Department of Obstetrics and Gynaecology, The University of Hong Kong, Pokfulam Road, Hong Kong, China.
BMC Cancer. 2013 Jan 18;13:25. doi: 10.1186/1471-2407-13-25.
Choriocarcinoma is a gestational trophoblastic tumor which causes high mortality if left untreated. MicroRNAs (miRNAs) are small non protein-coding RNAs which inhibit target gene expression. The role of miRNAs in choriocarcinoma, however, is not well understood. In this study, we examined the effect of miR-34a in choriocarcinoma.
MiR-34a was either inhibited or ectopically expressed transiently in two choriocarcinoma cell lines (BeWo and JEG-3) respectively. Its actions on cell invasion, proliferation and colony formation at low cell density were examined. The miR-34a putative target Notch ligand Delta-like 1 (DLL1) was identified by adoption of different approaches including: in-silico analysis, functional luciferase assay and western blotting. Real-time quantitative polymerase chain reaction was used to quantify changes in the expression of matrix proteinase in the treated cells. To nullify the effect of miR-34a ectopic expression, we activated Notch signaling through force-expression of the Notch intracellular domain in the miR-34a force-expressed cells. In addition, we studied the importance of DLL1 in BeWo cell invasion through ligand stimulation and antibody inhibition. Furthermore, the induction in tumor formation of miR-34a-inhibited BeWo cells in SCID mice was investigated.
Transient miR-34a force-expression significantly suppressed cell proliferation and invasion in BeWo and JEG-3 cells. In silicon miRNA target prediction, luciferase functional assays and Western blotting analysis demonstrated that miR-34a regulated DLL1 expression in both cell lines. Although force-expression of miR-34a suppressed the expression of DLL1 and NOTCH1, the extent of suppression was higher in DLL1 than NOTCH1 in both cell lines. MiR-34a-mediated DLL1 suppression led to reduced matrix metallopeptidase 9 and urokinase-type plasminogen activator expression. The effect of miR-34a on cell invasion was partially nullified by Notch signaling activation. DLL1 ligand stimulated while anti-DLL1 antibody treatment suppressed cell invasion. Mice inoculated with BeWo cells transfected with miR-34a inhibitor had significantly larger xenografts and stronger DLL1 expression than those with cells transfected with the control inhibitor.
MiR-34a reduced cell proliferation and invasiveness, at least, partially through its inhibitory effect on DLL1.
绒癌是一种妊娠滋养细胞肿瘤，如果不治疗会导致高死亡率。微小 RNA（miRNA）是一种小的非蛋白编码 RNA，可抑制靶基因的表达。然而，miRNA 在绒癌中的作用尚不清楚。在这项研究中，我们研究了 miR-34a 在绒癌中的作用。
分别在绒癌细胞系（BeWo 和 JEG-3）中转染 miR-34a 抑制剂或过表达 miR-34a，检测其对低细胞密度下细胞侵袭、增殖和集落形成的影响。采用不同方法（包括计算机分析、功能荧光素酶检测和 Western blot）鉴定 miR-34a 的潜在靶基因 Notch 配体 Delta-like 1（DLL1）。采用实时定量聚合酶链反应检测处理细胞中基质蛋白酶表达的变化。为了消除 miR-34a 过表达的影响，我们通过转染 Notch 胞内结构域激活 Notch 信号通路，从而过表达 Notch 信号通路。此外，我们通过配体刺激和抗体抑制研究了 DLL1 在 BeWo 细胞侵袭中的重要性。此外，我们还研究了 miR-34a 抑制的 BeWo 细胞在 SCID 小鼠中的肿瘤形成诱导作用。
瞬时 miR-34a 过表达显著抑制了 BeWo 和 JEG-3 细胞的增殖和侵袭。在硅基 miRNA 靶位预测、荧光素酶功能检测和 Western blot 分析中，miR-34a 在两种细胞系中均调控 DLL1 的表达。尽管过表达 miR-34a 抑制了 DLL1 和 NOTCH1 的表达，但在两种细胞系中，DLL1 的抑制程度均高于 NOTCH1。miR-34a 介导的 DLL1 抑制导致基质金属蛋白酶 9 和尿激酶型纤溶酶原激活物表达减少。 Notch 信号通路的激活部分消除了 miR-34a 对细胞侵袭的影响。DLL1 配体刺激，而抗 DLL1 抗体处理抑制细胞侵袭。与转染对照抑制剂的细胞相比，转染 miR-34a 抑制剂的 BeWo 细胞接种的小鼠的异种移植物明显增大，DLL1 表达增强。
miR-34a 通过抑制 DLL1 至少部分降低细胞增殖和侵袭能力。
