Study On The Role And Mechanism Of EMT-related PEDF In Breast Cancer

BackgroundIn recent years,the importance of epithelial-mesenchymal transition in cancer metastasis and the relationship between EMT and tumor microenvironment has been a research focus.The concept of EMT was first discovered when its related changes of mesenchymal cell phenotype status in adult and embryonic epithelium, said by Garry Greenburg and Elisabeth Hay,they found that EMT not only is in embryonic development and tissue differentiation process, but also participates in tissue repair, organ fibrosis and tumor invasion and metastasis. In addition to the changes on cell morphology, EMT can also increase cell invasion and metastasis, induce stem cell properties,prevent their apoptosis and senescence-induced immune suppression, and allows to producethe relevant components of the extracellular matrix (extracellular matrix, ECM) increased. A series of biological changes of epithelial cells during EMT process, such as cell polarity disappeared, lost connections between cells, cytoskeletal remodeling, cell migration, or scattered, and the deletion of the cytokeratin expression of epithelial cell phenotype markers such as E-cadherin, β-catenin, tight junction proteinsand others enhanced expression of mesenchymal cell markers:as vimentin, fibronectin, N-cadherin, which translates to mesenchymal stromal-like cells withInterstitial characteristics,what made them acquire invasive or movement phenotype, therefore, the cancer cells transformed from epithelial-mesenchymalare more invasive in situ and stronger metastasis, lower sensitivity to chemotherapy and targeted drugs.In recent years, with ever increasing incidence of breast cancer, a cancer treatment subsequently increase, but still about 25%-35% of breast cancer metastasis and lead to treatment failure. In the process of clinical treatment, the same cases of breast cancer diagnosis, the clinical prognosis is often the difference is very big, the cause of the prognosis of patients with the different is the biological heterogeneity of cancer. The study found that EMT marker expression in individual and group levels are associated with poor prognosis of breast cancer, the EMT induced mammary epithelial cells, could be expressed in a cancer stem cell phenotype, and tumors of EMT are associated with cancer chemotherapy drug resistance, promote multi-resistant genes (multidrug resistance gene 1, MDR1) expression. Studies have shown that breast cancer cells can be induced in the phenotype of basaloid EMT, include:increase of Vimentin, E-caherin to the N-Cadherin, cytoskeleton reorganization, etc; Breast cancer cells EMT is associated with high snail expression, and P13K/Akt signaling pathway and snail play an important place in the EMT in breast cancer cells; The study found that the NF-κB p65 exists abnormal activation in breast cancer tissue, high expression of the NF-κB have close relationship with the occurrence and development of breast cancer, in the process of the occurrence of breast cancer and malignant progression plays an important role in promoting.Pigment epithelium derived factor (PEDF) is a form of protein can effectively inhibit angiogenesis, PEDF not only as an anti angiogenesis factor to restrain the growth of the tumor, and exogenous or cell surface PEDF is directly interact with the extracellular matrix to prevent tumor cell metastasis. Recent studies have found that PEDF protein can inhibit interstitial protein hydrolysis and morphology change to inhibit melanoma tumors extravasation, show that PEDF and directly interact with the extracellular matrix to prevent tumor cell metastasis, in tumor microenvironment promotes the apoptosis of tumor cells resulted in the apoptosis of endothelial cells. Recent experimental studies show that PEDF protein by laminin receptor protein AKT/ERK pathway down fiber connections, to curb breast cancer metastasis, and showed that the PEDF protein may have a dual function to inhibit the growth and metastasis of breast cancer, which has opened up a new way of prevent breast cancer progression. Currently for the promotion of EMT related factor, transcription factors have been research, especially in terms of breast cancer, metalloproteinases, micrornas in the regulation of EMT has made certain