| Breast cancer(BC)is the most common malignant tumor and the leading cause of death for females worldwide.Compared with North American and European countries,BC shows features of younger onset age,higher proportion of negative hormone receptors,and lower incidence in developing countries including China.With the improvements in screening and comprehensive treatment,in China,the prognoses of BC patients has been significantly improved.However,BC is still the highest cause of female cancer death,especially in some developed countries and regions,and also a serious harm to women’s physical and mental health.Therefore,it is of great significance to investigation of mechanism of breast cancer carcinogenesis and progression,especially invasion and metastasis of BC,which provides important theoretical basis for finding new molecular markers and therapeutic targets of BC.The present studies have demonstrated that recurrence and metastasis are the main causes of cancer death due to unlimited cancer cells proliferation and migration.Therefore,it is of great significance for the prognosis of cancer research through studying the proliferation of tumor cells and predicting the biological behaviors of BC.Type Ⅱ transmembrane serine protease(TTSPs)plays an extremely important role in the epithelial-mesenchymal transition,invasion,metastasis and signal pathway in tumor cells.Transmembrane serine protease 4(TMPRSS4)is one of members of the TTSPs family.A number of domestic and international studies have shown that TMPRSS4 is highly expressed in a variety of malignant tumors,such as gastric cancer,pancreatic cancer,liver cancer and colon cancer,and is closely related to the invasion and metastasis of malignant tumor cells.Recent studies have also shown that TMPRSS4 could promote the degradation and tissue reconstruction of basal cell membrane and extracellular matrix(ECM)in tumor cells,and lead to epithelial-mesenchymal transition(EMT)induced by the down-regulation of E-cadherin protein expression in order to promote tumor invasion and metastasis.But there is a paucity of data available regarding the correlation between the expression of TMPRSS4 in breast cancer and clinical biological behavioral characteristics.In this study,we examined the expression of TMPRSS4 protein in BC tissues and normal breast tissues to explore its relationship with clinicopathological features and prognosis.The expression of TMPRSS4 mRNA and protein in BC cell lines was detected in vitro,the regulation of TMPRSS4 expression on the proliferation,invasion and migration of BC cells was observed,and the potential mechanism was explored.This study was divided into two parts,as the following:PART ONEExpression and correlation of TMPRSS4 and clinicopathological characteristics and prognosis in Breast CancerObjective:To investigate the expression of TMPRSS4 protein in clinical BC tissues and normal breast tissues and to explore its relationship with clinicopathological features and prognosis.Methods:The expression of TMPRSS4 in BC tissues was detected by immunohistochemistry staining.The relationships between TMPRSS4 expression and clinicopathological characteristics as well as overall survival and disease free survival were evaluated.Results:The positive expression rate of TMPRSS4 was 65.4%(70/107)in BC cases and 17.6%(9/52)in normal breast tissues.A signifcant statistical difference was found between the two groups(P<0.05).Furthermore,the expression of TMPRSS4 was increased along with grade,with the highest expression in grade III invision ductal cancinoma tissues and the lowest expression in grade I invision ductal cancinoma(IDC)tissues(P<0.05).(2)The expression level of TMPRSS4 was closely correlated with tumor size,histological grade of IDC,lymph node metastasis,clinical stage as well as poor survival(all P<0.05),but not related wigh age,menstrual status,histological type and the expressions of ER,PR and HER-2(all P>0.05).(3)The univariate analysis revealed that tumor size(OS,P=0.042;DFS,P=0.047),lymph node metastasis(OS,P=0.000;DFS,P=0.001),histological grade of IDC(OS,P=0.036;DFS,P=0.039),clinical stage(OS,P=0.009;DFS,P=0.012),and TMPRSS4 expression(OS,P=0.015;DFS,P=0.018)were prognostic factors for BC patients.Furthermore,Patients with high TMPRSS4 expression had shorter OS(P=0.036)and DFS(P=0.010)compared with the patients with low TMPRSS4 expression.(4)The multivariate analysis indicated that TMPRSS4 expression was one of the independent prognostic factors,along with clinical stage and lymph node metastasis.Conclusion:(1)TMPRSS4 was overexpressed in BC tissues.(2)High expression of TMPRSS4 was closely correlated with tumor size,histological grade of invision ductal cancinoma,lymph node metastasis,clinical stage as well as poor survival.(3)Patients with high expression of TMPRSS4 had shorter survival outcome.Overexpression of TMPRSS4 could be recognized as an independent prognostic factor for BC patients.(4)To conclude,TMPRSS4 may be a potential therapeutic target for cancer treatment.PART TWOThe Functional Study of TMPRSS4 Expression in the Modulation of Invasion and Metastasis of Breast Cancer and correlation of TMPRSS4 and epithelial-mesenchymal transitionObjective:To investigate the expression of TMPRSS4 in BC cells and to analyze the relationship between TMPRSS4 expression and EMT as well as the significant of TMPRSS4 expression in the development and progression of breast cancer.Methods:(1)RT-qPCR and Western blot analysis were used to determine the expressions levels of TMPRSS4 mRNA and protein in 5 BC cell lines(MCF-7,MCF-7/ADM,T47D,MDA-MB-231,and MDA-MB-468).(2)The specific TMPRSS4 overexpression plasmid was extracted and transfected into BC MDA-MB-468 and MDA-MB-231 cells and transfection effciency was evaluated.The MTS assay,EdU assay,and transwell invasion were used to study the mechanism of proliferation,invasion and metastasis in BC cells.(3)Two cell lines(MDA-MB-468 and MDA-MB-231)were transfected with a small interfering RNA(siRNA)duplex and transfection effciency was evaluated by RT-qPCR.Following siRNA transfection,the MTS assay,EdU assay,and transwell and Migration assays were used to study the mechanism of proliferation,invasion and metastasis in BC cells.(4)Following transfected with TMPRSS4 expression plasmid and siRNA,the expressions of key biomarkers of EMT(E-cadherin,Vimentin,Claudin-1,Slug,and ZEB1)were observed by RT-qPCR and Western blot analysis.Results:(1)RT-qPCR and Western blot analysis showed that TMPRSS4 was differentially expressed in five BC cell lines,with the highest expression in MDA-MB-468 cells.The determinations of overexpression and siRNA efficiency were consistent with the experimental requirements.(2)MTS and EdU assays showed that the cell proliferation rate was significantly increased in TMPRSS4 overexpression group.In contrast,the cell proliferation rate was decreased significantly when TMPRSS4 expression was inhibited(all P<0.05).(3)Transwell Chambers and Matrigel experiment showed that the cell migration and invasion abilities were increased significantly in TMPRSS4 overexpression group.However,the cell migration and invasion abilities were decreased significantly in TMPRSS4 inhibition group(all P<0.05).(4)RT-qPCR and Western blot analysis revealed that the expression of E-cadherin and Claudin-1 were significantly decreased,while the expression of Vimentin and Slug were inceased after the overexpression of TMPRSS4(P<0.05).The expression of E-cadherin and Claudin-1 in breast cancer cells were significantly increased after transfection with TMPRSS4 siRNA,while the expression of Vimentin and Slug was significantly decreased(P<0.05).Conclusion:(1)TMPRSS4 was overexpressed in BC MDA-MB-468 and MDA-MB-231cell lines.(2)Overexpression of TMPRSS4 could promote the proliferation,migration and invasion of BC cells,in contrast,silencing of TMPRSS4 expression could inhibit the proliferation,migration and invasion of BC cells in vitro.(3)The up-regulation or down-regulation of TMPRSS4 expression could regulate the expression of EMT related genes in BC cells.TMPRSS4 may be involved in the development of BC EMT. |
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