RNA干扰抑制ABCG2表达对子痫前期滋养细胞分泌hCG的影响

Effect of the SiRNA-Mediated ABCG2 Gene Silencing on the Secretion of hCG by Trophoblasts in Pre-Eclampsia

目的:应用RNA干扰技术,使胎盘滋养细胞ATP结合匣式转运子G2(ABCG2)基因表达沉默,检测干扰前后ABCG2的表达及人绒毛膜促性腺激素β亚单位(β-hCG)水平变化,研究ABCG2的表达对子痫前期滋养细胞分泌β-hCG功能的影响。方法:通过小干扰RNA(siRNA)预测工具确定3个ABCG2基因的siRNA靶位点,合成3对互补的短发夹RNA(shRNA)序列转化质粒,选取干扰有效的质粒包装慢病毒后感染原代培养正常及子痫前期胎盘滋养细胞,应用实时荧光定量聚合酶链反应(qPCR)及蛋白质印迹(Western blotting)检测干扰前、后滋养细胞ABCG2 mRNA及蛋白的表达水平,酶联免疫吸附测定(ELISA)检测干扰前、后不同分化状态细胞上清液中的β-hCG水平。结果:有效的RNA干扰后滋养细胞ABCG2 mRNA及蛋白的表达明显降低,无论干扰前、后子痫前期滋养细胞hCG分泌都高于正常,干扰后滋养细胞分泌hCG水平降低。结论:siRNA可有效干扰滋养细胞ABCG2基因的表达,ABCG2基因表达沉默后胎盘滋养细胞hCG分泌下降,推测子痫前期ABCG2表达升高可通过促进滋养细胞分泌hCG抑制子痫前期的发展。

Objective:To detect the expression of mRNA and protein of the active ATP-binding cassette family G2 transporters(ABCG2)and the level ofβ-hCG before and after the RNA interference of ABCG2 gene in primary cultured trophoblast cells from normal and pre-eclamptic placentas,explore the effect of ABCG2 on the function of trophoblasts in preeclampsia.Methods:Three target siRNA sites of ABCG2 genes were determined by siRNA prediction tools and three pairs of complementary shRNA sequence transformation plasmids were synthesized.The effective interfering plasmids were selected to package lentivirus and infected trophoblasts.The change of expression of ABCG2 mRNA and protein in the cells were detected by real time fluorescence quantitative PCR and Western blotting in cultivated villous trophoblasts of different differentiation states from normal and pre-eclamptic placentas to verify the effect of interference,β-hCG level was detected by ELISA before and after interference in cell culture supernatants.Results:After RNA interference,the expression of ABCG2 mRNA and protein decreased significantly in trophoblasts and hCG secretion of trophoblasts was reduced.The level of hCG in pre-eclampsia trophoblasts was higher than normal before and after interference.Conclusions:SiRNA could effectively interfere the expression of ABCG2 gene in trophoblast cells.Secretion of hCG was inhibited after the silence of ABCG2 gene expression in placental trophoblasts,hCG secretion in pre-eclampsia patient was higher than normal.ABCG2 might play a role in inhibiting the development of pre-eclampsia by stimulating the secretion of hCG of trophoblasts.