Expression Of Med19 In Astrocytic Tumors And Role Of Altered Med19 On The Oncogenic Behavior Of Malignant Astrocytomas

In 2010 , U.S.A added 22,020 new cases of brain tumor patients , in addition to 13,140 cases of brain tumor patients died ( www.cancer.org, Cancer Facts & Figures 2010). Astrocytomas are the most common primary intracranial tumors, which origin from neuroepithelial. Astrocytomas account for approximately 73%-86% of all adult intracranial tumors, among them, nearly 90% are malignant astrocytomas , which are prevalent of anaplastic astrocytoma and glioblastoma. After completly surgery, the recurrence rate of malignant astrocytomas is nearly 60%, with poor prognosis and low survival time. Nearly 5 years, with the development of basic and clinical research, it found that the average survival time of glioblastoma patients who receiving surgery, radiotherapy and chemotherapy, is increased from 10 months to 14 months. However, astrocytomas with the biological characteristics of a rich blood supply and infiltrating growth are easy to relapse.Previous studies showed that the malignant progression, invasion and recurrence of astrocytomas are are a matter of multi-gene, multi-stage and multi-step complex processes, which are the results of a variety of genes, proteins and cytokines. The genes involved in the development of astrocytomas are epithelial growth factor, endothelial growth factor, transforming growth factor, tumor oncogenes and tumor suppressor genes. However, the molecular mechanisms underlying the initiation, maintenance and progression of astrocytomas still remain largely unclarified. Hence, identification and characterization of the regulatory molecules that involved in the astrocytomas tumorigenesis may offer important targets for treatment strategies.Med19 gene, interacting with the RNA polymeraseⅡ, was found by means of differential display from two different metastatic potential of human lung carcinoma cell lines(95C and 95D ). In previous reports, Med19 was higher expressed in human breast cancer. Med19 played an important role in the proliferation of human breast cancer cells, which suggested that the lentiviruses delivering shRNA against Med19 could be a promising tool for breast cancer therapy. However, little was known about its role in malignant progression of astrocytomas tumorigenesis. Hence, further investigation of the functional role of Med19 in the carcinogenesis of astrocytomas may offer a better understanding of malignant behavior of astrocytomas. The aim of this study is to clarify the exactly role of Med19 in the oncogenic process of astrocytomas by examining its expression level in astrocytoma tissues of different grades and astrocytoma cell lines and knocking down or overexpressing its expression level in astrocytoma cell lines. This study is consisted of four main parts: the first part is to investigate the expression pattern of Med19 in astrocytomas of different grades and survival rate of the patients analysis; the second part is to study expression level of Med19 in different astrocytoma cell lines, including U87 , U251, U373 glioblastoma cell lines; the third part is to construct lentiviral-Mediated RNAi and overexpression system of Med19 gene. Finally, the effects of siRNA or overexpression targeting Med19 on tumor proliferation, colony formation, cell cycle and apoptosis are evaluated in vitro and in vivo.PartⅠExpression of Med19 in Astrocytic Tumors and Survival Analysis of the PatientsObjective: The purpose of this study is to investigate the expression level of Med19 gene in astrocytomas of different grades and normal brain tissues and to analysis the direct relationship between the expression level of Med19 and patients'life span.Methods: The expression levels of Med19 mRNA were evaluated by real-time quantitative PCR, and the protein levels of Med19 were assessed by using immunohistochemistry and western blot. Further, the direct relationship between the expression level of Med19 and patients'life span was evaluated by the product-limit estimate of the survival function (Kaplan-Meier method).Results: First of all, quantitative real time PCR analysis demonstrated elevated expression levels of Med19/β-actin in high-grade astrocytomas versus low-grade (p<0.01) or normal brain tissues (p<0.01). And secondly, statistical analysis showed increased Med19 protein levels in high-grade astrocytomas versus low-grade