Wang Ya-Xin, Zhao Jiu-Ru, Xu Yue-Ying, Wu Wei-Bin, Zhang Hui-Juan
*Departments of Pathology and Bio-Bank, International Peace Maternity and Child Health Hospital Affiliated to Shanghai Jiao Tong University School of Medicine; †Department of Ultrasound in Medicine, Shanghai Jiao Tong University Affiliated Sixth People's Hospital; and ‡Institute of Embryo-Fetal Original Adult Disease Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Int J Gynecol Cancer. 2017 Feb;27(2):364-374. doi: 10.1097/IGC.0000000000000861.
The aims of this study were to make clear whether miR-21 was dysregulated in hydatidiform mole (HM) tissues and choriocarcinoma (CCA) cells, to elucidate whether aberrant miR-21 expression would affect the function of CCA cells, and to find out whether there was a relationship between miR-21 and AKT, PDCD4, and PTEN in CCA cells.
Fresh and formalin-fixed, paraffin-embedded trophoblastic tissues (normal first trimester placentas and HMs) were retrieved from the biobank in the International Peace Maternity and Child Health Hospital, Shanghai Jiao Tong University. Choriocarcinoma JAR and JEG-3 cells were cultured. Expression of miR-21 in trophoblast cells and tissues was examined by quantitative real-time polymerase chain reaction. Location and distribution of miR-21 in trophoblast tissues were determinated by in situ hybridization and fluorescent in situ hybridization. The effect of miR-21 on JAR and JEG-3 cells was tested by miR-21 mimics and inhibitor transfection, followed by cell viability assay, flow cytometric analysis, and Transwell analysis. Interaction between miR-21 and its target genes in CCA cells was verified by quantitative real-time polymerase chain reaction, Western blot, and luciferase report system.
We originally found miR-21 was markedly upregulated in HM tissues compared with normal first trimester placentas. The expression of miR-21 was exclusively confined in trophoblastic layers. Furthermore, we discovered miR-21 was significantly increased in JAR and JEG-3 cells compared with normal primary human trophoblastic cells. Moreover, we demonstrated miR-21 could promote proliferation, migration, and invasion of CCA cells. We furthermore proved miR-21 negatively regulated PDCD4 and PTEN in CCA cells and targeted to PDCD4 3'UTR directly. In addition, we confirmed that miR-21 could activate Akt pathway by phosphorylating Akt at Ser 473.
Our results suggested miR-21 was responsible for aggressive phenotype of gestational trophoblastic disease and had the potential diagnostic and therapeutic values for gestational trophoblastic neoplasm.
本研究旨在明确微小RNA-21（miR-21）在葡萄胎（HM）组织和绒毛膜癌（CCA）细胞中是否失调，阐明miR-21异常表达是否会影响CCA细胞的功能，并探究miR-21与CCA细胞中AKT、程序性细胞死亡蛋白4（PDCD4）和第10号染色体缺失的磷酸酶及张力蛋白同源物（PTEN）之间是否存在关联。
从上海交通大学国际和平妇幼保健院生物样本库中获取新鲜的以及经福尔马林固定、石蜡包埋的滋养层组织（正常早孕胎盘和葡萄胎）。培养绒毛膜癌JAR和JEG-3细胞。采用定量实时聚合酶链反应检测滋养层细胞和组织中miR-21的表达。通过原位杂交和荧光原位杂交确定miR-21在滋养层组织中的定位和分布。用miR-21模拟物和抑制剂转染来检测miR-21对JAR和JEG-3细胞的影响，随后进行细胞活力测定、流式细胞术分析和Transwell分析。通过定量实时聚合酶链反应、蛋白质免疫印迹法和荧光素酶报告系统验证miR-21与CCA细胞中其靶基因之间的相互作用。
我们首次发现，与正常早孕胎盘相比，miR-21在葡萄胎组织中显著上调。miR-21的表达仅局限于滋养层。此外，我们发现与正常原代人滋养层细胞相比，miR-21在JAR和JEG-3细胞中显著增加。而且，我们证明miR-21可促进CCA细胞的增殖、迁移和侵袭。我们进一步证明miR-21在CCA细胞中负向调节PDCD4和PTEN，并直接靶向PDCD4的3'非翻译区（3'UTR）。此外，我们证实miR-21可通过将丝氨酸473位点的Akt磷酸化来激活Akt信号通路。
我们的结果表明，miR-21与妊娠滋养细胞疾病的侵袭性表型有关，对妊娠滋养细胞肿瘤具有潜在的诊断和治疗价值。
