The Expression Of PRR11 In Breast And Its Mechanism On Breast Cancer Growth And Metastasis

As the world’s most common malignancy tumor of women, breast cancer accounts for about 22% of all female malignant tumors, which is one of the main cause that jeopardize women’s health. As reported, there was a lower incidence of breast cancer in the past in China and other developing countries, but with changes in social, economic, environmental and many other factors, the incidence of breast cancer is increasing rapidly nowadays, the overall incidence of breast cancer increase nearly 10 times in 10 years world wide and is growing at an annual rate of 2.7% per year in China. Epidemiological data shows that amplification and abnormal changes in gene mutation and genetic susceptibility play an important role in the pathogenesis of breast cancer. In recent years, along with deepening research in the field of biological functions, some important oncogenes and tumor suppressor genes were found and the molecular mechanism of breast cancer development is gradually interpretation.With the proposed molecular type and tumor precise medical concepts, breast cancer research is changing gradually from the previous evidence-based treatment to the new whole genome sequencing and gene mutation for oriented individualized treatment, under this environment, explore new regulating cancer genes and finding the new regulatory mechanism and biological function has become the key points and difficulties in the field of breast cancer research recent years.Although some progress have been achieved in the diagnosis and treatment of breast cancer, due to characteristics of breast cancer distal recurrence, the prognosis of breast cancer patients remains poor. Better awareness of breast cancer pathogenesis and progression of molecular mechanism, mining breast cancer growth and metastasis in the invasion process of signs of cancer gene to develop a treatment for metastatic recurrence of breast cancer patients is one of the most important studies in china and abroad. PRR11 was reported playing an important role in a variety of tumor development. Proline-rich protein-PRR11(Proline-rich protein 11) gene is located on human chromosome 17q22 region, bioinformatics analysis showed PRR11 contains a bivalent nuclear localization signal, two proline-rich region, and a zinc finger domains. Proline-rich motif and SH3 domain can be interacted with protein and involved in cell signal transduction, causing a series of cancer-related events; zinc finger domains regulate gene transcription by binding to double-stranded DNA. The target of this protein may be membrane protein receptors, adhesion molecules, growth factors, transcription factor and tumor formation, proliferation, invasion and metastasis related protein and gene loci.In a previous study, PRR11 was considered to be a negative factor regulating tumor cells, but its role and clinical value in solid tumors are rarely known by people. In a recent study, immunohistochemical techniques were used to evaluate the expression level of PRR11 in 6 kinds of gastrointestinal tumors and the expression of PRR11 in hilar cholangiocarcinoma(HC) has an important impact on the patient’s condition. The expression of PRR11 was also reported in lung cancer, researchers found that the expression of PRR11 has related with cell cycle of lung cancer. Further research also found that PRR11 has a wide range of expression in solid tumors, which has the highest expression rate in esophageal squamous cell carcinoma(93.3%),and the lowest in liver cancer(53.3%). This result confirms previous studies on the high expression of PRR11 in squamous cell carcinoma of the lung. What’s more, among 5 digestive system cancer(esophageal squamous cell carcinoma, gastric carcinoma, node rectal cancer and hepatocellular carcinoma), of PRR11 expression analysis showed that PRR11 expression has obvious changes in the five tumors, also, PRR11 was closely related to the poor prognosis of gastric cancer. According to another report, by Ying Ji et al, PRR11 was regarded as a potential new target for diagnosis and treatment of lung cancer by regulating the important genes associated with cell cycle and tumor occurrence, and it was also reported involved in the transformation of epithelial cells of breast cancer. As a relatively new molecule,PRR11’s possible mechanism in the development of cancer remains unknown. More work is needed to assess the clinical significance of the protein and to clarify the underlying mechanism.The concept of Epithelial-mesenchymal transition(EMT) was proposed by Greenberg and Hay in 1982, refers to the specific