Estrogen Combine With Different Receptors Can Differentially Regulate The Expression Of Sema4D In Cell Strain SKOV-3

[Objective] To explore in the human ovarian serous papillary adenocarcinoma cell line SKOV-3, estrogen can differential regulate the expression of Sema4D by binding different estrogen receptor.[Methods] 1.construction of ER a and ER β high expression plasmid, cultured cell line SKOV-3. The cultured cells were divided into three groups, a, b and c. A group will infected ER a over expression vector by lentiviral into SKOV-3 cells; group B will infected ER β over expression vector by lentiviral infection into SKOV-3 cells; SKOV-3 cells in group C were untreated as a control group.2. Lenticiral infection in SKOV-3 cells for 24 hours, using RT-PCR and Western blot detection, a, B, C three groups of cells infected with ER a and estrogen receptor β mRNA and protein expression levels.3. lentivirus infected SKOV-3 cells after 24 hours, Western Blot to detected the expression level of Sema4D protein in three cells Sema4D protein.4.10-6 mol/L of 17 P estradiol stimulated cells in each group at 24h after, RT-PCR and Western blot detection a, B, C three groups of cells within the Sema4D mRNA and protein expression levels.5.to add 100 nm estradiol receptor antagonist(ici182780)agent treatment of cells in each group 6 hours after adding 10-6 mol/L of 17 β estradiol treatment for 12 hours, RT-PCR and Western blot, three groups cells were detected in Sema4D mRNA and protein expression levels.[Results]:1.lentivirus infection in SKOV-3 cells 24 hours after, RT-PCR was used to detect group a cellular ER α mRNA 2-△△Ct is 100.99+5.80, higher than group c 9.75 +1.01 increased significantly (P< 0.01), and group b was 8.47+0.74, compared with C group has no significant difference (P> 0.05). Group ER p mRNA 2-△△Ct is 58.45+ 2.95,had no significant difference with c group 59.64+4.20 (P>0.05),b group is 292.05+12.27, significantly higher than that in c group (P>0.01).2.Western blot detect a group of cellular ER α/β-actin is 100.99+5.80, higher than group C 975+1.01 increased significantly (P< 0.01), and group B was 8.47+0.74, compared with C group has no significant difference (P> 0.05). A group of ER β/ β-actin as 58.45+2.95 compared with C group 59.64+4.20 has no significant difference (P> 0.05), group B was 292.05+12.27. Compared with c group significantly increased (P< 0.01).3. Western blot was used to detect lentivirus infected cells for 24h, the expression of Sema4D/β-actin in each cell is 0.22+0.02,0.05+0.28,0.26+0.04, the difference was not statistically significant (P>0.05).4. After 10-6 mol/L of 17 β estradiol stimulated three groups of cells 24 hours, RT-PCR detect Sema4D mRNA2-△△Ct value of a group is 2.128+0.745,compared with c group 13.556+/-2.181 was significantly increased (P<0.01), group b was 0.000548+0.00009,compared with c group significantly decreased (P<0.01).5. After 10-6 mol/L of 17 β estradiol stimulated C three groups of cells 24 hours, a group of cells within the Sema4D/β-actin is 0.530+0.085, compared with c group 0.263+0.042 increased significantly (P<0.01) and b group Sema4D/β-actin as 0.140+0.046, compared with c group decreased significantly (P<0.01).6. After ICI180,782treat each group 6h, RT-PCR detect a, b, c three groups of intracellular Sema4D mRNA2-△△Ct value were 11.556+2.181,9.548+0.009,10.210 +0.632 comparison of three groups has no significant difference (P>0.05).7. Western blot was used to detect ICI180,782,treat each cell 6h,a, b, c groups of Sema4D/β-actin values were 0.303+0.083,0.257+0.061,0.298+0.042. Comparison of three groups no significant difference (P>0.05)[Conclusion]:1. We successfully established the high expression of ER a and ERβ in ovarian cancer cell lines, laid the foundation for the follow-up study.2.Non estrogen, lentivirus infection SKOV-3 can’t influence the Sema4D expression.3.High expression of ERa cells, estrogen can promote the expression of Sema4D mRNA and protein in cells.4.High expression of ER β cells, the expression of estrogen can inhibit Sema4D mRNA and protein in cells.5.Estrogen receptor antagonists can inhibit estrogen regulation of Sema4D.That means estrogen differentially regulated the expression of Sema4D was mediated by different estrogen receptor.6.The above conclusion confirmed that estrogen can different regulate the expression of Sema4D by combine different receptors. May provide a new theoretical basis for the pathogenesis of ovarian cancer and may also for ovarian cancer target treatment to provide new ideas.