Expression Of MACF1 Associated With Autophagy Promoted Migration And Invasion Of Glioblastoma Cells

Glioma is the most frequent primary central nervous system tumor,accounting for approximately 65%of primary intracranial tumors.Among all types of glioma,glioblastoma multiforme(GBM),also known as WHO IV Astrocytoma,is the most malignant,with high recurrent rate and considerable mortality.Despite of the current therapy including maximal surgical resection combined alkylating agent based-chemotherapy with temozolomide(TMZ)and ionizing radiation,the prognosis of GBM remains poor.The mean survival time of patients with GBM is only 15 months.Moreover,the overall 2-year survival rate of patients with GBM is only 27%.Tumor recurs and invades broadly into peripheral brain tissue after normative treatments is the main reason for the unfavorable prognosis.However,it is reported that hypoxia and ischemia of local tissue inward.tumor during the progression of GBM as well as stimulation of chemotherapeutics,TMZ,are related to the induction of autophagy of GBM cells,and thus promote survival and progression of these cells.Besides,Epithelial-Mesenchymal Transition(EMT)is reckoned to play important roles in the upregulation of migration and invasion of epithelial malignant tumors,including GBM.In our former researches,we have demonstrated that TMZ can induce the autophagy of GBM cells,and subsequently induce EMT as well as promoted migration and invasion.Besides,we found that TMZ remarkably upregulates the expression of Microtubule-Actin Crosslinking Factor 1(MACF1,also as ACF7).Moreover,it has been confirmed that MACF1 is related with migration of mouse epithelial cells.Thus we assumed that MACF1 may play a role in TMZ inducedautophagy and ensued EMT in GBM cells,and we paid some efforts to clarify it in U87 and U251 GBM cell lines.Chapter 1.Functional analysis of MACF1 in glioma in vitro.Method:siRNA specially targeted MACF1 was transfected into U87 and U251 cells.Meanwhile,lentiviral shRNA vectors were used to specifically and stably knock down the expression of MACF1 in U87 and U251 cells.The efficiency of siRNA and shMACFl was confirmed by qRT-PCR and western blot analysis.Cell growth was evaluated by cell counting and CCK-8 assays.Variation of cell mophorlogy was observed with microscope.Wound-healing assay,Transwell and Boyden chambers assay were performed to determine the function of MACF1 in glioma cell migration and invasion.The expression of EMT associated proteins were detected by Western Blot and Immunofluorescence assays.Results:Two siRNA were used to specifically knock down the expression of MACF1 in U87 cells.The efficiency of the two siRNA candidates was confirmed by qRT-PCR and western blot analysis.The efficiency of siMACF1 1#in U87 cells was highest(28.87%,P<0.001),and therefore was selected for further analysis in U87 and U251 cells.Cell counting and CCK-8 assays revealed that knockdown of MACF1 did not affect the glioma cell growth rate(P>0.05).Cell morphology was transformed into cells of shorter length with less as shorter protrusions after MACF1 suppressed.In wound-healing assay,quantitative analysis at 24h showed a significant reduction in wound closure in cells with silenced MACF1 expression compared with control cells(P<0.001).Moreover,in Transwell chamber assays,knockdown of MACF1 significantly decreased the percentage of migrated cells in shMACF1 cell group(P<0.001).Furthermore,Boyden chamber assays demonstrated that MACF1 depletion decreased the number of invaded cells in both U87 and U251 cells(P<0.05).Chapter 2.Autophagy upregulates expression of MACF1 in U87 cells.Method:The expression of MACF1 of rapamycin(RAPA)and TMZ and chloroquine(CQ)treated U87cells was determined by Western Blot.Results:After continuously treated with RAPA and TMZ separately,the expression of MACF1 of U87cells were upregulated(P<0.01).When treated with CQ plus RAPA or CQ plus TMZ,the expression of MACF1 of U87 cells were repressed compared to the negative control(P<0.05).Chapter 3.The role of MACF1 in autophagy promoted migration and invasion in U87 cells.Method:Lentiviral shRNA vectors were used to specifically and stably knock down the expression of MACF1 in U87 cells.Then shMACF1 and shNC U87 cells were treated with RAPA.Cell migration and invasion of these cells were determined by Transwell and Boyden chambers assays.Results:RAPA significantly promoted autophagy flux and the migration and invasion in shNC U87cells(P<0.01).In shMACF1 U87 cells,application of RAPA could not promote their function of migration and invasion,compared to shNC cells with RAPA treated(P<0.001).