tumors (p<0.001) or normal controls (p<0.01). Finally, Kaplan-Meier survival curves indicated that increased expression of Med19 was significantly associated with poor overall survival of astrocytoma patients (P<0.05).Conclusion: In summary, we demonstrate that Med19 plays an important role in the astrocytic tumors. Med19 is positive in brain astrocytomas. The Med19 expression levels of astrocytomas were significantly correlated with pathological grade and were negatively correlated with patients'life span. PartⅡExpression of Med19 in Astrocytic Tumors cell linesObjective: To investigate the expression pattern of Med19 in glioblastoma cell lines, U87, U251, and U373.Methods: Immunofluorescene labelling assay was employed to investigate the celluar location of Med19 protein in U251, U373, and U87 glioblastoma cell lines. The expression level of Med19 mRNA and protein were evaluated by real-time quantitative PCR and Western blotting respectively in U251, U373, and U87 glioblastoma cell lines.Results: (1) Med19 protein was predominantly detected in the cytoplasm of astrocytoma cells by immunofluorescene labelling assay. (2) Quantitative real time PCR analysis demonstrated similar expression levels of Med19 /β-actin in U251, U373, and U87 glioblastoma cell lines. (3) Western blot analysis showed Med19 protein expressed in U251, U373, and U87 glioblastoma cell lines.Conclusion: Med19 is present in glioblastoma cell lines, and its expression levels were examined at both mRNA and protein levels.PartⅢConstruction and identification of lentiviral-mediated RNA interference and overexpression system targeting Med19Objective: Med19 was detected in both astrocytoma tissues and glioblastoma cell lines. Its expression in astrocytoma samples was related to astrocytoma tumor grades. In this study, we constructed and identified the lentiviral-mediated RNA interference and overexpression system targeting Med19 for further study of its molecular function on the tumorgenesis of glioblastoma.Method: 1) siRNA targeting sequences were constructed, after screening, they were connected with lentivirus, fluorescence microscope, real-time PCR, Western blot were then applied in U373, U251 and U87 cells to confirm the effects of RNA interference 2) Plasmid overexpressing Med19 was synthesed, after sequencing, it was connected with lentivirus, fluorescence microscope, real-time PCR, Western blot were then applied in U373, U251 and U87 cells to confirm the effects of overexpression.Results: siRNA targeting sequences were constructed to knock-down Med19 expression, two of them were selected because of high interference efficacy of more than 75%. Med19 expression was significantly inhibited by these two siRNA sequences in U373, U87 and U251 cell lines at both mRNA and protein levels. The overexpression plasmid showed the same sequence with Med19, while Med19 expression was significantly increased by lentiviral-mediated overexpression plasmid in U373, U87 and U251 cell lines at mRNA and (or) protein levels.Conclusion: We successfully constructed lentivirus based RNAi and overexpression system, it was highly effective to change the expression of Med19 in glioblastoma cells.PartⅣLentiviral-mediated RNAi and overexpression targeting Med19 on oncogenic behavior of glioblastoma cells, in vitro and in vivo.Objective: Using lentiviral-mediated RNAi and overexpression targeting Med19, we investigated the role of Med19 on oncogenic behavior of gliomblastoma cells, both in vitro and in vivo.Method: MTT, colony formation, flow cytometry and nude mouse xenograft model were applied to assess the oncogenic behavior of gliomblastoma cell lines, U373 and U251. The fouces were on the effect of Med19 RNAi and overexpression on gliomblastoma cells growth both in vitro and in vivo.Results: (1)In cell-based experiments, knocking down of Med19 can suppress the proliferation, colony formation of U251 and U87 glioblastoma cell lines, while overexpressing Med19 can enhance the proliferation, colony formation of glioblastoma cell lines. (2) In Med19 RNAi and overexpression U251 glioblastoma cells, the cell cycle and apoptosis change. (3)The capability of invasion in Med19 overexpression glioblastoma cells was enhanced. (4) In a nude mouse xenograft model, Med19 RNAi lentivirus significantly delayed tumor growth and extended mice'life span. Conclusion: Med19 played an important role in tumorigenesis of glioblastoma.