The Role Of Smarcdl In Human Glioblastoma Progression,Invasion And Chemoresistance

Glioblastoma is the lethal primary brain tumor in the central nervous system(CNS),characterized by the high ability of proliferation,invasion and chemoresistance.Despite aggressive treatments,namely accurate surgery accompanied by chemotherapy and radiotherapy,the prognosis remains dismal with a median survival of less than 20 months and patients with inevitable recurrence usually survive less than 12 months.The 2016 World Health Organization classification of tumors in the CNS highlighted new entities of molecular features in defining the glioma grades.Therefore,comprehensive studies at the cellular and molecular levels are urgently needed to find better therapeutic,diagnostic and prognostic targets for glioblastoma.Recently,tumor epigenetics has became the prevalent of research.The role of chromatin remodeling protein in tumorigenesis has been gradually uncovered.Smarcdl is the non-catalytic subunit,but is indispensable in all the SWI/SNF family,which has been previously reported decreased in several human malignancies.Notch1 is deemed to be activated in primary glioblastoma,which is postulated to act as an oncogene and facilitate tumor malignant phenotype.Therefore,we focused on the role of smarcdl in glioblastoma cell proliferation,invasion and chemoresistance.The crosstalk between smarcdl and Notch1 contributed to the malignancy of glioblastoma and broke up of this feedback loop may be a potential therapeutic target for glioblastoma treatment.This thesis will include five partsPart I The expression of smarcdl in human glioblastoma tumor tissues and cell lines.Objective:To investigate the relative expression of smarcdl in patient glioma tissues and glioblastoma cell lines.To establish cell lines of knock-down and over-expression smarcdl by lentivirus transfection.Methods:Quantitative real-time PCR(qRT-PCR),western blot and immunofluorescence assays were employed to compare the relative expression of smarcd1:1)between normal brain tissues and glioma tissues;2)between normal human astrocytes and glioblastoma cell lines(U87 and U251).The lentivirus-mediated smarcd1 gene knock-down and over-expression approach was conducted.Results:Compared with normal brain tissues,the relative expression of smarcdl was significantly down-regulated in high grade gliomas.Compared with normal human astrocytes,the relative expression of smarcdl was decreased in U87 and U251 cells.Knock-down and over-expression of smarcdl in U87 and U251 cell lines were successfully constructed.Conclusion:The expression of smarcdl was decreased in glioblastoma both in tumor tissues and cell lines,which may act as a tumor suppressorPart Ⅱ The effects of smarcdl on cell proliferation and invasion in glioblastoma cell nes.Objective:To investigate the role of smarcdl in glioblastoma proliferation and invasion and to clarify the potential mechanisms.Methods:CCK-8 and colony forming assays were used to testify cell proliferation rate in smarcdl knock-down and over-expression cell lines in vitro.Tumor xenografts study was taken to demonstrate the role of smarcdl in glioblastoma growth in vivo.Flow cytometry was employed to analyze cell cycle.Transwell assays were utilized to evaluate the cell motility and invasive ability.The protein levels of CDK4,Cyclin D1,Vimentin,β-Catenin,N-Cadherin,E-Cadherin and ZO-1 were detected by western blot to evaluate cell cycle arrest and invasion ability.Results:Knock-down of smarcdl increased glioblastoma cell proliferation in vitro and in vivo,while over-expression of smarcd1 inhibited cell growth.High expression of smarcdl arrested cells in G1 phase and suppressed cell migration and invasion.Conclusion:The expression of smarcdl hinted glioblastoma cell proliferation,migration and invasion.Part Ⅲ The effects of smarcd1 on TMZ related chemoresistance in glioblastoma cell nes.Objective:To investigate the role of smarcdl in temozolomide(TMZ)induced glioblastoma apoptosis and the potential mechanisms.Methods:Flow cytometry was employed to analyze cell apoptosis after treating with TMZ in knock-down and over-expression glioblastoma cell lines.CCK-8 assay was used to detect cell viability after TMZ administration.The protein levels of Bcl-xl,Bax,Cleaved-Caspase3 and p21 were analyzed by western blot.Co-immunoprecipitation(co-IP)assay was operated to testify the connection between smarcdl and tumor suppressor p53.Results:The apoptotic cell ratio was deceased and the cell viability was increased after TMZ treatment in smarcdl knock-down cells compared with control cells.The anti-apoptotic proteins were upregulated and the pro-apoptotic proteins were downregulated in smarcdl low expression cells.The tumor suppressor protein p53 was directly connected with smarcdl and decreased when knock-down of smarcdl.Over-expression of smarcdl exhibited the reverse results above.Conclusion:Low expression of smarcdl in glioblastoma cells promoted chemoresistance after TMZ treatment,while over-expression of smarcdl exerted susceptibility to TMZ.The interaction with p53 might be the potential mechanism of smarcdl induced tumor chemosensibility.Part IV The role of crosstalk between smarcdl and Notch1 signaling pathway inglioblastoma biological behavior.Objective:To investigate whether the interaction between smarcdl and Notchl signaling pathway is involved in glioblastoma proliferation and invasion.Methods:QRT-PCR and western blot assays were operated to detect the expression of Notchl and its targeted genes Hesl,Hey1.CCK-8 and Transwell assays were employed to test cell proliferation and invasion after Notchl specific inhibitor DAPT treatment in smarcdl knock-down and control cells.Active Notchl(NICD)expression was inhibited by siRNA and DAPT and then the expression of smarcdl,Hes1,Hey1 were analyzed by qRT-PCR,western blot and immunofluorescence.Hesl siRNA was used to downregulate gene expression and then detect the expression of smarcdl by qRT-PCR,western blot and immunofluorescence.Results:The relative expression of Notchl,Hesl and Heyl were enhanced when knock-down of smarcdl,while over-expression of smarcdl inactivated Notchl signaling pathway.Inactivation of Notchl by DAPT can restore low expression of smarcdl induced proliferation and invasion abilities.In glioblastoma cells,inhibition of Notch1 and Hesl significantly increased the expression of smarcdl.Conclusion:Smarcdl and Notchl signaling pathway could regulate each other and form a feedback loop to promote the malignant phenotype of glioblastoma.Part Ⅴ The role of smarcdl in chromatin structure and gene transcription regulation.Objective:To explore whether smarcdl effects chromatin remodeling and regulates downstream gene expression.Methods:Immunofluorescence assay was conducted to evaluate the expression and distribution of histone H3K4me3,H3K27me3,H3ac and heterochromatin protein-la(HP-1α).Chromatin Immunoprecipitation and Next Generation Sequencing(CHIP-seq)technology were employed to analyze the potential transcriptional genes of smarcdl.QRT-PCR assay was used to testify the targeted gene expression.Western blot was used to analyze the expression of smarcdl downstream protein IL-9R and the involving STAT3 signaling pathway.Results:The immunofluorescence intensities of the "open chromatin" histones H3ac and H3K4me3 were enhanced,while the "closed chromatin" proteins H3K27me3 and HP-la were weakened in smarcdl knock-down glioblastoma cells compared with the over-expression smarcdl cells.Smarcdl directly interacted with IL-9R gene,while low expression of smarcdl could enhance the expression of IL-9R and the activation of STAT3 pathway.Conclusion:Smarcdl acted as the vital subunit of SWI/SNF chromatin remodeling family and facilitated chromatin condensation.Smarcdl also combined in the IL-9R encoding gene and repressed its transcription.SummaryThis original study found that smarcdl was down-regulated in both glioblastoma tissues and cell lines.We further demonstrated the crosstalk between smarcdl and Notch1 in glioblastoma cell lines through lentivirus-mediated knock-down and over-expression of smarcdl.This crosstalk facilitated glioblastoma cell proliferation,invasion and chemotherapy resistance.Moreover,smarcdl could condense chromatin structure and combine with IL-9R gene,thus reducing its expression.Hence,smarcdl could act as an important tumor suppressor and have the potential as a candidate for the treatment of glioblastoma.