physiological and pathological conditions of epithelial cells through a series of changes to the mesenchymal cell differentiation, characterized as the loss of epithelial polarity and the acquisition of mesenchymal phenotype, such as the decreased or disappeared connection and adhesion ability in cells, increased migration and invasion ability, the changing of cell morphology and marker expression, for instance, epithelial cell markers expression decreased, such as b-catenin, E-cadherin, mesenchymal phenotype markers expression increased, such as vimentin, N-cadherin, a-SMA. Also, EMT plays an important role in embryonic and tissue development, and exists in the pathogenesis of various chronic diseases or the invasion and metastasis of tumor. The molecular mechanism of regulation of cell EMT is more complex,which involved in several cell signal transduction pathways, including TGF-BMP pathway, PI3-K/Akt pathway, Wnt/b-catenin pathway, Src pathway and integrin pathway. However, the key gene which mediated EMT and correlated pathway is not clear, and its mechanism also remains to be elucidated.As reported, breast cancer cells originated from breast epithelial cells, and express the epithelial cell marker, and existing EMT phenomenon; In breast cancer cell lines, up-regulated mesenchymal cells markers--vimentin(VIM) can significantly improve the metastatic ability of breast cancer cell lines, and is closely related to the poor prognosis of the patients with breast cancer. All of the above studies suggest that EMT plays an important role in the process of breast cancer recurrence and metastasis and poor prognosis. In our previous study through overexpression or interference of PRR11 in MCF-7 breast cancer cell line and contracted cell lines at the level of gene and protein detection showed that the expression of PRR11 and β-catenin is the positive correlation, suggesting that PRR11 in breast cancer cell lines may positively regulate the Wnt /b-catenin pathway, and mediate EMT in breast cancer cells, thereby enhancing its ability of transformation, and affecting the prognosis of patients with breast cancer.Breast cancer is one of the biggest threats to women’s health. Its regulation mechanism is still unknown. There is lack of prognostic markers for clinical diagnosis, and also short of effective molecular targerts for treatment. Finding the novel targets and prognostic markers of breast cancer is of great importance in breast cancer research. PRR11, as an important tumor signal regulatory gene, has no any report on its regulation on growth and metastasis in breast cancer. This project will use a variety of characteristics of breast cancer cell model, and tumor metastasis model to study of role and biological function of PRR11 in the progress of breast cancer growth and metastasis, reveal the regulation and molecular mechanism, and explore the related clinical pathological and follow-up significance, to clarify the application prospect of PRR11 as a prognostic marker of breast cancer, ultimately discuss the possible new target for breast cancer treatment.Part 1. Expression of PRR11 in breast cancer cells and itsclinical significanceObjective: To detect the expression of PRR11 in breast cancer cells and tissues and to investigate the correlation between PRR11 expression and the clinipathological characteristics of breast cancer and its prognostic value.Methods: Real-time PCR, western blot and immunofluorescence were performed to detect the expression of PRR11 in breast cancer MCF-7 and MDA-MB-231 cells and normal breast epithelial MCF-10 A cells. Real-time PCR, western blot and immunohistochemistry were performed to detect the expression of PRR11 in breast cancerous and normal tissues. Correlation analysis was performed between PRR11 exrpression and the clinipathological characteristics of breast cancer, such as tumor stage, proliferation index, histological grade and molecular phenotype. Survival analysis and Cox proportional hazard model were performed to explore the prognostic significance of PRR11 in breast cancer.Results: munofluorescence assay showed that PRR11 was mainly located in cytoplasm ofberast cancer cells. RT-PCR and Western blot assay suggested that the expression of PRR11 in breast cancer cells and tissues was significantly higher compared with their non-cancerous counterparts both in m RNA and protein levels. The expression of PRR11 was significantly higher in breast cancers of high Ki67 index than that of low Ki67 index. PRR11 expression was positively correlated with stage of breast cancer. The overall survival of patients with high expression of PRR11 was significantly