research progress, but to look for a targeted gene research the regulation of EMT in breast cancer research at home and abroad.This is the first study to investigate the relationship between PEDF protein expression in breast cancer and breast cancer epithelial mesenchymal transformation and its role in the metastasis of breast cancer. And Vimentin, E-cadherin, Snail, the NF-κB joint detection will be more helpful in assessment of tumor biological behavior and prognosis, and this is important.for guiding the treatment of breast cancer and improve the curative effect. Observe whether PEDF is mediated through breast cancer epithelial-interstitial cells convert to participate in the metastasis of breast cancer, looking for new treatment and drug targets for breast cancer foundation.Part one Expression and Correlation of PEDF protein in Breast cancer and EMTObjective:1. To clarify PEDF gene regulation in breast cancer during EMT by study the expression of PEDF, Vimentin, E-cadherin Snail anf NF-κB in invasive ductal carcinoma of the breast tissue samples to detect whether PEDF gene involved in breast cancer epithelial-mesenchymal transition process and detected breast cancer level, the organization clinicopathological parameters, molecular typing of relations.Methods:1. Using immunohistochemical methods to detect the expression of PEDF gene, mesenchymal marker Vimentin, epithelial marker gene E-cadherin, Snail and the transcription factor NF-κB protein in 119 cases of primary breast cancer and 30 cases of tissues adjacent to breast cancer in order to compare the expression of the five indicators, to analyze the relationship between the expression of PEDF, E-cadherin, Vimentin, Snail, NF-κB proteins and clinical stage of breast cancer,between the expression of E-cadherin, Vimentin, Snail, NF-κB proteins and PEDF,between the expression of PEDF, E-cadherin, Vimentin, Snail, NF-κB proteins and mammary gland and invasive ductal carcinoma prognosis, PEDF, between Vimentin, E-cadherin, Snail, NF-κB protein correlated with clinicopathological features and clinicopathological features univariate and multivariate analysis.2. Statistical analysis:using SPSS13.0 (SPSS, Inc., Chicago, IL, USA) statistical software. Using the t test or analysis of variance parameters χ2±S represents. Pearson or Spearman correlation analysis using correlation analysis, P<0.05 was considered statistically significantResults:1. The positive expression of PEDF, E-cadherin, Vimentin, Snail and NF-κB protein in breast invasive ductal carcinoma respectively was 44.5%,49.5%,47.9%, 57.1%; high protein of PEDF and lower expression of E-cadherin protein,higher expression of Snail protein, NF-κB protein were in a statistically significant correlation (P<0.001, r=0.496, r=0.34, r=-0.337, r=0.383).2. The expression of PEDF protein in invasive breast cancer was connected with tumor size (P<0.05); but not with the patient’s age, TNM stage, histological type, ER receptor, PR receptors and axillary lymph node metastasis (P>0.05).The expression of Vimentin protein in invasive breast cancer was connected with TNM stage, tumor size, axillary lymph node metastasis, ER receptor (P<0.001 and P=0.009; P<0.001 and P=0.009), but not with age, PR receptors, histological type (P>0.05). The expression of E-cadherin protein in invasive breast cancer was connected with tumor size, TNM stage, PR receptors, axillary lymph node metastasis (P<0.05); but not with age, histological type, ER receptor-independent (P>0.05); Snail protein expression was connected with tumor size, axillary lymph node metastasis, TNM stage, but not with age, histological type, ER receptor, PR receptor-independent (P≤0.001); NF-κB protein expression was connected with tumor size, histological grade, axillary lymph node metastasis status, but not with age, TNM stage, ER receptor, PR receptor-independent (P<0.001).3. In the 119 cases of invasive ductal carcinoma, the application of Spearman correlation analysis showed that high expression of Vimentin, Snail and NF-κB and low E-cadherin expression was negatively correlated (P<0.001). Vimentin expression of nuclear overexpression Snai, NF-κB expression was positively correlated with both said (P<0.001, r=0.428; P=0.002, r=0.291). In addition, PEDF expression and low expression of E-cadherin positive