ABSTRACT
AIMS: In this study, we examined the expression of GINS2 in glioma and determined its role in glioma development. METHODS: The protein expression of GINS2 was assessed in 120 human glioma samples via immunohistochemistry. Then, we suppressed the expression of GINS2 in glioma cell strains U87 and U251 using a short hairpin RNA lentiviral vector. In addition, RNA sequencing and bioinformatics analysis were performed on glioma cells before and after GINS2 knockdown. Subsequent co-immunoprecipitation and western blot experiments indicated possible downstream regulatory molecules. RESULTS: The present results showed that GINS2 can accelerate the growth of glioma cells, whereas the suppression of GINS2 expression decreased the proliferation and tumorigenicity of glioma cells. Mechanism research experiments proved that GINS2 can block the cell cycle by regulating certain downstream molecules, such as MCM2, ATM, and CHEK2. CONCLUSION: GINS2 is closely related to the occurrence and development of glioma, and is likely to become a prognostic marker for glioma patients, as well as a potential therapeutic target in the treatment of glioma.
Subject(s)
Brain Neoplasms/genetics , Brain Neoplasms/pathology , Carcinogenesis/genetics , Cell Proliferation , Chromosomal Proteins, Non-Histone/genetics , Glioma/genetics , Glioma/pathology , Animals , Apoptosis/genetics , Biomarkers, Tumor/genetics , Cell Cycle/genetics , Female , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Humans , Immunohistochemistry , Mice , Mice, Inbred BALB C , Neoplasm Transplantation , RNA, Small Interfering/genetics , Tumor Cells, CulturedABSTRACT
OBJECTIVE: To investigate the role of microtubule-actin crosslinking factor 1 (MACF1) in the response of glioma cells to temozolomide (TMZ). METHODS: TMZ was applied to a human gliomablastoma cell line (U87) and changes in the protein expression and cellular localization were determined with Western blot, RT-PCR, and immunofluorescence. The responses of the cells with MACF1 expression knockdown by RNA interference to TMZ were assessed. TMZ-induced effects on MACF1 expression were also assessed by immunohistochemistry in a nude mouse model bearing human glioblastoma xenografts. RESULTS: TMZ resulted in significantly increased MACF1 expression (by about 2 folds) and changes in its localization in the gliomablastoma cells both in vitro and in vivo (P<0.01). Knockdown of MACF1 reduced the proliferation (by 45%) of human glioma cell lines treated with TMZ (P<0.01). TMZ-induced changes in MACF1 expression was accompanied by cytoskeletal rearrangement. CONCLUSION: MACF1 may be a potential therapeutic target for glioblastoma.
ABSTRACT
OBJECTIVE: To investigate the effect of small interfering RNA (siRNA)-mediated silencing of PC4 and SFRS1 interacting protein 1 (PSIP1) on invasion and migration of human glioma U87 cells. METHODS: Chemically synthesized siRNA targeting PSIP1 gene was transfected into U87 cells via lipofectamine, and the gene silencing effect was determined using real-time PCR. The changes in the invasion and migration abilities of the transfected cells were assessed with Transwell assay and wound healing assay, respectively. Western blotting was used to analyze the expression of N-cadherin, ß-catenin and the transcription factor Slug. RESULTS: The mRNA and protein level of PSIP1 was significantly reduced in U87 cells after transfection with PSIP1 siRNA (P<0.0001). PSIP1 knockdown in U87 cells resulted in significant suppression of cell invasion and migration abilities (P<0.01) and also reduced N-cadherin, ß-catenin and Slug expressions. CONCLUSION: s Silencing of PSIP1 impairs the invasion and migration abilities of glioma cells and lowers the expressions of N-cadherin, ß-catenin and Slug, suggesting that PSIP1 may regulate Slug by classical Wnt/ß-catenin signaling pathway to modulate epithelial-mesenchymal transition and promote the invasion and migration of glioma cells.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Cell Movement , Glioma/pathology , Neoplasm Invasiveness , RNA Interference , Transcription Factors/metabolism , Adaptor Proteins, Signal Transducing/genetics , Antigens, CD/metabolism , Cadherins/metabolism , Cell Line, Tumor , Epithelial-Mesenchymal Transition , Humans , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , Real-Time Polymerase Chain Reaction , Snail Family Transcription Factors , Transcription Factors/genetics , Transfection , Wnt Signaling Pathway , beta Catenin/metabolismABSTRACT
Glioblastoma remains among the most devastating cancers with a median survival of less than 15 months and virtually no survival beyond five years. Currently, the treatment of glioma includes surgery, radiation therapy, chemotherapy, and comprehensive treatment. Intrinsic or acquired resistance to TMZ, is one of the greatest obstacles in successful GB treatment, and is thought to be influenced by a variety of mechanisms. The EZH2 gene, which is expressed in various solid tumors, can regulate gene transcription and promote the generation and progression of tumors. Our aim was to investigate the relationship between EZH2 and multidrug-resistance of human glioblastoma cells. In this study, we established TMZ-resistant U251 and U87 clones (U251/TMZ and U87/TMZ cells), which expressed high level of EZH2. Using RNA interference, we demonstrated that the downregulation of Ezh2 expression in U251/TMZ and U87/TMZ cells resulted in apoptosis and a cell cycle arrest in the G1/S phase. Furthermore, the reduced expression of Ezh2 altered the MDR, MRP and BCRP mRNA and protein levels. These findings suggest that EZH2 plays an important part in the development of multidrug resistance and may represent a novel therapeutic target for multidrug-resistant glioblastoma.
