The Role And Its Mechanism Of Notch Pathway In Proliferation And Migration Of SH-SY5Y Cell Induced By MEHP

Di-(2-ethylhexyl)phthalate(DEHP)is a common environmental endocrine disruptor(EEDs),which is widely observed in thehuman living environment.As the most commonly used phthalate esters(PAEs)plasticizer,DEHPhas been detected in children’s toys and decoration materials.DEHP can enter the body of infants and young children through breathing,diet,and skin contact.The DEHP that enters the body can be firsthydrolyzed in the liver and small intestine cells and produce its metabolite,mono-2-ethylhexyl phthalate [mono-(2-ethylhexyl)phthalate,MEHP],and MEHPhas a main toxic effect on the body.Neuroblastoma(NB)is the most common extracranial malignant tumor in children,which seriously endangers the life andhealth of children.So far,the pathogenesis of NB is unclear.Studieshave found that some environmental endocrine disruptors can promote the proliferation and migration of neuroblastoma.As an environmental endocrine disruptor with neurotoxic effects,DEHP may affect the proliferation and migration of neuroblastoma.In this study,SH-SY5Y neuroblastoma cells were cultured in vitro and exposed to MEHP to clarify the effect of MEHP exposure on the proliferation and migration of SH-SY5Y cells.By inhibiting the Notch signaling pathway,it was explored that the Notch signaling pathway affects SH-SY5Y after MEHP exposure.The mechanism of action in cell proliferation and migration can provide a scientific basis for the prevention and treatment of neuroblastoma.Part I The effect of MEHP on the proliferation of SH-SY5Y cells and its mechanism Objective:Clarify the effects of MEHP on the proliferation,cycle and apoptosis of SH-SY5Y cells,and explore the role of key genes in the cell cycle and apoptosis.Methods:SH-SY5Y cells were routinely cultured inhigh-sugar DMEM medium.SH-SY5Y cells were exposed to MEHP with gradient concentration;CCK8 method was used to detect the survival rate of SH-SY5Y cells;according to the results of CCK8,the exposure dose and experimental group were divided into blank control group,solvent control group(0.1% DMSO),and low-dose group(10 μM MEHP),medium-dose group(50 μM MEHP),high dose group(250 μM MEHP),the exposure time was 12h and 24h.Flow cytometry was used to detect cell cycle and cell apoptosis;real-time PCR method was used to detect cell cycle and apoptosis key genes C-MYC,Cyclin D1,CDK4,P27,Bax,Bcl-2 m RNA expression level;western blot method to detect the expression levels of cell cycle and apoptosis key proteins C-MYC,Cyclin D1,CDK4,P27,Bax,and Bcl-2.Statistical analysis was performed using IBM SPSS 24.0,one way ANOVA was used to compare the differences between the MEHP exposed group and the control group,LSD test was used to compare the differences between the two groups,and ranksum test was used for non-normal data.The test level was α =0.05.Results:1.After 12h and 24h MEHP exposure,the survival rate of SH-SY5Y cells in each dose group was significantlyhigher than that in the control group(P < 0.05); with the increase of exposure time,the survival rate of 32,64,128,250,500 and 1000 μM MEHP groups was significantly increased(P < 0.05).2.After 12h and 24h of MEHP exposure,the number of cells in G1 phase in MEHP exposure group was significantly decreased(P < 0.05),while the percentage of cells in S phase in MEHP group was increased(P < 0.05).3.The apoptosis level of SH-SY5Y cells in MEHP exposed group was significantly decreased after 12h and 24h exposure(P < 0.05);The apoptosis level of 250 μM MEHP exposed group at 24h was lower than that in 12h group(P < 0.05).4.After 12h and 24h of MEHP exposure,the expression levels of C-MYC and Cyclin D1 m RNA and protein in the medium andhigh dose groups increased(P<0.05);the m RNA and protein expression levels of CDK4 and P27 did not change significantly(P> 0.05).5.After 12h and 24h of MEHP exposure,the expression levels of Bax m RNA and protein in SH-SY5Y cells in the medium andhigh dose groups decreased(P<0.05);the expression levels of