| Background:Breast cancer is the most common cancer in women worldwide,accounting for 16 percent of all female cancer incidence.The occurrence of breast cancer is affected by a variety of environmental and genetic factors.Genetic traits,age and hormones are considered to be the most important risk factors for susceptibility.Female breast cancer includes multiple types of tumors,which differ in morphology,biology and molecular features,leading to different clinical outcomes and patient survival.Currently,we have a number of better methods and molecular markers for the diagnosis and prognosis of breast cancer,such as tumor staging and grading,p53,Bcl-2,Ki-67,hormone receptors.However,there is an urgent need for us to identify the molecular subtypes of breast cancer(e.g.luminal A and B,basal cell-like type,triple negative breast cancer type),which are closely related to the treatment of breast cancer.Different molecular subtypes have different prognosis and response to the same treatment.However,a subset of breast cancers with positive estrogen receptor(ER),progesterone receptor(PR),and human epidermal growth factor 2 receptor(Her2/neu)proteins was effectively controlled by hormone therapy or molecular target therapy In contrast,triple negative breast cancer(TNBC)lacking ER,PR,and Her2 expression is a biologically aggressive neoplasm and has poor prognosis,frequent relapses and visceral metastasis due to a lack of effective treatment options.Thus,further investigations into the underlying molecular mechanisms responsible for TNBC progression and metastasis could help medical biologist effectively control these deadly diseases.Recurrence and metastasis of breast cancer is the main cause of death in breast cancer patients,up to 40% patients can have recurrence and metastasisevery year.Metastatic breast cancer is difficult to treat with poor prognosis and low average survival rate.In the study of new molecular markers of breast cancer,it has been confirmed that the MMP family is closely related to the invasion and metastasis of breast cancer.MMP family is a zinc-dependent peptide chain endonuclease,which belongs to the superfamily.MMP1 is involved in key events in physiological processes(e.g.tissue reconstruction,stem cell differentiation and proliferation,apoptosis)and pathological processes(e.g.inflammation,degeneration and malignancy,and cancer).The MMP family contains many types of proteases,decomposing most of the extracellular matrix components and some proteins,producing important growth factors for oncogenesis and progression,which indicate a good target for tumor biomarkers.There are at least 24 kinds of MMP genes in humans,producing 23 proteins,including 17 soluble secretory proteins and 6 membrane related proteases.MMPs consists of multiple domains,the substrate-specific and tissue-specific expression patterns are different.MMPs are named according to their dominant substrates in extracellular matrix.collagen-cleaving MMPs(1,8,13)are called collagenases,gelatin-cleaving MMPs(2,9)are called gelatinases,MMPs with broad-spectrum degradation of ECM protein are called stromelysins(3,10,11)or matrilysin(7).With the discovery of other collateral homology,the MMP family including membrane-related MMPs continues to grow.MMPs can be used as markers for molecular biological markers and cancer metastasis,and can be helpful for diagnosis and prognosis.Many studies have found that an increase in the expression of MMPs protein and mRNA is a key event leading to the occurrence and progression of breast cancer,and that MMPs may promote metastasis of breast cancer.On the other hand,recent studies have highlighted the fact that some MMPs,such as MMP-8,may provide an active defense mechanism instead of stimulating tumor proliferation,and these proteases may play a protective biological role in the process of cancer.MMP-1,as a collagen lyase,can be widely expressed in the intercellularsubstance and degrade I,II,and type III collagen,which is involved in the physiological and pathological processes of the body.Studies have shown that the high expression of MMP-1 is associated with the poor prognosis of gastric,bladder and prostate cancer.The molecular biological role of MMP-1 in breast cancer has been studied and analyzed.Many experiments in vivo and in vitro have been carried out to study the role of MMP-1 in the pathogenesis of breast cancer.It has also been found that the expression of MMP-1 in breast cancer and its stroma are related to the progression and poor prognosis of breast cancer.However,the expression of MMP-1and the corresponding regulatory mechanisms are still unclear and controversial.The expression of MMP-1 in molecular classification of breast cancer,its interaction with other proteins,and whether MMP-1 can become a new molecular target for the treatment of breast cancer,especially for breast cancer prone to metastasis,are worthy of further study.Objective: The purpose of this study was to investigate the expression of MMP-1 protein and mRNA in ER positive group,HER-2(3+)group and TNBC group,and to investigate the relationship between MMP-1 protein