Estrogen Combine With Different Receptors Can Differentially Regulate The Expression Of MiR-214 In Cell Strain A2780

Objectives:To explore in the human ovarian serous papillary adenocarcinoma cell line A2780, estrogen can differential regulate the expression of miR-214 by binding different estrogen receptor.Methods:1.construction of ER a and ER (3 high expression plasmid, cultured cell line A2780. The cultured cells were divided into three groups, a, b and c. A group will infected ERa over expression vector by lentiviral into A2780 cells; group B will infected ERβ over expression vector by lentiviral infection into A2780 cells; The control group c were untreated.2. Lenticiral infection in A2780 cells for 24 hours, using RT-PCR and Western blot to detect, a, b, c three groups of cells infected with ER a and ER β mRNA and protein expression levels.3. lentivirus infected A2780 cells after 24 hours, using RT-PCR to detect the expression level of miR-214 in three groups.4.10-6 mol/L of 17-β estradiol stimulated cells in each group at 24h after, using RT-PCR to detect the expression level of miR-214 in three groups.5.adding 100 nM estradiol receptor antagonist(ici182、780)agent treatment of cells in each group 6 hours.then adding 10-6 mol/L of 17-β estradiol treatment for 12h, using RT-PCR to detect the expression level of miR-214 in three groups.Results:1.lentivirus infection in A2780 cells 24 hours after, RT-PCR was used to detect group a cellular ERa mRNA 2-ΔΔCt is 7931.25±65.81, higher than group c 53.76±6.09 significantly (P< 0.01). Group b cellular ER β mRNA 2-ΔΔCt is 100.66±15.72, significantly higher than that in group c (P< 0.01).2.Using Western blot to detect group a of cellular ERα/β-actin is 1.77±0.25, higher than group c 1.14±0.18 significantly (P< 0.01).The group b was 0.82 ±0.04,significantly higher than that in group c 0.65 ± 0.06(P< 0.01).3. Western blot was used to detect lentivirus infected cells for 24h, the expression of miR-214 in each cell is 0.18+0.05,0.17+0.03,0.14+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, using RT-PCR to detect miR-214 2-ΔΔCt value of group a is 0.038±0.014,compared with group c 0.144±0.045 was significantly decreased (P< 0.05), group b was 1.325±0.643,compared with c group significantly increased (P< 0.05).5.After ICI180,782treat each group 6h, RT-PCR detect a, b, c three groups of intracellular miR-214 2-△△Ct value were 0.305±0.024,0.292±0.017,0.266 ±0.013 comparison of three groups has no significant difference (P> 0.05).Conclusions:1.We successfully established the high expression of ERβ and ERβ in ovarian cancer cell lines,laid the foundation for the following study.2.Non estrogen, lentivirus infection A2780 can’t influence the miR-214 expression.3.High expression of ERa cells,estrogen can inhibit the expression of miR-214 in cells.4.High expression of ERβ cells,estrogen can promote the expression of miR-214 in cells.5.Estrogen receptor antagonists can inhibit estrogen regulation of miR-214.That means estrogen differentially regulated the expression of miR-214 was mediated by different estrogen receptor.6.The above conclusion confirmed that estrogen can regulate the expression of Sema4D differentially by combine with different receptors. This May provide a new theoretical basis for the pathogenesis of ovarian cancer and may also provide new ideas for ovarian cancer target treatment.