Duan Hong-Yu, Ma Dan, Zhou Kai-Yu, Wang Tao, Zhang Yi, Li Yi-Fei, Wu Jin-Lin, Hua Yi-Min, Wang Chuan
Department of Pediatric Cardiology, West China Second University Hospital, Sichuan University, Chengdu, Sichuan 610041; The Cardiac Development and Early Intervention Unit, West China Institute of Women and Children's Health, West China Second University Hospital, Sichuan University, Chengdu, Sichuan 610041, China.
Department of Pediatric Rehabilitation, West China Second University Hospital, Sichuan University, Chengdu, Sichuan 610041, China.
Chin Med J (Engl). 2017 Jun 5;130(11):1352-1360. doi: 10.4103/0366-6999.206352.
Placental multidrug resistance-associated protein 2 (MRP2), encoded by ABCC2 gene in human, plays a significant role in regulating drugs' transplacental transfer rates. Studies on placental MRP2 regulation could provide more therapeutic targets for individualized and safe pharmacotherapy during pregnancy. Currently, the roles of epigenetic mechanisms in regulating placental drug transporters are still unclear. This study aimed to investigate the effect of histone deacetylases (HDACs) inhibition on MRP2 expression in the placental trophoblast cell line and to explore whether HDAC1/2/3 are preliminarily involved in this process.
The human choriocarcinoma-derived trophoblast cell line (Bewo cells) was treated with the HDAC inhibitors-trichostatin A (TSA) at different concentration gradients of 0.5, 1.0, 3.0, and 5.0 μmol/L. Cells were harvested after 24 and 48 h treatment. Small interfering RNA (siRNA) specific for HDAC1/HDAC2/HDAC3 or control siRNA was transfected into cells. Total HDAC activity was detected by colorimetric assay kits. HDAC1/2/3/ABCC2 messenger RNA (mRNA) and protein expressions were determined by real-time quantitative polymerase chain reaction and Western-blot analysis, respectively. Immunofluorescence for MRP2 protein expression was visualized and assessed using an immunofluorescence microscopy and ImageJ software, respectively.
TSA could inhibit total HDAC activity and HDAC1/2/3 expression in company with increase of MRP2 expression in Bewo cells. Reduction of HDAC1 protein level was noted after 24 h of TSA incubation at 1.0, 3.0, and 5.0 μmol/L (vs. vehicle group, all P < 0.001), accompanied with dose-dependent induction of MRP2 expression (P = 0.045 for 1.0 μmol/L, P = 0.001 for 3.0 μmol/L, and P < 0.001 for 5.0 μmol/L), whereas no significant differences in MRP2 expression were noted after HDAC2/3 silencing. Fluorescent micrograph images of MRP2 protein were expressed on the cell membrane. The fluorescent intensities of MRP2 in the control, HDAC2, and HDAC3 siRNA-transfected cells were week, and no significant differences were noticed among these three groups (all P > 0.05). However, MRP2 expression was remarkably elevated in HDAC1 siRNA-transfected cells, which displayed an almost 3.19-fold changes in comparison with the control siRNA-transfected cells (P < 0.001).
HDACs inhibition could up-regulate placental MRP2 expression in vitro, and HDAC1 was probably to be involved in this process.
人胎盘多药耐药相关蛋白2（MRP2）由ABCC2基因编码，在调节药物经胎盘转运速率中起重要作用。胎盘MRP2调控的研究可为孕期个体化和安全的药物治疗提供更多治疗靶点。目前，表观遗传机制在调节胎盘药物转运体中的作用仍不清楚。本研究旨在探讨组蛋白去乙酰化酶（HDACs）抑制对胎盘滋养层细胞系中MRP2表达的影响，并探索HDAC1/2/3是否初步参与该过程。
将人绒毛膜癌来源的滋养层细胞系（Bewo细胞）用不同浓度梯度（0.5、1.0、3.0和5.0 μmol/L）的HDAC抑制剂曲古抑菌素A（TSA）处理。处理24小时和48小时后收获细胞。将针对HDAC1/HDAC2/HDAC3的小干扰RNA（siRNA）或对照siRNA转染到细胞中。通过比色测定试剂盒检测总HDAC活性。分别通过实时定量聚合酶链反应和蛋白质免疫印迹分析测定HDAC1/2/3/ABCC2信使核糖核酸（mRNA）和蛋白表达。分别使用免疫荧光显微镜和ImageJ软件对MRP2蛋白表达进行免疫荧光观察和评估。
TSA可抑制Bewo细胞中的总HDAC活性和HDAC1/2/3表达，同时增加MRP2表达。在1.0、3.0和5.0 μmol/L的TSA孵育24小时后，HDAC1蛋白水平降低（与溶剂对照组相比，均P < 0.001），同时伴随MRP2表达的剂量依赖性诱导（1.0 μmol/L时P = 0.045，3.0 μmol/L时P = 0.001，5.0 μmol/L时P < 0.001），而HDAC2/3沉默后MRP2表达无显著差异。MRP2蛋白的荧光显微图像在细胞膜上表达。在对照、HDAC2和HDAC3 siRNA转染细胞中MRP2的荧光强度较弱，这三组之间未观察到显著差异（均P > 0.05）。然而，在HDAC1 siRNA转染细胞中MRP2表达显著升高，与对照siRNA转染细胞相比显示出近3.19倍的变化（P < 0.001）。
HDACs抑制在体外可上调胎盘MRP2表达，且HDAC1可能参与该过程。