and clinicopathological features of breast cancer.To investigate the changes of biological characteristics and the effect on signal pathway of breast cancer cells after MMP-1gene silencing.We studied the role of MMP-1 in the occurrence and development of breast cancer,MMP-1 may be a molecular target for breast cancer treatment,and we will provide theoretical basis for clinical treatment of TNBC.Methods:1.Immunohistochemical method was used to detect the expression of MMP-1 protein in 99 cases of breast cancer and 15 cases of paracancerous tissues.,including ER positive group(51 cases),HER-2(3+)group(22 cases),TNBC group(26cases).To observe the expression of MMP-1 protein in breast cancer cells and stroma cells,to analyze the relationship with the clinicopathological parameters and the relationship with the P53 protein and the Ki67 index.2.Western-blot method was used to detect the expression of MMP-1 protein in 28 fresh samples of breast cancer.Among them,ER positive group(14 cases),HER-2(3+)group(7 cases),TNBC group(7 cases).To observe the expression level of MMP-1protein in breast cancer cells and analyze the difference of MMP-1 protein expression in different groups and its relationship with clinicopathological parameters.3.RT-PCR method was used to detect the expression of MMP-1mRNA in 17 fresh samples of breast cancer.There were 4 cases in ER positive group,6 cases in HER-2(3+)group and 7 cases in TNBC group.The expression of MMP-1mRNA was observed,and the expression difference in different groups of breast cancer was analyzed.4.According to the principle of siRNA design,we designed the shRNAs targeting MMP-1 gene,and constructed MMP1-shRNA#1,MMP1-shRNA#2,MMP1-shRNA#3 eukaryotic expression vector,and transfected into MCF-7 cells.The transfection efficiency was detected by Western-blot experiment.5.The MMP1-shRNA#2-GV102 plasmid with the highest silencing efficiency was used to transfect MCF-7 cells and MDA-MB-231 cells.The expression of MMP-1 protein was detected by immunohistochemistry,and the expression of MMP-1protein in the two groups after MMP-1 gene silencing was analyzed.6.The proliferation of MCF-7-MMP1 shRNA,MDA-MB-231-MMP1 shRNA and their corresponding control group was detected by MTT assay,and the proliferative ability of the two groups after MMP1 gene silencing was analyzed.7.The clones of MCF-7-MMP1 shRNA,MDA-MB-231-MMP1 shRNA and their corresponding control cells were detected by plate clone forming assay.The clone forming ability of two groups of cells after MMP1 gene silencing was analyzed.8.The cell cycle of MCF-7-MMP1 shRNA,MDA-MB-231-MMP1 shRNA and their corresponding control group was detected by flow cytometry.The cell cycle changes of the two groups after MMP1 gene silencing were analyzed.9.The invasion and migration of MCF-7-MMP1 shRNA,MDA-MB-231-MMP1 shRNA and their corresponding control cells was detected by Transwell invasion assay and migration assay.The ability of invasion and migration of two groups of cells after MMP1 gene silencing was analyzed.10.The migration of MCF-7-MMP1,shRNA-MDA-MB-231-MMP1 shRNA and their corresponding control cells was detected by scratch healing test.The migration ability of two groups of cells after MMP1 gene silencing was analyzed.11.The expression of c-myc,p-AKT and AKT proteins in MCF-7-MMP1 shRNA,MDA-MB-231-MMP1 shRNA and their corresponding control cells was detected by Western-blot method.The changes of c-myc,p-AKT and AKT protein levels in the two groups after MMP1 gene silencing were analyzed.12.The expression of Bcl-2,BAX and Cleaved Caspase 3 proteins in MCF-7-MMP1 shRNA,MDA-MB-231-MMP1 shRNA and their corresponding control cells was detected by Western-blot method.The changes of Bcl-2,BAX and caspase 3protein levels in the two groups after MMP1 gene silencing were analyzed.13.All the data analysis and statistical processing were carried out by spss17.0statistical software.We used different statistical methods such as the χ2 statistics test,the Spearman correlation test,the Kruskall-Wallis H test,the independent sample T test,basing on different data samples.The difference was statistically significant in terms of p < 0.05.Results:1.In breast cancer tissue,MMP-1 protein was expressed in the cytoplasm of cancer cells and in the stroma of cancer tissues.The positive expression rate in cytoplasm of cancer cells and stroma was higher in lymph node metastasis group than in non-lymph node metastasis group.MMP-1 protein was not expressed in breast cancer adjacent tissues.2.The expression rate of MMP-1 protein in ER positive group,HER-2(3+)group and TNBC group was significantly different.In TNBC group,the positive expression rate of MMP-1 protein in the cytoplasm of cancer cells was 76.92%(20/26)and the positive expression rate of MMP-1 protein in the stroma was 92.31%(24/26),which were higher than that in ER positive group and HER-2(3+)group.3.The expression level of MMP-1 in the cytoplasm of cancer cells and tumor stroma was positively correlated with the expression level of P53.The expression level of MMP-1 in cytoplasm of cancer cells and tumor stroma was positivelycorrelated with