IKK ε Expression In Human Gliomas And Its Molecular Mechanisms In Promoting Glioma Cell Proliferation And Invasion

Gliomas is the one of the most incurable tumors. Despite the development of neurosurgery and radiation therapy technology has played an important role in promoting the treatment of gliomas, the effect is not ideal, the prognosis remains poor.[1].Especially the glioblastoma (GBM),because of its high proliferation and invasive biological behaviour, although surgery combined with radiation and chemotherapy the prognosis has not been significantly improved. The average survival time of only12-15months[2].Pleomorphic glioblastoma cell tumor (glioblastomamultiforme, GBM) patients, the survival of less than1year[3].How to control malignant proliferation and invasion of glioma is the focus in current research. Traditional drug are more concentrated in the active proliferation of tumor cells or resulting in abnormal proliferation of material such as DNA, proteins and genes,which lack of specificity of the glioma cells. It is recognized that glioma is a disorders represented multi-gene and subtile molecular mechanism.involving a variety of abnormal cytokines in cell signaling pathways[4].Therefore looking for genes lead to the glioma, then understanding of the pathogenesis of glioma and further explore new strategies and targets of glioma therapy has become an important mission in glioma research areas. In recent years, RNA interference technology is developing rapidly, and provides a new idea for the study of glioma. It not only be used to find thegene proliferation and invasion-related targets, but also as a potential treatment for glioma therapy. IKK/NF-KB pathway is an important cancerigenic signaling pathway[5]. The phosphorylation of1KB (inhibitor of NF-KB) is involved the NF-KB pathway activation, while the latter depends on the regulation of IKK kinase family (kinases, IKKs) as a key member of the NF-KB signaling pathway,IκBs are phosphorylated by IKK complexes-which leads to the ubiquitination and proteasomal degradation of IκBs. NF-K B is then released and translocated into the nucleus to regulate the expression of targeted more than400kinds of gene transcription involved in immune response, proliferation, differentiation, apoptosis and tumor formation[6-8]. Recently IKKε as a member of the IKK family discovered are highly expressed and plays an important role in malignant transformation in many tumors. And most studies have confirmed that IKKs activating NF-K B pathway is an important carcinogenicity mechanism.Cells and animal studies found that knockdown IKKs can suppress canceration in cancers such as breast cancer[9],ovarian cancer[10],prostate cancer[11], but little studied was involved in the expression and mechanisms of IKKε in glioma.Our previous study examined9glioma cell line (D32, D37, M067, H4, U138, U251, U87, U373, TP483),3normal brain tissue and10glioma tissue,the result suggest IKKε is overexpression in most glioma tissue and glioma cells(only TP483no expression),and almost no expression in normal brain tissue. These preliminary findings is similar to the role of IKKε oncogenes in breast cancer. We believe that these findings provide important clues for the further study of the pathogenesis of glioma molecular pathology, it is necessary to conduct preliminary studies on glioma IKKε/NF-K B pathway.The first part we detected IKKs mRNA expression of51cases of different types of glioma clinical specimens and7normal brain tissues and7cell lines by RT-PCR. IKKε gene expression at the protein level were detected by immunohistochemical methods and western blotting and the correlation between IKKε expression and tumor grade were studyed.The silencing effect of RNA interference on IKKε expression was evaluated by RT-PCR and Westernblot. The cell proliferation rate and cell cycle kinetics were evaluated by MTT assay and cell colony assay and flowcytometry respectively. And cell migration and the cell invasive ability was evaluated by scratch test and transwell assay. Cell apoptosis was detected by flow cytometry. Using Western blot and PCR were used to detect the members involved in proliferation, invasion, apoptosis-related protein in NF-κB pathway. NF-κB transposition was detected in the cells transfected with IKKs siRNA by cell immunohistochemistry and cell immunofluorescence. The NF-κB components P65was further validated by western blot.In the third part subcutaneous U251glioma model was established in nude mice for in vivo study, then a mixture of oligofectamine and siRNA was injected into tumor. every two days measuring tumor volume tumor growth. the expression of key members of the NF-K B pathway was detected upon the IKKs silencing by immunohistochemisty.ResultsIn the first part of experiment the tumor specimens showed that IKKs expression40/51cases and IKKε high expression in23/27III/IV grade. And the tumor level was positively correlated. Also detected in4/7cell lines IKKs overexpression. Glioma IKKε expression intensity increases with the degree of malignancy.IKKs siRNA dramatically down-regulated the expression of IKKε in glioma cells. flow cytometry detected the cell cycle SPF was declined and blocked in the G0/G1, MTT assay confirmed that cell proliferation was inhibited. And cell invasion and migration ability is significantly reduced; knockdown IKKε increased the levels of IκBα. In addition, the nucleus transposition of NF-K B is significantly reduced when IKKε was knockdowned.However, our experiment shows there seems no significant correlation between IKKε and apoptosis.Subcutaneous U251glioma model was established in nude mice for in vivo study. a mixture of oligofectamine and siRNA was injected into tumor. the tumor growth is inhibited. Compared with the group transfected with NC siRNA,immunohistochemisty indicated that proliferation and invasive proteins in the downstream of NF-K B were descended in the group transfected with IKKe siRNAConclusions1IKKε was overexpressed in human gliomas and positively related to tumor grade, it may be involved in the progression of glioma.2The malignant progression of tumors can be achieved by activating the pathway of IκB/NF-K B via IKKε in glioma.3The findings of in vivo study is in accordance with those in vitro study, which confirmed that the synthetic ikks siRNA can effectively inhibit the biological behavior of gliomas4IKKs can be a candidate therapeutic target for gene treatment,and RNA interference is an effective gene silencing technology for the potential clinical application.