shortened than patients with low expression of PRR11. PRR11 might serve as a poor prognostic marker for breast cancer.Conclusion: The expression of PRR11 was significantly higher in cancerous breast cells and tissues than its expression in non-cancerous breast epithelial cells and normal tissues. PRR11 expression was associated with Ki67 expression and the clinical stage of breast cancer. PRR11 might serve as a poor prognostic marker for breast cancer.Part 2. PRR11 regulates proliferation and apoptosis of breastcancer cellsObjective: To investigate the effects of PRR11 regulation in breast cancer cell proliferation and apoptosis.Methods: The lentivirus-mediated PRR11 upregulating and silencing system were used to modulate PRR11 expression in breast cancer MCF-7 and MDA-MB-231 cells. MTT, Ed U, colony formation and TUNEL assays were performed to detect the effects of PRR11 modulation on cell proliferation and apoptosis. Breast cancer MCF-7 cells with stable expressing or silencing of PRR11 were injected subcutaniously into nude mice to explore the effects of PRR11 up- or down-regulation on breast cancer growth.Results: PRR11 upregulation resulted in enhanced cell viability, DNA synthesis and colony formation efficiency in breast cells. PRR11 downregulation significanted the above mentioned biological behaviors of breast cancer cells and induced apoptosis. In vivo studies suggested that PRR11 overexpression significantly increased, while PRR11 silence reduced the tumor volume, quality and Ki67 expression of MCF-7 cells xenografts.Conclusion: PRR11 upregulation promotes breast cancer cell proliferation in-vitro and growth in-vivo. PRR11 downregulation inhibits breast cancer cell growth and induces apoptosis.Part 3. PRR11 regulates the migration and invasion of breastcancer cellsObjective: To investigate the effects of PRR11 regulation in breast cancer cell migration and invasion.Methods: The lentivirus-mediated PRR11 upregulating and silencing system were used to modulate PRR11 expression in breast cancer MCF-7 and MDA-MB-231 cells. Scratches wound healing and transwell assays were performed to detect the effects of PRR11 modulation on cell migration and invasion. The expression of epithelial mesenchymal transition markers, such as E-cadherin, fibronectin and vimentin, was detected in PRR11 overexpressed breast cancer MCF-7 cells.Results: PRR11 upregulation resulted in enhanced scratch wound healing rate and cell invasion. When PRR11 was downregulated, breast cancer migration and invasion was inhibited. PRR11 expression induced EMT of MCF-7 cells. The expression of E-cadherin was inhibited, while the expression of fibronectin and vimentin was upregulated.Conclusion: PRR11 upregulation promotes breast cancer migration and invasion and induces EMT. PRR11 downregulation inhibits breast cancer cell migration and invasion.Part 4.The molecular mechanism of PRR11 in regulating breastcancer cell growth and metastasisObjective: To investigate the molecular mechanism of PRR11 in regulating breast cancer lell growth and metastasis.Methods: The impact of PRR11 modulation on the expression of β-catenin and its downstream targets including cyclin D1, c-myc, E-cadherin and vimentin, was detected by western blot and q PCR. TOP/FOP-flash assaywas performed to detect the transcriptionalactivity of β-catenin in PRR11 overexpressed MCF-7 cells. Cell migration and invasion were also evalued upon PRR11 upregulation to determine the role of β-catenin signaling pathway in PRR11-induced breast cancer cell proliferation and invasion. Immunohistochemistry was performed to determinethe correlation between PRR11 and β-catenin expression in breast cancer clinical specimens.Results: PRR11 overexpression significantly increased the expression and nuclear translocation of β-catenin in breast cancer MCF-7 cells. The downstream targets of β-catenin, including cyclin D1, c-myc and vimentin, were also increased, while the expression of E-cadherin was decreased. TOP/FOP-flash assay suggested that the transcriptional activity of β-catenin in PRR11 overexpressed MCF-7 cells. On the contrary, downregulation of PRR11 resulted in the opposite effects. Moreover, PRR11-induced upregulation of cyclin D1, c-myc and vimentin, as well as the increased TOP/FOP ratio were abolished by si RNA mediated β-catenin silencing. Further more, PRR11 enhanced cell proliferation and migration were significantly abrogated by si RNA mediated β-catenin silencing. The expression of PRR11 was positively correlated withβ-catenin in clinical breast cancer specimens.Conclusion: PRR11 promotes breast cancer cell growth and metastasis via β-catenin signaling.