Bcl-2 m RNA and protein increased(P<0.05).Conclusion:1.MEHP exposure can regulate SH-SY5Y cell cycle,inhibit cell apoptosis,and promote SH-SY5Y cell proliferation.2.MEHP exposure can up-regulate the expression of key cell cycle proteins CMYC and Cyclin D1,accelerate the cell cycle,and promote the proliferation of SHSY5 Y cells.3.MEHP exposure can regulate the expression of apoptosis-related genes Bcl-2and Bax,inhibit SH-SY5Y cell apoptosis,and promote neuroblastoma proliferation.Part Ⅱ The effect of MEHP on the migration of SH-SY5Y cells and its mechanismObjective:Clarify the effect of MEHP on the migration of SH-SY5Y cells,and explore the role of key genes and chemokines for cell migration.Methods:The experiment grouping and exposure are the same as the first part.ELISA were used to detect the secretion levels of chemokines VEGF,PDGF,TGF-β1 and CXCL-8;scratch test to detect cell migration level;transwell invasion test was used to detect cell invasion ability;real-time PCR method was used to detect cell migration key gene ECadherin,N-Cadherin,SNAIL,TWIST,Vimentin,HIF-1α,GPR81 and Versican m RNA expression levels;western blot method was used to detect the expression levels of key proteins in cell migration.The data processing and statistical analysis methods are the same as the first part.Results:1.MEHP exposure can increase the migration distance of SH-SY5Y cells(P<0.05).After 24h of MEHP exposure,the migration distance of SH-SY5Y cells increased significantly(P<0.05).2.MEHP exposure can increase the invasion ability of SH-SY5Y cells.The invasion ability of medium andhigh dose MEHP exposure groups was significantly increased(P<0.05).3.MEHP exposure can increase the secretion levels of VEGF,PDGF and TGF-β1in SH-SY5Y cells(P<0.05).The levels of VEGF,PDGF and TGF-β1 in SH-SY5Y cells in the MEHP exposure group for 24h were significantlyhigher than 12h Group(P<0.05).4.After 12h and 24h of MEHP exposure,the expression levels of E-Cadherin gene m RNA and protein in SH-SY5Y cells in the medium andhigh dose groups were significantly lower than those in the control group and DMSO group(P<0.05);in thehigh dose group N-Cadherin m RNA and protein expression levels were significantlyhigher than the other groups(P<0.05);compared with the 12h exposure group,NCadherin m RNA expression levels increased in the 24hhigh dose group SH-SY5Y cells(P<0.05)The expression levels of Vimentin m RNA and protein in the medium dose group were significantly increased(P<0.05),and the expression levels of Vimentin m RNA and protein in thehigh dose group were significantlyhigher than those of the control group,DMSO group and low dose group(P<0.05);and in comparison of the 12h exposure group,the expression level of Vimentin m RNA in each dose group at 24h increased(P<0.05);the expression of SNAIL gene m RNA and protein increased in the medium andhigh dose groups(P<0.05);the expression levels of HIF-1α m RNA and protein in the medium andhigh dose MEHP exposure groups were significantly increased(P<0.05).Compared with the 12h exposure group,the expression level of HIF-1α m RNA in the 24hhigh dose group washigher(P<0.05);the expression levels of Versican m RNA and protein in the medium andhigh dose MEHP exposure groups were significantlyhigher than those in the control group and the DMSO group(P<0.05)in the 24h group;compared with the 12h exposure group,the expression level of Versican m RNA in 24hhigh dose SH-SY5Y cells increased(P<0.05).Conclusion:1.MEHP exposure can promote the migration and invasion of SH-SY5Y cells.2.MEHP exposure can regulate the expression of key migration genes E-Cadherin,N-Cadherin,SNAIL,Vimentin,HIF-1α and Versican,and promote the migration and invasion of SH-SY5Y cells.3.MEHP exposure may enhance the migration ability of SH-SY5Y cells by upregulating the secretion of chemokines VEGF,PDGF and TGF-β1.Part Ⅲ The effect of MEHP on Notch signaling pathway in SH-SY5Y cells Objective:To clarify the influence of MEHP on the Notch signaling pathway of SH-SY5Y cells,and to explore the relationship between the