced.6.OTUD3 affects cisplatin-induced apoptosis of wild-type p53 breast cancer cells by deubiquitination enzyme activity:The sensitivity of OTUD3 transfected cells to cisplatin induced apoptosis was significantly increased.However,after transfection with OTUD3C76A,the sensitivity of the cells to cisplatin induced apoptosis was significantly decreased.Compared with negative control cells,the sensitivity of OTUD3 stable knockdown cells to cisplatin-induced apoptosis was significantly reduced.In MCF-7 and DU4475 cells with stable OTUD3 knockdown and without shRNA interference,the apoptosis rate of OTUD3 cells significantly increased,while that of OTUD3C76A cells decreased.7.OTUD3 was positively correlated with the expression of p53 and p21:WB detection indicated that the expression of OTUD3 in breast cancer tissues was lower than that in paracancer tissues,but it was not related to histological grading.Both OTUD3 and p5 3 were expressed in breast cancer and paracancer normal tissues.The expression level of OTUD3 protein was positively correlated with the expression level of p5 3 protein(r=0.8849,p<0.0001),and the expression level of OTUD3 was positively correlated with the expression level of p21(r=0.6427,p<0.0001).8.OTUD3 positively regulates the stability of p53:Overexpression of OTUD3,p53 and downstream p21 and BAX in Luminal type MCF7 cells and triple-negative DU4475 cells expressing wild-type p5 3 increased the levels of OTUD3,p5 3 and downstream p21 and BAX proteins,and significantly decreased the levels of p5 3 proteins when OTUD3 in MCF7 and DU4475 cells was knocked down,while the levels of p53 proteins increased after MG132 treated cells inhibited proteasome function.In the half-life experiment of p53 protein,OTUD3 was knocked down,and the half-life of p53 was shortened.The half-life of p53 was prolonged by rotation of OTUD3.Overexpression of MDM2 decreased the level of p53 protein.After overexpression of OTUD3,p5 3 protein level was saved.9.OTUD3 interacts with p53:Luminal type MCF7 cells and triple-negative DU4475 cells expressing wild-type p5 3 were selected to make cell lysates,respectively,and immunoco-precipitation experiment(co-ip)and interaction experiment were conducted to confirm the in vivo interaction between OTUD3 and p53.The truncated mutant of OTUD3 was constructed,and the truncated CoIP experiment verified that both OTUD3 and DI could interact with p53,and there was no significant difference in binding capacity between them and p5 3,indicating that the OUT domain in OTUD3 mediates its interaction with p53.The truncated mutant of p53 was constructed.The truncated mutant CoIP experiment verified that p5 3 and T2 and T3 truncated mutants could interact with OTUD3,but T1 and T4 could not bind to OTUD3,proving that the T2 and T3 structural domains of p53 mediated its binding to OTUD3.The interaction between OTUD3 and p53 was detected by truncated GST pull-down experiment.The results suggested that the OUT domain of OTUD3 could interact directly with p53.The T2 and T3 structural domains of p5 3 can directly interact with OTUD3,which again verifies the results of the CoIP experiment of OTUD3 truncated body and the CoIP experiment of p53 truncated body,and further indicates that the interaction between OTUD3 and p5 3 is a direct interaction.10.OTUD3 stabilized p53 by deubiquitination enzyme activity:Knocking down OTUD3 can significantly increase the ubiquitination level of p53.When MDM2 was overexpressed alone,p5 3 ubiquitination level was high,while OTUD3 was overexpressed at the same time,which could effectively antagonize the increased p53 ubiquitination level caused by MDM2.However,when Flag-OTUD3C76A was induced to express the active mutant of OTUD3 enzyme,p5 3 ubiquitination level did not decrease,that is,the mutant OTUD3C76A could not antagonize the effect of MDM2.ConclusionsOTUD3 is an important tumor suppressor DUBs of breast cancer and an important deubiquitination enzyme of p53,which has important biological functions for wild-type p53 breast cancer cells.1.The expression of OTUD3 in breast cancer was decreased,and its expression level was independent of clinical staging,molecular typing and histological grading.2.OTUD3 can be used as an independent prognostic factor for breast cancer.Breast cancer patients with high expression of OTUD3 have better prognosis than those with low expression.3.In human breast cancer tissues,OTUD3 is highly positively correlated with the expression of p53 and p2 1 downstream of p53.4.OTUD3 affects the biological functions of p5 3 wild-type breast cancer cells through deubiquitination enzyme activity,which can inhibit cell proliferation,inhibit cell cloning and increase cell sensitivity to cisplatin-induced apoptosis.5.OTUD3 is a deubiquitination enzyme of p53,which can maintain the stability of p5 3 protein.The regions that mediate the interaction between them are the OUT domain of OTUD3 and the T2 and T3 domains of p53,respectively,and the interaction is dependent on the deubiquitination enzyme activity of OTUD3.Research significanceThe deubiquitination enzyme of p5 3 in breast cancer was supplemented. |
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