Role And Mechanism Of ERα In Proliferation And Migration Of SH-SY5Y Cell Induced By MEHP

Objective:To clarify the effect of MEHP exposure on the proliferation and migration of SHSY5Y neuroblastoma cells,explore the role of ERα in proliferation and migration of SH-SY5Y cells induced by MEHP,and provide scientific basis for elucidating the mechanism of MEHP exposure promoting tumor cell proliferation and metastasis,provide new ideas for finding the prevention and treatment targets of neuroblastoma.Methods:SH-SY5Y cells were routinely cultured in DMEM medium containing 10% fetal bovine serum.SH-SY5Y cells were exposed to MEHP with gradient concentration,CCK-8 method was used to detect the survival rate of SH-SY5Y cells,and the exposure dose was determined according to the results of CCK-8.The experiment was divided into blank control group(0 μM MEHP),solvent control group(0.1% DMSO),low-dose group(10 μM MEHP),medium-dose group(50 μM MEHP),high-dose group(250 μM MEHP)and estradiol control group(100 n M E2),cells and their supernatant were collected after 12 h and 24 h exposure.SH-SY5Y cells were transfected with ERαsi RNA,and the silencing efficiency of ERα was verified by Q-PCR.Set up blank control group(0 μM MEHP),solvent control group(0.1% DMSO),silent control group(Transfect + si RNA(si NC)),silent exposure group(si RNA + 250 μM MEHP),exposure group(250μM MEHP)and estradiol control group(100 n M E2),the cells and their supernatant were collected 24 h later.Flow cytometry was used to detect cell cycle;scratch test and Transwell test were used to detect cell migration;Q-PCR and Western Blot methods were used to detect mRNA and protein expression levels of ERα,cell proliferation and migration-related genes.Statistical analysis was performed using IBM SPSS 24.0,one-way analysis of variance was used to compare the differences of indicators between different exposure dose groups,and the LSD method was used for pairwise comparisons between groups;the t-test was used to compare the differences of indicators in different time periods.The difference is statistically significant when P<0.05.Results:1.After 12 h of MEHP exposure,the survival rate of SH-SY5Y cells in each MEHP exposure group was significantly higher than that of the control group(P<0.05);after 24 h of MEHP exposure,the cell survival rate of 125～1000 μM MEHP in each exposure group was significantly higher than that of the control group(P<0.05);and the 24 h cell survival rate of each exposure group of 8～1000 μM was significantly lower than that of the 12 h group(P<0.05).2.After 12 h and 24 h of MEHP exposure,with the increase of MEHP exposure time,percentage of cells in S phase and G2 phase cells in the medium dose group increased significantly(P<0.05),the percentage of cells in G1 phase decreased(P<0.05).3.The migration distance and migration number of SH-SY5Y cells in the highdose group were significantly higher than those in the medium-dose and low-dose groups after MEHP exposure for 12 h and 24 h(P<0.05);the relative migration number of cells in the middle-dose group was significantly higher than that of the control group(P<0.05);the cell migration distance and number of cells in the medium-dose and lowdose groups were significantly smaller than those in the E2 group(P<0.05),after 24 h of MEHP exposure,the cell migration distance of the low-dose,high-dose and E2 groups was significantly higher than that of MEHP for 12 h(P<0.05).4.After MEHP was exposed for 12 h,the ERα mRNA expression level of SHSY5Y cells in the high-dose group was significantly higher than that of the other groups(P<0.05),and the ERα mRNA expression level increased with the increase of MEHP concentration(P<0.05),the expression level of ERα protein in each exposure group and E2 group was significantly higher than that of the control group(P<0.05),and the expression level of ERα increased with the increase of MEHP concentration(P<0.05);MEHP exposure 24 h,the expression levels of ERα mRNA and protein in each MEHP exposure group and E2 group was significantly higher than those in the control group(P<0.05),and the expression levels increased with the increase of MEHP concentration(P<0.05).After 24 h of MEHP exposure,the expression levels of ERα mRNA in the medium and low-dose groups and E2 groups were significantly higher than the expression levels of 12 h after MEHP exposure(P<0.05).5.After 12 h of MEHP exposure,the PCNA mRNA expression level of SH-SY5Y cells in the high-dose group was significantly higher than that of the other groups(P<0.05),and its expression level increased with the increase of MEHP concentration(P<0.05);the expression level of MMP-2 mRNA in each exposure group was significantly lower than that of the control group(P<0.05),but its expression level increased with the increase of MEHP concentration(P<0.05);the expression level of MMP-9 mRNA in high-dose group and E2 group was significantly higher than that of the medium-dose,low-dose and control groups(P<0.05);the expression levels of TIMP-2 mRNA in the low-dose and medium-dose groups were significantly higher than that of the control group(P<0.05),the expression levels in the high-dose group and E2 group were significantly lower than those of the control group(P<0.05).After 12 h of MEHP exposure,the PCNA protein expression levels in the mediumdose,high-dose and E2 groups were significantly higher than those in the low-dose and control groups(P<0.05);the MMP-2 protein expression levels in each exposure group and E2 group were significantly higher than those in the control group(P<0.05),and its expression level increased with the