The Influence Of ERα/βover Expression To The Expression Plexin-B1 In SKOV-3 Cells

Objective:To study the effect of ERa/β(estrogen receptora/β) over expression in ovarian serous carcinoma SKOV-3 cells, we detected multiple receptors of estrogen-bonded in regulation of Plexin-B1.Methods:1, In order to build the ERa/β over expression SKOV-3 cell strain, we transfected the over expression ERaand ERβvectors to SKOV-3 cell strain via lentiviral transfection.2, The SKOV-3 cells had been infected by lentiviral for 24 hours. RT-PCR and Western Blotting were used to detect the expression of ERaand ERβin SKOV-3 cells in over expression ERagroup and over expression ERβgroup.3, The MTT (Methyl Thiazolyl Tetrazolium) assay was used to detect the SKOV-3 cell viability in control group, over expression ERagroup and over expression ERPgroup which had been treated through 17β-estradiol (10-6mol/L) and estrogen receptor antagonist ICI180,782 (100 nM) individually or jointly for 24 hours.4, After infecting under lentiviral for 24h, the Western Blotting and RT-PCR were used to detect the Plexin-B1 expression of protein and mRNA in control group, ERagroup and ERPgroup.5, After the stimulation of 17β-estradiol(10-6mol/L)onto each group for 24h. The Plexin-B1 expression in protein and mRNA in control group, over expression ERagroup and over expression ERβgroup were detected via Western Blotting and RT-PCR. And the cell migration ability was tested by Transwell chamber assay.6, After pretreating each group via estrogen receptor antagonist ICI 180,782 (100 nM)for 6h, the 17β-estradiol(10-6mol/L)was supplied to each group for 24h. Western Blotting and RT-PCR were used to detect the Plexin-B1 expression of protein and mRNA in the three groups. And the cell migration ability was tested by Transwell chamber assay.Results:1, The efficiency of infection was observed through fluorescence microscope after 24h infection of lentiviral.70% of the vision was occupied by green fluorescence which proved the infection is successful, and the cell survival rate is beyond 95%.2, The cells were cultured for 24h after passaging the transfected SKOV-3 cells. Western Blotting and RT-PCR results showed that the transfected ERa and ERβcan stay on the high stable expression level in SKOV-3 cells.3, After 24h stimulation of 17β-estradiol(10-6mol/L)in control group, ERa group and ERβgroup, we detected the Plexin-B1 expression of protein and mRNA in each group. We found (1) The Plexin-B1 expression in protein and mRNA and mRNA in over expression ERa group was significantly higher than control(P<0.05);(2) The cell migration capacity in over expression ERa group was significantly higher than control(P<0.05); (3) The Plexin-B1 expression of protein and mRNA in over expression ERβgroup was significantly lower than control(P<0.05); The cell migration capacity in over expression ERβ group was significantly lower than control(P<0.05);4, After 6h pretreatment of estrogen receptor antagonist ICI 180,782 (100nM), the 17β-estradiol (10-6mol/L) was supplied to each group for 24h. Western Blotting and RT-PCR were used to detection. (1) The Plexin-B1 expression of protein and mRNA in the three groups didn’t show statistically significant difference (P>0.05); (2) The cell migration capacity of SKOV-3 didn’t show statistically significant difference either (P>0.05)Result:1, We have built the high expression ERaand ERβovarian carcinoma cell strain through lentiviral induced ERaand ERβvector over expression to infect SKOV-3 cells. It has laid the fundament for the relevant future studies.2, After infecting SKOV-3 cells via lentiviral induced ERa and ERβvector over expression, we cultured and passaged the cells. The ERaand ERβcan keep stabe expression of transfected SKOV-3 cells.3, The over expression ERaand estrogen in cells can promote the Plexin-B1 expression in protein and mRNA as well as increase the cell migration.4, The over expression ERβ and estrogen in cells can inhibit the Plexin-B1 Expression in protein and mRNA, and decrease the cell migration either.5, Estrogen receptor antagonist can inhibit the regulation of estrogen in Plexin-B1. It proves that estrogen regulation difference for Plexin-B1 is induced by different estrogen receptors.