| Background: Breast cancer is the highest incidence of malignant tumor in C hinese female,and the incidence rises year by year.Every year the morbidity and mortality is respectively 12.2% and 9.6% in the world.If the event of distant metastasis of breast cancer occur,the 10 year survival rate is less than 10%.It is hot for research that target therapy is considered to be the most promising treatment in the future,and at present there has been no clear targets and effective drugs,so looking for new targets to block the invasion and metastasis of breast cancer is an urgent problem to solve.Previous study showed that the development and metastasis of breast cancer is the result of joint action of many factors,which is closely related to protein degradation disorder Ubiquitin proteasome system,which is one of the main system for protein degradation.Many studies have shown that ubiquitin-proteasome system upregulated in malignant tumors and played an important role in the process of metastasis.Objective: Ubiquitin specific protease(USP)is the main member of ubiquitin enzyme family.Research shows that USP is closely related to the occurrence and metastasis of breast cancer.A variety of USP were confirmed increased in breast cancer tissue,and involved in the proliferation and metastasis of breast cancer,and was positively correlated with invasion and poor prognosis of breast cancer.USP4 has been found high expressed in a wide variety of tumor tissues and it is called oncogene.ONCOMINE and Kaplan Meier-plotter database are known as a public database currently,it integrated the cancer chip database and combined with the network data platform.It contains all kinds of literature which has been published and RNA and DN A sequence data of TCGA sources.And the aim is to promote genome-wide expression analysis.The database testing about 48 million genes.It compares the main types of cancer and analysis of the gene expression differences between the normal tissue and cancer.The classification of variety of cancer subtype and clinical and pathological analysis are all included.It can promote the discovery of new biomarkers and therapeutic targets.The database study the role of cancer genes.Because the breast cancer molecular classification is normal in clinical,so we study USP4 gene expression in breast cancer through ONCOMINE database and look for the relationship between USP4 and molecular classification of breast cancer to guide the treatment of patients and to predict the prognosis of patients with breast cancer.TGF-β/Smad signaling pathways of abnormal activation is closely related to the development of tumor,especially play an important role in the process of tumor invasion and metastasis.A new study has found that TGF-β decorated by ubiquitin and influence the activation of signal pathway,USP family also participate in the TGF-β signaling pathway to ubiquitin modification.A new study has found that USP4 could be regulated by transforming growth factor receptor(TβR1),and can enhance TGF-β signaling pathway activity,then induce epithelial-mesenchymal transition(EMT)and invasive migration in breast cancer.Some study also found that USP4 can enhance SMAD2 phosphorylation and promote TGF-β gene transcription.But how USP4 regulate TGF-β signaling pathways,and adjust the specific mechanisms of invasion and metastasis of breast cancer is unclear.Relaxin in recent years is found that in addition to a variety of effects on the reproductive system and cardiovascular system,while the effects on the development of tumor was found gradually.Studies have found that the extracellular matrix of solid tumors by blocking tumor outside the drug diffusion concentration affect the effectiveness of the treatment,by detecting the tumor in t he expression of polypeptide hormone relaxin can improve the degradation of extracellular matrix and improve the effectiveness of the by bead sheet resistance to treatment.When the relaxin march this bead sheet resistance to treatment,can inhibit tumor growth.Studies have found that relaxin is mediated TGF-beta 1 extracellular matrix synthesis and secretion inhibitor,and play a role like fibroblasts activation.In addition,it reduced MMP-2,MMP-9 secretion through the TGF-β1 signaling pathways.Studies have found that the concentration levels of relaxin was increased in serum of breast cancer patients,especially in the metastatic patients.The expression of relaxin levels in patients was correlated with the prognosisof breast cancer,and its excessive expression is positively correlated to cell proliferation,TGF–β regulated fibrosis and so on all.Because the mechanisms of how USP4 promote invasion and metastasis of breast cancer is still unclear,we’re going to continue to study with the relationship between USP4,relaxin and TGF-β1/Smad2 /MMP-9 pathway,then provide new ideas for clinical treatment of breast cancer patients.Methods: 1、We analyze USP4 gene expression differences in ONCOMINE database in breast cancer tissue and normal breast tiss ue.We also analyze the relationship between USP4 genes and 5 years,10 years OS by Kaplan Meier-plotter web.2、We detect USP4 expression in quantity both in breast cancer cell line MDA-MB-231 cells and normal breast epithelium MCF10 A by Real-time fluorescent quantitative PCR.3 、 We successfully establish MDA-MB-231 cells with silence USP4 by si RN A interference technology;Then establish T47 D cells with USP4 genes overexpressed in plasmid transfection technique,then using Western Blot to test its protein expression and RT-PCR to test the RN A level respectively.We use Transwell invasion experiment and scratch test to test the USP4 genes expressed in silence or effects on breast cancer cell invasion and migration ability.Then silence USP4 gene was detected in MDA-MB-231 cell lines and T47 D cells express USP4 gene expression of MMP-9 