| Triple-negative breast cancer(TNBC),a special molecular subtype of BC,refers to one that lacks estrogen receptor(ER),progesterone receptor(PR)and human epidermal growth factor receptor-2(HER-2)target expression.Unique molecular typing of triple-negative breast cancer determines its special clinicopathological characteristics,including earlier onset age,higher histological grade,higher invasiveness,higher recurrence and metastasis rate,shorter median survival time and higher mortality.At present,the treatment methods of breast cancer mainly include surgery,chemotherapy,radiotherapy,endocrine therapy for ER and PR and targeted therapy for HER-2 target.Due to the lack of ER,PR and HER-2 expression in triple-negative breast cancer,there is no other effective treatment excepting surgery,chemotherapy and radiotherapy.To look for an effective molecular target for triple-negative breast cancer treatment is an important strategy to explore the future triplenegative breast cancer treatment.Micro RNA(miRNA),a kind of endogenous non-coding single-stranded RNAs,consists of approximately 20~25 nucleotides,existing widely in eukaryotic organisms.It plays the role of gene silencing and post-transcriptional modification in regulating gene expression,mainly through partial pairing,especially between the m RNA 3?-untranslated region and the miRNA 5?-end.At present,many kinds of miRNAs have been shown to be involved in the regulation of carcinogenesis.MiR-153,as a conserved miRNA,is considered as a potential molecular marker of triple-negative breast cancer.However,the effect of miR-153 on clinical biological behavior and cell biological behavior of triple-negative breast cancer is not clear.Zinc finger E-box-binding homeobox 2(ZEB2)is a nuclear transcription factor,which has many important functions in eukaryotes.The most important function of ZEB2 is to induce tumor cells to transform from epithelial phenotypes with weaker malignant potential into mesenchymal phenotypes stronger malignant potential,resulting in the loss of epithelial markers and the acquisition of mesenchymal markers,which is known as epithelial–mesenchymal transition(EMT).In this process,cellular structure,polarity,adhesion and migration change promote malignant progression of tumors,including triple-negative breast cancer.In this study,triple-negative breast cancer tissues and triple-negative breast cancer cell lines were taken as the research objects.By using a number of techniques and methods,the effect of miR-153 on clinical biological behavior and cell biological behavior as well as molecular mechanism of triple-negative breast cancer were analyzed and discussed,providing theoretical basis for exploring the biological target of clinical diagnosis and treatment of triple-negative breast cancer.The three parts of this study are as follows:Part One Study of the Relationship between miR-153 and Clinicopathological Characteristic as well as prognosis of Triple-Negative Breast Cancer PatientsObjective: 1.To explore the expression level of miR-153 in triple-negative breast cancer tissues and their corresponding adjacent tissues and to analyze the results as well as to discuss the significance of its expression change.2.To detect the correlation between the expression level of miR-153 and the clinicopathological characteristic of triple-negative breast cancer patients,and to analyze the possible risk factors affecting the prognosis of triple-negative breast cancer patients and the relationship between the expression level of miR-153 and the disease-free survival period and the overall survival period of triple-negative breast cancer patients,and to provide a theoretical basis for the effect of miR-153 on prognosis of triple-negative breast cancer patients.Methods:1.The expression level of miR-153 in triple-negative breast cancer tissues and their corresponding paracancerous tissues were detected by RT-q PCR.2.The relationship between the expression level of miR-153 and age,tumor location,family history of breast cancer,tumor size,histological grade,lymph node metastasis,TNM stage and tumor thrombus was analyzed by χ2 test.3.Univariate and multivariate analysis was used to assess the possible risk factors for the prognosis of triple-negative breast cancer patients according to Cox’s proportional risk regression model.4.Kaplan-Meier method was used to estimate the correlation between the expression of miR-153 and disease-free survival as well as overall survival of triple-negative breast cancer patients.Results: 1.The expression level of miR-153 in triple-negative breast cancer tissues was significantly