correlation (P<0.001, r=0.496), but high expression of Vimentin, Snail expression, NF-κB expression was negatively correlated (P<0.001, r=0.337; P<0.001, r=0.34; P<0.001, r=0.383).4. Using univariate Cox regression model analysis confirmed that PEDF, Vimentin,, E-cadherin, Snail, and NF-κB expression (P=0.006, P<0.001, P<0.001, P=0.001, P<0.001), lymph nodes metastasis (P=0.015), tumor size (P=0.012), pathological stage (P=0.012) and PR receptor cases (P=0.003) were independent prognostic factor; age, menopausal status, histological grade, ER status were no prognostic significance. Multivariate analysis confirmed Vimentin and E-cadherin expression and overall survival (OS; P=0.016 and P=0.004) were independent prognostic factor, PEDF, tumor diameter was no relapse rate (DFS) independent prognostic factor.Fart two Influence of breast cancer cells in vitro invasion of epithelial-mesenchymal transition and metastasis by the interference of suppression influence PEDF protein expressionObjective:To detect the possible role of PEDF during epithelial-mesenchymal transition by Sk-Br-3 breast cancer cell lines transfected with stable interference PEDF gene, the proliferation, invasion and apoptosis of which were detected.Methods:1. Designing siRNA so that to inhibit the expression of PEDF expression (PEDF-siRNA) in Sk-Br-3 breast cancer cells; designing lentivirus, in Sk-Br-3 cells were packaged and amplified (Lentivirus-PEDF-vector), Effective interference sequences were then transfected breast cancer Sk-Br-3 cells, using effects on the cytoskeleton morphology PEDF expression was detected by immunofluorescence staining down/amplification; analysis of transfected PEDF-siRNA/Lentivirus-PEDF-vector plasmid Changes descendants Sk-Br-3 breast cancer cells PEDF protein expression;2. Transwell chamber invasion assay was used to detect invasion ability ofPEDF-siRNA/Lentivirus-PEDF-vector plasmid group,normal control group and negative control group of breast cancer cells in order to analyze the influence of PEDF-siRNA/Lentivirus-PEDF-vector plasmid for human breast cancer Sk-Br-3 cell proliferation.3. Using RNA interference (RNAinterference, RNAi) technology to silence PEDF gene so that to observe the proliferation, invasion, the changes of adhesion properties of breast cancer cells, and changes of epithelial marker E-cadherin and Vimentin by Western blotting in human breast cancer Sk-Br-3 cells to explore the changes of PEDF gene during mesenchymal transition process in the breast epithelium;4. Application Packaging lentiviral helper plasmid was amplified PEDF gene transfection Sk-Br-3 breast cancer cells, breast cancer cells were observed in vitro proliferation, invasion, adhesion properties, and changes in Western blotting was detected in human breast cancer Sk-Br-3 cells, epithelial marker E-cadherin, Vimentin expression changes in interstitial mark explore PEDF gene mesenchymal transition process between the breast epithelium;Result1. Inhibiting and increasing the expression of PEDF in Sk-Br-3 cells in order to do transwell experiments, the result shown that EDF may inhibit the invasion and migration of breast cancer cells. In addition,with the overexpression of PEDF, immunofluorescence staining Sk-Br-3 cells from mesenchymal-like morphology of the skeleton into epithelioid.2. The expression of PEDF between normal control group and negative control group was significantly higher than siRNA-transfected plasmid cell group,but significant lower than the Lentivirus-vector plasmid groups by Western blotting (P< 0.05), in which siRNA transfected plasmid group minimum protein level.3. With the overexpression of PEDF,the epithelial cell marker molecule E-cadherin expression was significantly up-regulated in Sk-Br-3 cells;while the expression of mesenchymal cell markers Vimentin are significantly reduced.However,the expression of molecular markers of epithelial were significantly reduced on PEDF-siRNA transfected group, while mesenchymal marker molecule expression was significantly increased.4. Statistical analysis:Using SPSS 13.0 (SPSS, Inc., Chicago, IL, USA) statistical software. Using the t test or analysis of variance parameters χ2±S represents. Between multiple sets of parameters using single factor analysis of variance, P<0.05 was considered statistically significant.