protein,and add MMP-9 inhibitor BB94 to the USP4 overexpressed T47 D cell,then test its invasion and migration ability of change by transwell invasion experiment and scratch test.4、Genes expressed in a USP4 T47 D cells using si RNA technology import Si Relaxin 2 respectively,Si TGF-β1,Si Smad2,by Western Blot test after the silence of the three genes relaxin 2,TGF-β1,Smad2,MMP-9 protein expression level change together.By transwell invasion experiment,scratch test and migration test of cell invasion were admit on USP4 T47 D of silent Relaxin-2,TGF-β1,Smad2 gene respectively.Results: 1 、 The genome-wide ONCOMINE database analysis shows that USP4 genes is significantly higher expressed in breast cancer tissue than in normal breast tissue(p < 0.001).The Kaplan Meier-plotter web analysis shows that the five years OS of the high USP4 gene in HER-2 positive patients with breast cancer was related to statistically significant poor prognosis(p = 0.042),and other types of molecular subypes of breast cancer did not see that survival disadvantage.2、The expression of USP4 is higher in breast cancer cells,and is lower in normal breast epithelium,p < 0.05,the difference is statistically significant.3 、 We successfully established USP4 gene silence MDA-MB-231 cells,and we Confirmed it by Western blot and RT-PCR experiments.4、It is showed in scratch experiments that the width of MDA-MB-231 cells with silent USP4 genes after 48 h was greater than the negative control group(p < 0.05).Invasive experiments showed that the amount of invading cells in silent USP4 genes MDA-MB-231 cells after 24 h was significantly less than the negative control group(p < 0.05).5、Confirmed by RT-PCR and Western blot experiments,we successfully established USP4-overexpressed T47 D cells through gene transfection,scratch experiments results showed that scratch width of T47 D cells of overexpression USP4 gene after 48 h is significantly less than the negative control group(p < 0.05),invasive experiments results showed that invading cells of T47 D cells with USP4 gene overexpression after 24 h was significantly more than the negative control group(p < 0.05).6、We test the protein expression of Relaxin2,TGF-β1,Smad2,MMP-9 in T47 D cells of overexpressed USP4 gene,the results showed that the expression of Relaxin2,TGF-β1 and Smad2 obviously increased,the protein expression of MMP-9 is also increased.In MDA-MB-231 cells with silence USP4 gene,we test the Relaxin2,TGF-β1,Smad2 and MMP-9 protein expression,the results showed that the protein expression of Relaxin2,TGF-β1 and Smad2 decreased obviously,and the MMP-9 protein expression has dropped significantly.7、We knock down the gene of Smad2 or TGF-β1 by si RN A with the technique of RN A interference in T47 D cells with USP4 overexpression.Western blot experiments confirmed the results that the expression of the Smad2 and MMP-9 protein is significantly reduced in Si Smad2 group compared with control group,but it did not influence the protein expression of Relaxin2.Western blot experiments confirmed the results that the protein expression of TGF-β1,Smad2 and MMP-9 is significantly reduced in Si TGF-β1 group compared with control group,but it did not influence the protein expression of Relaxin2 either.8、We knock down the gene of Relaxin2 by si RNA with the technique of RN A interference in T47 D cells with USP4 overexpression.Western blot experiments confirmed the results that the protein expression of TGF-β1,Smad2 and MMP-9 is significantly reduced compared with control group.9、The MMP-9 protein expression of MDA-MB-231 cells with silent USP4 genes in the negative control group decreased significantly(p < 0.05)in Western blot test,while expression of MMP-9 protein in T47 D cells with USP4 increased significantly compared with negative control group of protein expression(p < 0.05).We applied BB94 which is inhibitors of MMP-9 to USP4 overexpressed T47 D cells,the result of scratch test showed that after adding BB94,the scratch width of T47 D cells was significantly greater than the untreated group(p < 0.05).Transwell invasion of the experimental results also showed that adding BB94 to USP4 genes overexpressed T47 D cells significantly reduce the invading cells than the control group(p < 0.05).It is suggested that USP4 can promote breast cancer cell invasion ability,and can also be inhibited by MMP-9 inhibitors.MMP-9 can promote breast cancer cell invasion and migrationwith USP4.10、We knock down the Relaxin2 or TGF-β1 or Simad2 si RN A with the technique of RNA interference in T47 D cells with USP4 overexpression,the scratch experimental results showed that the scratch width of Si Relaxin2 group,Si TGF-β1 group and Si Smad2 group is larger than the control group and the migration ability markedly reduced(*p<0.05).The width of Si Relaxin2 group is largest(**p<0.01).Transwell invasion test results showed that the amount of invasive cell numbers in Si Relaxin2 group,Si TGF-β1 group and Si Smad2 group is more than the control group and the invasive ability markedly reduced(*p<0.05).The amount of Si Relaxin2 group is most(**p<0.01).Conclusions: 1、USP4 can promote migration and invasion of breast cancer cells.2、MMP-9 play a role in the process of the breast cancer cell invasion and migration of the USP4;3、USP4 can promote the expression of MMP-9 by activating the TGF-β1 / Smad2 signal pathway;USP4 can promote breast cancer cell invasion and migration through the TGF-β1 / Smad2 / MMP-9 signaling pathway;4、USP4 influence the expression of relaxin2,and USP4 can promote breast cancer migration through relaxin2 TGF-β1 / Smad2 / MMP-9 signaling pathway;5、Inhibition of TGF-β1 signaling pathways,inhibition the function of MMP-9,and knocking down the USP4 gene is expected to become new method s for the treatment of breast cancer. |
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