increase of MEHP concentration(P<0.05);the expression level of MMP-9 protein in the high-dose group and E2 group was significantly higher than that of the low-dose group and the control group(P<0.05);the expression levels of TIMP-2 protein in the low-dose and medium-dose groups were significantly higher than that of the control group(P<0.05),the protein expression levels of the high-dose group and E2 group were significantly lower than those of the control group(P<0.05),and the expression levels of TIMP-2 mRNA and protein decreased with the increase of MEHP concentration(P<0.05).With the increase of exposure time,after 24 h of MEHP exposure,PCNA and TIMP-2 mRNA expression levels in the low-dose group,MMP-2,MMP-9 and TIMP-2mRNA expression levels in the middle-dose group,and TIMP-2 mRNA and protein expression levels in the high-dose group significantly increased(P<0.05).6.After 24 h of MEHP exposure,the expression level of PCNA mRNA in SHSY5Y cells of each MEHP exposure group and E2 group was significantly higher than that of the control group(P<0.05),and its expression level increased with the increase of MEHP concentration(P<0.05);the expression level of MMP-2 mRNA in medium dose,high dose and the E2 groups were significantly higher than that of the low-dose group(P<0.05);the expression level MMP-9 mRNA in high dose and the E2 groups were significantly higher than that of the other groups(P<0.05);the expression level of TIMP-2 mRNA in each MEHP exposure group was significantly higher than those in the control group(P<0.05).After 24 h of MEHP exposure,the expression levels of PCNA and MMP-9proteins in SH-SY5Y cells in the high-dose and E2 groups were significantly higher than those in the low-dose,medium-dose and control groups(P<0.05);the expression level of MMP-2 protein in the medium-dose,high-dose and E2 groups was significantly higher than that of the low-dose and the control groups(P<0.05);the expression level of TIMP-2 protein in the low-dose and the middle-dose groups were significantly higher than that of the control group(P<0.05),and its expression level in the high dose and E2 groups were significantly lower than that of the control group(P<0.05),and the expression levels of TIMP-2 mRNA and protein decreased with the increase of MEHP concentration(P<0.05).7.After ERα gene silencing,compared with the control group,SH-SY5Y cells exposed to 250 μM MEHP for 24 h,the percentage of cells in the G1 and G2 phases was significantly reduced(P<0.05),and the percentage of cells in the S phase was significantly increased(P<0.05);compared with normal cells exposed to MEHP for 24 h,the percentage of cells in G2 phase was significantly reduced(P<0.05);after ERαgene silencing,the percentage of cells in G1 and G2 phases in SH-SY5Y cells was significantly increased(P<0.05),the percentage of cells in S phase was significantly reduced(P<0.05).8.After ERα gene silencing,the relative migration distance and migration number of SH-SY5Y cells were significantly reduced,and the migration ability was significantly reduced(P<0.05);compared with normal cells exposed to MEHP for 24 h,the relative migration distance of cells in the ERα gene silenced group was significantly shortened,the migration number was reduced,and the migration capacity was significantly reduced(P<0.05).9.The expression levels of the genes PCNA,MMP-2 and MMP-9 mRNA in SHSY5Y cells silenced by the ERα gene were significantly reduced(P<0.05),and the expression level of TIMP-2 mRNA was significantly increased(P<0.05);after ERαgene silencing,the cells were treated with MEHP for 24 h,the expression levels of the PCNA and MMP-9 mRNA in cells were significantly lower than before the ERα gene silencing(P<0.05),and the expression level of the TIMP-2 mRNA was significantly increased(P<0.05);when SH-SY5Y cells with ERα gene silenced,the cells were treated with MEHP for 24 h,the expression levels of PCNA and MMP-2 protein were significantly reduced(P<0.05),and the expression level of protein TIMP-2 was significantly increased(P<0.05);after ERα gene silencing,the cells were treated with MEHP for 24 h,the expression levels of PCNA and MMP-9 protein in the cells were significantly lower than before ERα gene silencing(P<0.05).Conclusion:1.MEHP exposure can promote the proliferation of SH-SY5Y cells.2.MEHP exposure can up-regulate the expression levels of PCNA mRNA and protein of SH-SY5Y cell proliferation-related genes;ERα silencing can cause cell cycle arrest and reduce the expression of PCNA protein.It shows that ERα plays a regulatory role in the proliferation of SH-SY5Y cells induced by MEHP exposure.3.MEHP exposure can promote SH-SY5Y cell migration,ERα silencing can shorten the migration distance and reduce migration number of cells,and weaken the migration ability of cells,indicating that ERα plays a regulatory role in SH-SY5Y cells induced by MEHP exposure.4.MEHP exposure can promote the invasion of SH-SY5Y cells,and ERα silencing can reduce the number of cell invasions and reduce the invasiveness of cells,indicating that plays a regulatory role in the invasion of SH-SY5Y cells induced by MEHP exposure.MEHP exposure may promote SH-SY5Y cell migration by up-regulating the expression levels of MMP-2 and MMP-9,and down-regulating the expression level of TIMP-2;ERα silencing can inhibit the expression level of MMP-2 and MMP-9 while increasing the expression level of TIMP-2;it shows that ERα plays a regulatory role in the metastasis of SH-SY5Y cells induced by MEHP exposure.5.MEHP can activate ERα through estrogen-like effects,and promote the proliferation and metastasis of SH-SY5Y cells.