lower than that in the corresponding paracancerous tissues(P < 0.01).2.The expression level of miR-153 was correlated with tumor size,lymph node metastasis and TNM stage(P < 0.05),but not with age,tumor location,family history of breast cancer,histological grade and tumor thrombus(P > 0.05).3.Univariate analysis displayed that tumor size,lymph node metastasis,TNM stage,tumor thrombus and miR-153 expression were risk factors of disease-free survival of patients with triple-negative breast cancer(P< 0.05);Multivariate analysis showed that TNM stage and miR-153 expression affected the disease-free survival of triple-negative breast cancer patients(P< 0.05).4.Univariate analysis displayed that tumor size,lymph node metastasis,TNM stage,tumor thrombus and miR-153 expression were risk factors of overall survival of patients with triple-negative breast cancer(P< 0.05);Multivariate analysis showed that TNM stage and miR-153 expression affected the overall survival of triple-negative breast cancer patients(P< 0.05).5.The disease-free survival of triple-negative breast cancer patients with high expression of miR-153 was better than those with low expression(P< 0.05).6.The overall survival of triple-negative breast cancer patients with high expression of miR-153 was better than those with low expression(P< 0.05).Summary:The expression level of miR-153 decreased in triple-negative breast cancer tissues.The expression level of miR-153 in triple-negative breast cancer was correlated with tumor size,lymph node metastasis and TMN stage.The disease-free survival and overall survival of triple-negative breast cancer patients with high expression of miR-153 was better than those with low expression.Part Two Study of the Effect of miR-153 on the Proliferation Ability,Invasion Ability and Migration Ability of Triple-Negative Breast Cancer CellsObjective: 1.To explore the expression level of miR-153 in triple-negative breast cancer cell lines and normal breast epithelial cell lines and to analyze the results as well as to discuss the significance of its expression change.2.To compare the expression level of miR-153 in triple-negative breast cancer cells transfected with miR-153 mimics or miR-NC,and to observe the changes of proliferation ability,invasion ability and migration ability of triple-negative breast cancer cells after over-expression of miR-153,and to explore the effect of miR-153 on the malignant biological behavior of triple-negative breast cancer cells.Methods: 1.The expression level of miR-153 in triple-negative breast cancer cell lines and normal breast epithelial cell lines was detected by RT-q PCR.2.The expression level of miR-153 in triple-negative breast cancer cells transfected with miR-153 mimics or miR-NC was detected by RT-q PCR.3.CCK-8 was used to detect the change of proliferation ability of triplenegative breast cancer cells with over-expression of miR-153.4.Transwell invasion assay was used to detect the change of invasion ability of triple-negative breast cancer cells with over-expression of miR-153.5.Wound healing assay was used to detect the change of migration ability of triple-negative breast cancer cells with over-expression of miR-153.Results: 1.The expression level of miR-153 in triple-negative breast cancer cells was significantly lower than that in normal breast epithelial cells(P < 0.01).2.The expression level of miR-153 in triple-negative breast cancer cells transfected with miR-153 mimics was significantly higher(P < 0.001).3.The proliferation ability of triple-negative breast cancer cells with over-expression of miR-153 was weakened(P < 0.01).4.The invasion ability of triple-negative breast cancer cells with over-expression of miR-153 was weakened(P < 0.01).5.The migration ability of triple-negative breast cancer cells with over-expression of miR-153 was weakened(P < 0.01).Summary:The expression level of miR-153 decreased in triple-negative breast cancer cells.The proliferation ability,invasion ability and migration ability of the triple-negative breast cancer cells transfected with miR-153 was were significantly weakened.Part Three Study on the Mechanism of miR-153 Effect on Biological Behavior of Triple-Negative Breast CancerObjective: 1.To predict the molecular targets downstream of miR-153 by bioinformatics website,to detect the expression level of ZEB2 protein in triple-negative breast cancer tissues and their corresponding paracancerous tissues,and to analyze the relationship between miR-153 and ZEB2 from the tissues level.2.To analyze the relationship between miR-153 and ZEB2 from the cell level,and to further analyze the