Effect And Mechanism Of Notchl Signaling Pathway On Proliferation And Apoptosis In Human Glioma U87Cells

Backround and ObjectiveGlioma,a lethal type of intracranial tumor of invasive growth characteristics,is aprimary central nervous system tumor with the highest morbidity,fatality andrecurrence rate.Presently,although integrated treatment withsurgery,radiotherapy,chemotherapy,immune and other means are taken to deal withglioma,which has made a great progress in the diagnosis and treatment,the overalltherapeutic efficacy of glioma is still very poor and its pathogenesis is not clear whilethere is short of specific therapy.Recent studies have shown that the development oftargeted therapies for certain cell signal transduction pathways of molecular targets isan important means to improve the overall efficacy of the malignant tumor.Notchsignaling pathway play a key role in the development of central nervous system andprogression of glioma,decisiving the growth,proliferation,apoptosis in gliomacell,which is closely related to a variety of diseases including glioma while it beingactivated abnormally.Apoptosis,regulated by a number of apoptosis-related genes,isan initiative cell death process to maintain the balance the body’s internalenvironment.Notch signaling and its downstream effectors play an important role inthis process,however, the precise regulatory role of the Notch signaling pathway hasnot been clarified,especially the effect of abnormally activated Notch singal in tumorcell proliferation,as well as the effect on the downstream target genes and theapoptotic process is not clear.In this experiment,human brain glioblastoma U87celllines were clutured in vitro,and Notch signaling activator rhNF-κB and inhibitorDAPT were added to respectively.Through observing the effect of rhNF-κB andDAPT on U87cell proliferation and apoptosis,we try to discuss the regulatory roleand the possible mechanism of the Notch signaling system in human U87glioma cellline proliferation and apoptosis process,so as to provide experimental and theoreticalbasis for the molecular level treatment of human brain glioma and development ofanticancer drugs. Methods1The cultivation of human brain glioblastoma tumor U-87MG cell linesU87cells were cultured and passaged in medium containing10%FBS at37℃5%CO₂humid incubator,taking the logarithmic growth phase cells for experiments.Cells were divided into three groups,the rhNF-κB group,DAPT group and blankcontrol group respectively,and collected for testing after48h’s cultivation.2The impact of Notch signaling on proliferation and apoptosis of U87cellTrypan blue staining and MTT assay were employed to determin the impact ofrhNF-κB and DAPT on proliferation activity in U87cells,within inverted phasecontrast microscope to observe the changes of cell morphology and flow cytometry todetect apoptosis rate.3The regulation mechanism of Notch signaling on U87cell proliferation andapoptosisRT-PCR were employed respectively to evaluate the gene expression of Notch1Hes1and Bcl-2in each U87cells group, immunofluorescence staining and Westernblot were used to detect protein expressions,measuring the total gray value andanalysising each protein relative gray value quantitative comparatively.4Statistical treatmentAll data were used mean±SD.SPSS19.0statistical software was used forunivariate analysis of variance, chi-square test for statistical analysis,P<0.05wasconsidered statistically significant.Results:1Human brain glioblastoma U-87cells were successfully cultured,RT-PCRassay results showed that Notch1and the downstream target gene of Notch signalingpathway Hes1were expressed in the all U87cell lines,suggesting that Notchl/Heslsignaling pathway exist in U87glioma cells.2The influence of Notch signaling on U87cell proliferation and apoptosisMTT assay results showed that after24,48,72and96h cultivation,the A values ofrhNF-κB group cells were0.185±0.007,0.398±0.012,0.735±0.015,1.083±0.031,while DAPT group were0.102±0.003,0.130±0.004,0.161±0.006,0.194±0.003,and control group were0.167±0.005,0.265±0.008,0.496±0.011,0.737±0.016. Compared with the control group, rhNF-κB group cells had a higher vitality,while DAPT group and control group had more poor cell viability withvarying degrees of apoptosis （P<0.05）.With flow cytometry,the apoptosis rate ofrhNF-κB group,DAPT group and control group cell were0.96%±0.17%,26.51%±3.74%,8.76%±1.4%,respectively.Compared with the control group, DAPT groupapoptosis more and rhNF-κB group less,the difference was significant （P<0.05）3The regulation mechanism of Notch signaling on proliferation and apoptosisin U87cellsRT-PCR, immunofluorescence,Western-blot determination showed thatNotch1,Hes1,and Bcl-2expressed in all rhNF-κB group,DAPT group and controlgroup cells. And the mRNA and protein expression levels of Notch1wererespectively0.974±0.110,0.910±0.082,0.263±0.054and0.302±0.09,0.642±0.072,0.578±0.101.Hes1were0.833±0.106,0.896±0.145,0.241±0.047and0.423±0.090,0.402±0.088,0.696±0.115.Bcl-2were0.230±0.075,0.726±0.113,0.098±0.024and0.252±0.064,0.151±0.052,0.503±0.092. Compared with controlgroup,the mRNA expressions of each group increased,while DAPT group decreased.Through the Western Blot detection,the Notch downstream target gene Hes1andanti-apoptotic protein Bcl-2expression increased significantly after the induction ofrhNF-κB,while DAPT group significantly decreased （all P<0.05）,indicating thatrhNF-κB can induce the U87cells to evade apoptosis,while DAPT role on thecontrary, promoting apoptosis.Conclusion:1Notch1signaling pathway exists in human glioma U87cells and has played animportant role in the process of glioma cell proliferation and survival.Notch signalingis activated after rhNF-κB induction,while DAPT suppressed.2Notch1signal has played an important regulating role in the process ofproliferation and apoptosis in human U87glioma cell lines. After the activation ofNotch signaling by rhNF-κB induction,the U87cells were promoted to proliferate andto evade apoptosis.And Notch signaling are inhibited after DAPTinduction,promoting apoptosis and inhibiting the proliferation in U87cells.3The possible mechanism of Notch signaling system regulating the proliferation andapoptotic process of glioma U87cells: after the activation or inhibition of Notch1 signaling,it regulates target gene Hes1and anti-apoptotic protein Bcl-2expressionthrough Notch1/Hes1-Bcl-2pathway to regulate cell proliferation and apoptosis.