protein expression level of Notch signaling pathway and the proliferation and migration of SH-SY5Y cells and the expression of related genes.Methods:The experiment grouping and exposure are the same as the first part.Real-time PCR method was used to detect the m RNA expression level of Notch signaling pathway genes in cells;western blot method was used to detect the protein expression level of Notch signaling pathway genes in cells.The correlation test of Notch signal pathway protein expression with SH-SY5Y cell proliferation and migration and related proteins was tested by Pearson correlation analysis method.The rest of the data processing and analysis methods are the same as the first part.Results:1.MEHP exposure can increase the expression levels of Notch-1,Notch-2,Notch-3,Notch-4,Dll-1 and Jagged-2 m RNA in SH-SY5Y cells in the medium andhigh dose groups,and decrease the expression level of Dll-4 m RNA(P<0.05).2.MEHP exposure can increase the expression levels of Notch-1,Notch-2,Notch-3 and Jagged-2 proteins in SH-SY5Y cells in the medium andhigh dose groups,and decrease the expression levels of Dll-4 protein(P<0.05).3.After 12h and 24h MEHP exposure,the cell cycle and apoptosis levels of SHSY5 Y were significantly correlated with the protein expression levels of Notch-1,Jagged-2 and Dll-4(P < 0.05).4.After MEHP exposure for 12h and 24h,the expression level of cell cycle key protein CDK4 was significantly correlated with Dll-1 protein expression levels(P<0.05);Cyclin D1 and C-MYC protein expression level was significantly correlated with Notch-1,Notch-2,Notch-4,Jagged-2,Dll-1 and Dll-4 proteins expression levels(P<0.05).After MEHP exposure for 12h and 24h,the expression level of apoptosisrelated protein Bax was significantly correlated with the protein expression levels of Notch-1,Notch-4 and Dll-4,and the expression level of Bcl-2 was significantly correlated with Dll-4(P<0.05).5.After 12h and 24h of MEHP exposure,the migration and invasion ability of SH-SY5Y cells was significantly correlated with the protein expression levels of Notch-1,Jagged-2,Dll-1 and Dll-4(P<0.05).6.After 12h and 24h of MEHP exposure,the expression level of cell migration related protein E-Cadherin was significantly correlated with the expression levels of Notch-1 and Jagged-2(P<0.05);the expression level of N-Cadherin protein was significantly correlated with Notch-1,Jagged-2(P<0.05);the protein expression levels of SNAIL were significantly correlated with Notch-4,Jagged-2 and Dll-4(P<0.05);the protein expression levels of Vimentin and HIF-1α were significantly correlated with Notch-1,Jagged-2 and Dll-4(P<0.05);Versican protein expression levels were significantly correlated with Notch-1,Notch-4 and Jagged-2(P<0.05).7.After MEHP exposure for 12h and 24h,the expression level of chemokine VEGF was significantly correlated with the protein expression levels of Notch-2 and Dll-4(P<0.05);the expression level of PDGF was correlated with Notch-1,Notch-4,Dll-1 and Jagged-2(P<0.05);TGF-β1 expression levels were significantly correlated with Notch-3 and Dll-4(P<0.05);CXCL-8 expression levels were significantly correlated with the expression level of Jagged-2 protein(P<0.05).Conclusion:1.MEHP exposure can up-regulate the protein expression levels of Notch-1,Notch-2,Notch-3,and Jagged-2 in the Notch signaling pathway,and down-regulate the protein expression levels of Dll-4.2.The proliferation and migration of SH-SY5Y cells induced by MEHP exposure is closely related to the expression of Notch pathway proteins.3.The Notch signaling pathway may play a role in MEHP promoting the proliferation and migration of SH-SY5Y cells by regulating the expression of key genes for cell proliferation and migration.4.The Notch signaling pathway may play a role in MEHP promoting the proliferation and migration of SH-SY5Y cells by regulating the secretion of chemokines.Part Ⅳ The role and mechanism of Notch signaling pathway in MEHPaffecting the proliferation and migration of SH-SY5Y cells Objective:To identify the role of Notch signaling pathway