TAZ Expression In Human Glioma And Its Impact On Glioma Cell Proliferation In Vivo And In Vitro

Glioma is the most common primary intracranial tumor,especially glioblastoma multiforme(GBM)is the most frequent,aggressive and fatal type of glioma in the adultsaccounting for 50% of all gliomas.Despite advances in the combination of treatment with surgical resection,radiotherapy,chemotherapy and biological therapy,the prognosis remains poor,with a median survival less than one year.GBM is characterized by rapid diffusely infiltrative growth and high level of cellular heterogeneity associated with therapeutic resistance.At present,it has been demonstrated that glioma is genetic disorder associated with multiple genetic abberations.Multiple oncogenes,tumor suppressor genes and signaling pathways are involved in the molecular pathogenesis of gliomas.Therefore research focus on the underlying mechanism of molecular genetics leading to the glioma has become one of the important hotspots in glioma studies,and will be helpful for understanding of the pathogenesis of glioma and exploring new strategies and targets of glioma therapy.The Hippo pathway was firstly discovered in Drosophilaa and identified as evolutionally conserved signaling cascade.Itplays an important role inorgan and tumor development.It regulates organ size by inhibiting cell proliferation and promoting cell apoptosis.TAZ is a major downstream effector of the Hippo pathway.The Hippo pathwayphosphoralate TAZ through a series of phosphorylation events.The p-TAZwas combined with 14-3-3 in cytoplasm and leads to inhibition of proliferative and antiapoptotic activity of TAZ.Dysregulation of the Hippo-TAZ signaling pathway can promotecell proliferation.TAZ plays animportant role in a variety of cancers, such as breast cancer, liver cancer, colon cancerand wasvalidated to be associated with cancer metastasis and prognosis.So far,only a few studieson the significance of TAZ expression in gliomahave been reported.We have detected glioma cell lines and glioma samples of different WHO grades.The results showed that TAZ significantly overexpressed in most of malignant glioma,cell lines and glioma samples in comparison to that of the normal brain tisues.This evidence suggested that TAZ have been associated with the development and progression of gliomas.In order to clarify the relationship of TAZ and gliomas,Our study aims to explore the effects and underlying mechanisms of TAZ in modulating the biological behavior of glioma cells.This project is divided into three parts:Part Ⅰ:The TAZ expression level was detected in 41 glioma samples of different WHO grades,3 normal brain tissues and 9 gliomas cell lines by Real-time PCR and Western blotting analysis.We also used immunohistochemal and immunefluorescence staining toexamine the expression leveland localization of TAZ in glioma tissues.The correlation of TAZ expressionlevel and gliomagrades was also observed.Part Ⅱ: TAZ expression was konckeddown in LN229 and SNB19 cell lines with a higher expression level of TAZ by TAZ-si RNA,Simultaneously,the TAZ plasmid was transfected into U118 and U87 cell lines,respectively,whichshowed thelowest expression of TAZ.Real-time PCR and Western blottingting were used to detect TAZexpression in gliomas cell lines after transfection.The cell proliferation was determined by MTT assay.The cell invasive ability was evaluated by scratch and transwell assays.Cell apoptosis and cycle was detected by Annexin V-FITC staining and flow cytometric analysis.The relation between TAZ and TEAD4 was detected by Co-Immunoprecipitation.Part Ⅲ: Establishing intracranial LN229 gliomamodel in nude mice,bioluminescent imaging was used to detect tumor growth,and the survivalperiod of nude mice was observed;Biological characteristicsof xenografts were examined by immunohistochemal staining.Apoptosis was evaluated by TUNEL method.The results weresummarizedas follows:1.TAZ expression in 38 cases of 41 glioma tissues was increased than that in normal brain tissues,and positively correlated with tumor grade,TAZ expression in grade Ⅲ/Ⅳ was significantly higher than that of grade Ⅰ/ⅡTAZ expression was also upregulated in 7/8 malignant glioma cell lines.The highest expression was in LN229 and SNB19 cell lineswhile the lowest in U87 and U118 cell lines.The results of Western blotting analysis were consistent with those of Real-time PCR.Immunohistochemical and immunoflurorescence staining were applied to detect the localization of TAZ and p-TAZ expression in glioma cells,The results demonstrated that the expression of TAZ in both cytoplasm and nucleus were increased,whereas the nuclear TAZ expression increased with the ascending of tumor grades,but p-TAZ only could be detectedin cytoplasm.2.The TAZ expression could be significantly knocked down after TAZ si RNA transfection in LN229 and SNB19 cells.Then cell proliferation activity and cell survival ratewere inhibited,cell cycle was arrested in G0/G1 phase.Cellmigration and invasion ability was significantly inhibited and cell apoptosis increased.The TAZ expression was upregulated by TAZ plasmid transfection in U87 and U118 cell lines,cell proliferation activity and cell survival rate wereincreased significantly,cells in G0/G1 phase were decreased while cells in S phase increased.The invasion ability of cells was enhanced obviously,whereas the number of apoptosis cells was reduced.Examining with Co-immunoprecipatation showed that TAZ and TEAD4 were combined with each other,and the expression of c-myc was changed together with TAZ.3.LN229 cell was transfected with Lenti TAZsi,TAZ expression wasknocked down.Compared with control group,theluminescent signal intensity of xenograftglioma transfected with Lenti TAZsi was lower in regular interval examinations..Kaplan-Meier survival analysis showed that the survival of the mice in Lenti-TAZ si group’was significantly prolonged than that in Lenti-NC group.Immuno-histochemistry and TUNEL examination demonstrated that when TAZ expression was knocked down,the tumor cell proliferation and invasion were reduced,but cell apoptosis increased.Conclusions:1.TAZ expression is up-regalutled in malignant glioma cell lines and glioma specimens and positively correlated to tumor grade,suggesting that TAZ contributes to the glioma formation and progression.2.TAZ upregulation promotes glioma cell proliferation and invasion hile reducescell apoptosis.TAZ downregulation inhibits glioma cell proliferation,invasion ability,and increases cell apoptosis.TAZ may play an important role through combined with TEAD4 acted on c-myc3.The growth of intracranial xenografted LN229 glioma in nude miceis inhibited when TAZ is knocked down,and survival period of xenografted mice is prolonged in TAZ knockdown group than that of control roup.4.Based on the study of the regulation of TAZ on glioma cell growth,we confirm that TAZ play an active role in the pathogenesis of glioma as aoncogenic gene,The results of in vivo study are in coincident with those in vitro ones..So this study provides the evidence that TAZ could be a candidate target for gene therapy of human glioma.