| Objective:To study the effects of estradiol on the proliferation,migration and invasion in HEC-1A endometrial carcinoma cells and to study the activation effect of estradiol on sphingosine kinase as well as the mechanism for promoting the proliferation,migration,and invasion of HEC-1A cells by GPER/SphK/1/2 pathway activation.Methods:1.The effects of estradiol on the proliferation,migration and invasion in HEC-1A cells(1)The effect of E2 on the proliferation in HEC-1A cellsThe MTT assay was used to detect the effect of different concentrations of E2 on the proliferation in HEC-1A endometrial carcinoma cells at different time.Experimental groups:(1)control group;(2)1×10~-99 M E2 group;(3)1×10~-88 M E2group;(4)1×10~-77 M E2 group;(5)1×10~-66 M E2 group;(6)1×10~-55 M E2 group.The cell proliferation was detected after 24 hours,48 hours and 72 hours of E2 treatment.(2)The effect of E2 on the migration in HEC-1A cellsThe effect of 1×10~-77 M E2 on cell migration was detected by wound healing assay at different time.Experimental groups:(1)control group;(2)E2 group.The cellmigration was detected after 24 hours,48 hours and 72 hours of E2 treatment.(3)The effect of E2 on the invasion in HEC-1A cellsThe cell invasion was detected by transwell assay after 48 hours of E2 treatment.Experimental groups:(1)control group;(2)E2 group.2.GPER/SphK mediated the non-transcriptional effect of estradiol on activation of the ERK1/2 pathway(1)The expression of SphK1/2 in HEC-1A cellsThe protein expression of SphK1 and SphK2 were detected by western blot after24 hours,48 hours and 72 hours of 1×10~-77 M E2 treatment.(2)The activity change of SphKUsing sphingosine kinase Activity Assay Kit to determine the effect of1×10~-77 M E2 on the SphK activities at different time points(0,5,15,30,60min).And the cells were pretreated with GPER inhibitor G-15(5?M)for 30 minutes,then treated with E2 for 15 minutes.The SphK activities were detected.Experimental groups:(1)control group;(2)E2 group;(3)G-15 group;(4)G-15+E2 group.(3)The phosphorylation change of ERK1/2Western blot was used to detected the effect of 1×10~-77 M E2 on the proteinphosphorylation of ERK1/2 at different time points(0,5,15,30,60,120 min).Then we pretreated cells with GPER inhibitor G-15(5?M),SphK inhibitor DMS(10?M)and ERK1/2 inhibitor PD98059(20?M)and observed their effects on E2-induced phosphorylation of ERK1/2.Experimental groups:(1)control group;(2)E2 group;(3)G-15 group;(4)G-15+E2 group;(5)PD98059+E2.(4)The protein expression of Cyclin D1,Cyclin E1 and MMP-9Using western blot to detect the protein expression of Cyclin D1,Cyclin E1 and MMP-9 after cells were treated with E2,GPER inhibitor G-15,SphK inhibitor DMS and ERK1/2 inhibitor PD98059 for 24 hours.Experimental groups:(1)control group;(2)E2 group;(3)G-15 group;(4)G-15+E2 group;(5)PD98059+E2.(5)Cell proliferation,migration and invasionThe MTT assay was used to detect the effect on cell proliferation after cells were treated with E2,GPER inhibitor G-15,SphK inhibitor DMS and ERK1/2 inhibitorPD98059 for 24 hours.The cell migration and invasion were detected by the wound healing and transwell assay respectively after cells were treated with E2,GPERinhibitor G-15,SphK inhibitor DMS and ERK1/2 inhibitor PD98059 for 48 hours.Experimental groups:(1)control group;(2)E2 group;(3)G-15 group;(4)G-15+E2group;(5)PD98059+E2.Results:1.The effects of estradiol on the proliferation,migration and invasion in HEC-1A cells(1)The effect of E2 on the proliferation in HEC-1A cellsThe proliferation ability of HEC-1A cells in the E2 treatment group wassignificantly increased compared with the Ctrl group,and we selected 1×10~-77 M E2for the follow-up experiments.(2)The effect of E2 on the migration in HEC-1A cellsCompared with the control group,the migration ability of HEC-1A cells in the E2 treatment group was significantly increased.(3)The effect of E2 on the invasion in HEC-1A cellsE2 could promote HEC-1A cells invasion.2.GPER/SphK mediated the non-transcriptional effect of estradiol on activation of the ERK1/2 pathway(1)The expression of SphK1/2 in HEC-1A cellsThe protein expression of SphK1 and SphK2 in HEC-1A cells were no significant difference after E2 treatment.(2)The activity change of SphKE2 could stimulate cells to cause a rapid and short-time SphK activity increase,and the SphK activity reached the peak at the point of 15 min.Then cells were treated with GPER inhibitor G-15 and the SphK activities were determined.The SphKactivity in the E2 group was significantly increased,and G-15 group significantly reduced compared with the Ctrl group.There was a remarkably decrease in theG-15+E2 group compared with the E2 group.It indicates that the E2 SphK activation could be blocked by G-15 and E2 rapidly activates SphK through thenon-transcriptional pathway of GPER.(3)The phosphorylation change of ERK1/2The protein expression of p-ERK1/2 significantly increased at the time of 5 min and 15 min,in which the expression reached the peak at the point of 5 min.Itsuggests that E2 could rapidly activate the ERK1/2 signal pathway in a short time.Then cells were treated with G-15,DMS and PD98059 and the ERK1/2 activities were determined.Compared with the Ctrl group,the protein expression of p-ERK1/2had a significant increase in the E2 group and compared with the E2 group,theprotein expression had a significant decrease in inhibitors groups.These resultssuggest that the E2 ERK1/2 activation could be blocked by G-15,DMS and PD98059and E2 rapidly activates ERK1/2 signal molecules through the GPER/SphK pathway.(4)The protein expression of Cyclin D1,Cyclin E1 and MMP-9Compared with the Ctrl group,the protein expression of Cyclin D1,Cyclin E1and MMP-9 had a significant increase in the E2 group and compared with the E2group,the protein expression of three proteins had a significant decrease in all inhibitors groups.(5)Cell proliferation,migration and invasionThe proliferation,migration and invasion ability of cells were significantly increased when treated with E2 as well as all inhibitors could suppress cellproliferation,migration and invasion.Conclusion:E2 could promote the proliferation,migration and invasion in HEC-1A endometrial carcinoma cells.The GPER/SphK pathway is involved in theproliferation,migration and invasion of estradiol-mediated in HEC-1A endometrial carcinoma cells,which is related to the activation of the ERK1/2 signal pathway and the protein expression of Cyclin D1,Cyclin E1,MMP-9. |
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