Study On The Biological Function And Mechanism Of Syntenin In Breast Cancer

Part I:Expression of syntenin in breast cancer cells and tissues and construction of stable cell linesObjective:To detect the mRNA and protein expression levels of syntenin in breast cancer cell lines and27pair’s breast cancer samples. To establish syntenin-overexpressing stable cell lines and syntenin-knockdown stable cell lines, providing cell lines for the study of biological function of syntenin in breast cancer.Methods:(1) mRNA extracted from the cells and tissues using Trizol reagent and reverse-transcriped into cDNA for real-time quantitative PCR;(2) Western Blot was used to detect cell and tissue protein expression levels;(3) Full-length syntenin was amplified from human cDNA library, and constructed into the lentvirus cloning vector.293T cells were used to package lentiviruses. Puromycin was used to filter stable cell lines after virus infection of MDA-MB-231cells;(4) Construction of syntenin interference lentivirus vectors and packaged viruses in293T cells. MDA-MB-231HM cells were infected with lentiviruses and puromycin was used to filter stable cells.(5) Real-time quantitative PCR and Western Blot were used to detect the effect of overexpression or knokdown.Results:(1) Syntenin mRNA and protein expression levels were up-regulated in high-metastasis breast cancer cell lines;(2) Syntenin mRNA and protein expression levels were elevated in breast cancer tissues;(3) Successfully constructed syntenin stable overexpressing cell line231-SYN, its mRNA and protein levels were significantly higher than that of control cells;(4) Successfully constructed the syntenin stable interference expression cell lines231HM-262and231HM-622, the mRNA and protein expression levels in these cells were significantly lower than control cells.Conclusions:Syntenin mRNA and protein expression levels were up-regulated in high-metastasis breast cancer cell lines and breast cancer tissues, suggesting an important role of syntenin in breast cancer progression and metastasis. Successfully constructed syntenin overexpression and interference cell lines, providing an ideal experimental cell models to study syntenin biological function in human breast cancer cell lines. Part II:Effects of syntenin on breast cancer cell migration and invasion and its related mechanismObjective:To investigate the effects of syntenin on cell cycle, proliferation, migration and invasion of breast cancer cells and its related mechanism.Methods:(1) Flow cytometry was used to evaluate the effect of syntenin on cell cycle;(2) CCK8was used to investigate the effect of syntenin on cell proliferation in vitro;(3) Scratch assay and Transwell migration assay was utilized to evaluate the effect of syntenin on migration;(4) Cell invasion was assessed by Transwell invasion assay;(5) Western blot was used to study the impact of syntenin expression on the MAPK signaling pathway;(6) Western blot and function blocking integrin β1antibody were used to study the effect of integrin β1on ERK1/2activation;(7) ERK1/2specific inhibitor U0126was used to study the effect of ERK1/2activation on syntenin induced migration and invasion.Results:(1)The flow cytometry results showed that alerting syntenin expression has no effect on the proportion of each period of cell cycle;(2) CCK8proliferation assay showed that syntenin expression has no effect on migration ability of breast cancer cell lines;(3) Scratch assay and Transwell migration assay results showed that migration ability of syntenin-overexpressing cells was significantly enhanced compared to the control cells. In contrast, syntenin-knockdown cells had significantly decreased migration ability relative to the control group;(4) Overexpression of syntenin promoted the invasion ability of MDA-MB-231cells; whereas syntenin knockdown inhibited the invasion ability of MDA-MB-231HM cells.(5) Syntenin overexpression induced activation of ERK1/2, but not JNK and p38MAPK in breast cancer cells;(6) Integrin β1activation was involved in ERK1/2activation in breast cancer cells;(7) ERK1/2specific inhibitor U0126significantly inhibited the ERK1/2activation and syntenin-induced cell migration and invasion.Conclusions:Syntenin has no effect on breast cancer cell cycle and proliferation, but affect the migration and invasion of breast cancer cells in vitro. Activation of integrin β1and ERK1/2signaling pathway is essential for syntenin-induced breast cancer cell migration and invasion. Part III:Effects of syntenin on breast tumor growth and metastasis and its relationship with clinical parametersObjective:To investigate the effects of syntenin on breast tumor growth and lung metastasis in orthotropic xenograft nude mice model, and the expression of syntenin in breast cancer samples, clarifying the relationship between syntenin expression and clinical parameters.Methods:(1) Orthotropic xenograft nude mouse model was used to study the effect of syntenin expression in breast tumor growth and metastasis;(2) HE staining was used to observe the number of lung metastasis;(3) IHC was utilized to lable neovascularization of xenograft (CD31), count micro vessel density (MVD);(4) Expression of syntenin in239breast cancer tissues was assessed by IHC;(5) Expression of syntenin in breast cancer tissues were classified into two categories, syntenin negative expression and syntenin positive expression. Spearman’s correlation analysis was used to study the relationship between syntenin expression and clinical parameters;(6) Kaplan-Meier survival analysis was used to analysis sytenin expression correlation with patient’s overall survival and disease-free survival.(7) Cox proportional hazards regression analysis was used to study whether syntenin is an independent prognostic factor in breast cancer.Results:(1) Syntenin overexpression promote the growth of xenografts and syntenin knockdown inhibit breast tumor growth in nude mice;(2) Syntenin overexpression promoted lung metastases of xenograft tumors, the opposite was syntenin interference expression inhibited the formation of lung metastases;(3) Microvessel density of syntenin overexpressinon in nude xenograft was significantly higher than that in the control group, while the microvascular density of syntenin knockdown expression formed in nude mice was significantly reduced relative to the control group;(4) IHC results of human breast cancer tissues showed that in239patients with breast carcinoma,88cases of patients with syntenin positive expression, expression ratio of36.82%. Syntenin positive expression was correlated with tumor size, lymph node metastasis and recurrence;(5) syntenin positive expression was correlated with overall survival and disease-free survival of patients;(6) the results of multivariate analysis shows, syntenin was an independent prognostic factors for overall survival and disease-free survival in patients with invasive breast cancer.Conclusions:syntenin overexpression promotes breast cancer tumors growth, lung metastases and angiogenesis; the status of syntenin is a potential prognostic indicators in breast cancer patients.