Ki-67 index.4.Western-blot analysis showed that the expression of MMP-1 protein was significantly different in ER positive group,HER-2(3+)group and TNBC group,and the expression level of MMP-1 protein was the highest in TNBC group(33.90 ±7.56).5.The expression of MMP-1 protein was related to tumor size,clinical stage and lymph node metastasis.The expression of MMP-1 in tumor diameter > 2 cm group,clinical stage Ⅲ Ⅳ group,lymph node metastasis group was higher than that in tumor diameter ≤ 2 cm group,clinical stage Ⅰ Ⅱ group and no lymph node metastasis group,and the difference was statistically significant.6.The expression of MMP-1 mRNA was detected by RT-PCR in all three groups of breast cancer.The expression of MMP-1m RNA in TNBC group was higher than that in ER positive group and HER-2(3+)group,the difference was statistically significant.7.The eukaryotic expression vector of MMP1-shRNA-GV102 was successfully constructed by using RNAi vector GV102 and stably transfected MCF-7cells.Western-blot assay showed that the expression level of MMP-1 protein was significantly decreased after transfection in each experimental group,and the silencing efficiency of MMP1-shRNA#2-GV102 plasmid was the highest.8.The results of cellular immunohistochemistry showed that after the MMP-1gene was silenced,the expression of MMP-1 protein in the MCF-7 and MDA-MB-231 cells was significantly lower than that in the corresponding control group.9.MTT results showed that the proliferation ability of MCF-7 MDA-MB-231 cells was significantly decreased after MMP-1 gene silencing.10.The results of plate cloning test showed that after MMP-1 gene silencing,the clone formation of MCF-7 and MDA-MB-231 cells was significantly lower than that of the corresponding control group.11.The results of flow cytometry showed that after MMP-1 gene silencing,thenumber of MCF-7 and MDA-MB-231 cells in G0/G1 phase increased significantly and the number of cells in S phase and in G2 / M phase decreased significantly compared with the corresponding control group.12.The results of Transwell invasion assay showed that after MMP-1 gene silencing,the invasiveness of MCF-7 and MDA-MB-231 cells was significantly lower than that in the control group.13.The results of transwell migration assay showed that after MMP-1 gene silencing,the migration ability of MCF-7 and MDA-MB-231 cells was significantly lower than that in the control group.14.The results of scratch healing test showed that after MMP-1 gene silencing,the migration ability of MCF-7 and MDA-MB-231 cells was significantly lower than that of control group.15.The results of Western-blot assay showed that after MMP-1 gene silencing,the protein expression of c-myc,p-AKT,AKT was lower in MCF-7 and MDA-MB-231 cells than the control group.16.The results of Western-blot assay showed that after MMP-1 gene silencing,the expression of Bcl-2 protein in MCF-7 and MDA-MB-231 cells was lower than that in control group.the expression of BAX and Cleaved Caspase 3 in breast cancer cells was higher than that in control group.Conclusion: 1.The positive expression of MMP-1 protein in cancer cells and stroma was correlated with lymph node metastasis,and the expression of MMP-1protein was positively correlated with the expression of p53 and ki-67 index.The MMP-1 protein content was related to tumor size,clinical stage and lymph node metastasis.It suggests that MMP-1 protein can promote the progression and metastasis of breast cancer.2.The expression of MMP-1 protein and mRNAwas significantly different in ER positive group,HER-2(3+)group and TNBC group,The expression of MMP-1and mRNA in TNBC group was significantly higher than that in ER positive group and HER-2(3+)group.3.The expression of MMP-1ShRNA eukaryotic expression vector MMP1-shRNA-GV102 was successfully constructed,and was transfected into MCF-7and MDA-MB-231 cells.After transfection,the expression of MMP-1 protein was significantly decreased.4.MMP-l gene silencing can inhibit the proliferation and the ability of form clones in MCF-7 and MDA-MB-231 cells.MMP-l gene silencing caused the number of cells in the G0/G1 phase increased significantly,and the cells in the S phase and G2/M phase decreased significantly,which hindered the normal process of DNA synthesis.5.MMP-l gene silencing can reduce the migration and invasion of MCF-7 and MDA-MB-231 cells.6.The expression of c-myc,p-AKT and AKT in MCF-7 and MDA-MB-231 cells decreased after MMP-1 gene silencing,suggesting that MMP-1 gene silencing may inhibit the proliferation and invasion of breast cancer cells by regulating AKT/c-myc signaling pathway.7.After MMP-1 gene silencing,the expression of Bcl-2 protein in MCF-7 and MDA-MB-231 cells decreased,while the expression of BAX and Cleaved Caspase3 protein increased,suggesting that MMP-1 gene silencing may inhibit the proliferation of breast cancer cells by promoting cell apoptosis.8.MMP-1 can be used as a biomarker of lymph node metastasis and poor prognosis of breast cancer,and it may be the target gene for the treatment of refractory breast cancer such as TNBC. |
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