effect of miR-153 on ZEB2 and EMT markers expression level and the effect of over-expression of miR-153 and ZEB2 on the malignant biological behavior and EMT of triple-negative breast cancer,to provide experimental basis for the study on the mechanism of miR-153 effect on biological behavior of triple-negative breast cancer.Methods: 1.The immediate target gene downstream of miR-153 was forecasted by bioinformatics website.2.The expression of ZEB2 was tested by immunohistochemistry in triple-negative breast cancer tissues and their corresponding paracancerous tissues,and to analyze the relationship between the expression level of miR-153 and the expression level of ZEB2 protein in triple-negative breast cancer tissues.3.The predicted target gene was verified by double luciferase reporter gene experiment.4.The change of expression level of ZEB2 of triple-negative breast cancer cells was detected by RT-q PCR and Western blot respectively.5.The change of expression levels of E-cadherin,N-cadherin and Vimentin of triple-negative breast cancer cells were detected by RT-q PCR and Western blot respectively.6.CCK-8 assay was used to observe the change of proliferation of triple-negative breast cancer cells overexpressing miR-153 and ZEB2 simultaneously.7.Transwell invasion assay was used to observe the change of invasion of triple-negative breast cancer cells overexpressing miR-153 and ZEB2 simultaneously.8.Western blot was used to observe the change of expression levels of E-cadherin,N-cadherin,Vimentin and ZEB2 of triple-negative breast cancer cells overexpressing miR-153 and ZEB2 simultaneously.Results: 1.ZEB2 was predicted to be the target gene downstream of miR-153.2.ZEB2 protein positive staining was mainly located in the cytoplasm and cell membrane of triple-negative breast cancer tissues.The expression level of miR-153 was negatively correlated with the expression level of ZEB2 protein(P < 0.001).3.MiR-153 inhibited the luciferase activity of wild-type ZEB2 plasmid(P < 0.01).4.MiR-153 inhibited the expression level of ZEB2 in triple-negative breast cancer cells(P< 0.01).5.MiR-153 inhibited the expression level of N-cadherin and Vimentin,and promoted the expression level of E-cadherin in triple-negative breast cancer cells(P< 0.01).6.Over-expression of ZEB2 reversed the proliferation ability of triple-negative breast cancer cells with over-expression of miR-153(P< 0.01).7.Over-expression of ZEB2 reversed the invasion ability of triple-negative breast cancer cells with over-expression of miR-153(P< 0.01).8.Over-expression of ZEB2 reversed the expression levels E-cadherin,N-cadherin,Vimentin and ZEB2 in triple-negative breast cancer cells with over-expression of miR-153(P< 0.05).Summary:ZEB2 is predicted to be the target gene downstream of miR-153,which may negatively regulate the expression of ZEB2 in triple-negative breast cancer.Over-expression miR-153 could inhibit the expression level of ZEB2 and EMT process in triple-negative breast cancer cells,over-expression ZEB2 could reverse the inhibition effect of miR-153 on malignant biological behavior of triple-negative breast cancer cells.Conclusions: 1.The expression level of miR-153 was declined in triple-negative breast cancer tissues.The expression level of miR-153 is correlated with tumor size,lymph node metastasis and TMN stage of triple-negative breast cancer tissues.The disease-free survival and overall survival of triple-negative breast cancer patients with high expression of miR-153 was better than those with low expression.It is speculated that over-expression of miR-153 can improve the prognosis of triple-negative breast cancer patients.2.The expression level of miR-153 decreased in triple-negative breast cancer cells.The proliferation ability,invasion ability and migration ability of the triple-negative breast cancer cells transfected with miR-153 was were significantly weakened.It is speculated that over-expression of miR-153 can inhibit the malignant biological behavior of triple-negative breast cancer cells.3.ZEB2 is predicted to be the target gene downstream of miR-153,which may negatively regulate the expression of ZEB2 in triple-negative breast cancer.Over-expression miR-153 could inhibit the expression level of ZEB2 and EMT process in triple-negative breast cancer cells,over-expression ZEB2 could reverse the inhibition effect of miR-153 on malignant biological behavior of triple-negative breast cancer cells.It is speculated that miR-153 may inhibit the biological behavior of triple-negative breast cancer by targeting ZEB2 / EMT axis. |
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