miR-221/222及其靶基因在妊娠期肝内胆汁淤积症发病机制中作用的研究

Role of miR-221/222 and its target genes in the pathogenesis of intrahepatic cholestasis of pregnancy

摘要:

目的：研究微小核糖核酸（MicroRNA，miRNA）221/222及其靶基因在正常孕妇和妊娠期肝内胆汁淤积症（intrahepatic cholestasis of pregnancy，ICP）孕妇胎盘中表达的差异，并通过细胞实验过表达方法研究miR-221/222对靶基因表达影响，探讨其在ICP发病机制中的作用。方法：搜集2015年9月到2016年3月在重庆医科大学附属第二医院妇产科行剖宫产分娩的正常孕妇和ICP孕妇的胎盘组织各20例，直接提取miRNA，采用实时荧光定量逆转录PCR（qRT-PCR）检测miR-221/222在2组胎盘组织中的表达情况；预测miR-221/222的靶基因是顶端钠依赖性胆酸转运体（apical sodium-dependent bile acid transporter，ASBT，又称SLC10A2）；Western blot检测ASBT在ICP和正常胎盘组织中的表达情况；用Lipofectaine2000脂质体包裹合成的miR-221/222 mimic转染正常滋养细胞HTR-8；qRT-PCR检测miR-221/222表达水平；蛋白质印迹法检测转染后ASBT蛋白表达。结果：①经过内参U6的校正，miR-221在ICP胎盘表达量为1.066±0.044，正常胎盘表达量为0.053±0.009；miR-222在ICP胎盘表达量为13.724±4.355，正常胎盘表达量为0.833±0.189。可见miR-221/222在ICP胎盘中的表达明显上调（P<0.05）。②SLC10A2在ICP组中的表达量为0.328±0.102，在正常组中的表达量为0.604±0.119，其在ICP组的表达较正常组的表达下调（P=0.000）。③经过内参ACTIN校正，ASBT在转染miR-221组细胞内表达量为0.338±0.064，对照组表达量为0.583±0.040；ASBT在转染miR-222组细胞内表达量为0.371±0.024，对照组表达量为0.624±0.031，可见ASBT在转染组中较对照组表达下调。结论：miR-221/222在ICP胎盘组织中表达升高，而其预测靶基因ASBT的表达则下降；细胞实验中，miR-221/222转染组靶基因ASBT表达下降。两者之间可能存在负调控关系。可能由此影响了孕妇胆汁酸在肠道的重吸收及在肝脏的转运而导致妊娠期肝内胆汁淤积。

Abstract:

Objective：To investigate the difference in the expression of microRNA（miR）-221/222 and its target gene in the placenta between normal pregnant women and pregnant women with intrahepatic cholestasis of pregnancy（ICP）， the effect of miR-221/222 on the expression of target gene according to a cell experiment，and the role of miR-221/222 in the pathogenesis of ICP. Methods：A total of 40 placenta samples were collected from normal pregnant women and pregnant women with ICP，all of whom underwent cesarean section in The Second Affiliated Hospital of Chongqing Medical University from September 2015 to March 2016，with 20 samples from each group. miRNA was extracted and qRT-PCR was used to measure the expression of miR-221/222 in the placenta. Apical sodium-dependent bile acid transporter（ASBT），also known as SLC10A2，was predicted to be the target gene of miR-221/222，and Western blot was used to measure the expression of ASBT in the placenta from normal pregnant women and pregnant women with ICP. Normal HTR-8 cells were transfected with miR-221/222 mimic wrapped with Lipofectaine 2000；qRT-PCR was used to measure the expression of miR-221/222，and Western blot was used to measure the expression of ASBT protein after transfection. Results：After correction with the internal control U6，the expression of miR-221 in the ICP and normal placenta was 1.066±0.044 and 0.053±0.009，respectively，and the expression of miR-222 in the ICP and normal placenta was 13.724±4.355 and 0.833±0.189，respectively，suggesting that miR-221/222 was significantly upregulated in the ICP placenta（P <0.05）. The ICP group had significantly lower expression of SLC10A2 than the normal group（0.328±0.102 vs. 0.604±0.119，P=0.000）. After correction with the internal control ACTIN，the expression of ASBT was 0.338±0.064 in the miR-221 transfection group and 0.583±0.040 in the control group；the expression of ASBT was 0.371±0.024 in the miR-222 transfection group and 0.624±0.031 in the control group，suggesting that the transfection group had significantly lower expression of ASBT than the control group. Conclusion：There is an increase in the expression of miR-221/222 in the ICP placenta and a reduction in the expression of its predicted target gene ASBT. The cell experiment shows a reduction in the expression of ASBT in the miR-221/222 transfection group. There may be a negative regulation between them， which affects the reabsorption of bile acid in the intestinal tract and its transport in the liver among pregnant women， thus leading to ICP.