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(intrahepaticcholestasis 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。
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 low
作者
王林若
刘建
Wang Linruo;Liu Jian(Department of Obstetrics and Gynecology,The Second Affiliated Hospital of Chongqing Medical University)
出处
《重庆医科大学学报》
CAS
CSCD
北大核心
2019年第5期662-667,共6页
Journal of Chongqing Medical University