in MEHP promoting SH-SY5Y cell proliferation and migration and relative mechanism.Methods:The exposure dose and grouping were as follows: blank control group,solvent control group(0.1% DMSO),DAPT group(50μM DAPT),MEHP group(250μM MEHP)and MEHP+DAPT group(50μM DAPT+250μM MEHP).Each group was exposed for 24h.The Notch signaling pathway was inhibited by DAPT,and the expression level of Hes-1 was detected by Western blot to investigate the inhibitory efficiency of Notch signaling pathway.After inhibiting Notch signaling pathway,western blot was used to detect the expression levels of cell proliferation and migration related proteins.The other experimental methods and statistical analysis methods were as the same as the first part.Results:1.After DAPT exposure of SH-SY5Y cells for 24hours,the cell survival rate of each dose group did not change significantly(P>0.05);The Hes-1 protein expression level in SH-SY5Y cells treated with 50 μM DAPT was significantly reduced(P<0.05),and the Notch signaling pathway was effectively inhibited.2.The cell cycle results showed that compared with the control group and the DMSO group,the DAPT group and DAPT+MEHP cell cycle did not change significantly(P>0.05);the number of cells in the S phase of the MEHP group was significantlyhigher than that of the control group,DMSO group and DAPT group(P<0.05).3.Apoptosis results showed that MEHP group and DAPT+MEHP group significantly decreased the apoptosis level(P<0.05).4.The results of cell migration showed that the cell migration distance of MEHP group and DAPT+MEHP group increased significantly(P<0.05);compared with MEHP group,the cell migration distance of the DAPT+MEHP group was reduced,and the difference was not statistically significant(P>0.05).5.The cell invasion results showed that the MEHP group and DAPT+MEHP group cell invasion level increased significantly(P<0.05);compared with the MEHP group,the level of cell invasion in the DAPT+MEHP group was reduced,and the difference was not statistically significant(P>0.05).6.The chemokine results showed that the levels of VEGF,PDGF,TGF-β1 and CXCL-8 increased significantly in the MEHP group and the DAPT+MEHP group(P<0.05);the levels of VEGF and TGF-β1 in DAPT+MEHP group were significantly lower than those in the MEHP group(P<0.05).7.The cell cycle and apoptosis-related protein results showed that the expression levels of C-MYC and Bcl-2 protein in the MEHP group and DAPT+MEHP group were significantlyhigher than those in the control group,DMSO group and DAPT group(P<0.05);the expression levels of Bax protein in the MEHP group and DAPT+MEHP group were significantly lower than those in the control group,DMSO group and DAPT group(P<0.05);The expression levels of C-MYC,Cyclin D1,Bcl-2 protein in DAPT+MEHP group were significantly lower than those in MEHP group(P<0.05).8.The results of cell migration related proteins showed that the expression levels of Vimentin,SNAIL,HIF-1α,and Versican in the MEHP group and DAPT+MEHP cells were significantlyhigher than those in the control,DMSO and DAPT groups(P<0.05);the expression levels of E-Cadherin protein in the MEHP group and DAPT+MEHP groups were significantly lower in the control group,DMSO group and DAPT group(P<0.05);the expression levels of HIF-1α and N-Cadherin protein in the DAPT+MEHP group were significantly lower than those in the MEHP group(P<0.05).Conclusion:1.The Notch signaling pathway plays a positive regulatory role in MEHP promoting the proliferation and migration of SH-SY5Y cells.2.The Notch signaling pathway may promote the expression of anti-apoptotic protein Bcl-2 by regulating the key proteins of the cell cycle Cyclin D1 and C-MYC play a role in MEHP affecting the proliferation of SH-SY5Y cells.3.The Notch signaling pathway may promote the expression of key proteins NCadherin for cell migration,and play a role in MEHP’s influence on the migration of SH-SY5Y cells.4.Notch signaling pathway may affect the secretion of tumor cell chemokines VEGF and TGF-β1 and play a role in MEHP affecting SH-SY5Y cell migration.