Department of Obstetrics and Gynaecology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand.
Gynecol Oncol. 2013 Apr;129(1):209-15. doi: 10.1016/j.ygyno.2012.12.034. Epub 2012 Dec 30.
Despite their distinct biology, granulosa cell tumours (GCTs) are treated similarly to other ovarian tumours. Predominantly expressed in granulosa cells, the transcription factor Forkhead Box L2 (FOXL2) is near absent in juvenile-type GCTs. This research aimed to investigate miRNAs as a mechanism of suppression of FOXL2 expression in juvenile-type GCTs.
The miRNA abundance of two GCT cell lines COV434 and KGN was profiled using Affymetrix miRNA GeneChip arrays. Luciferase assays were used to confirm miRNA binding to the 3'UTR of FOXL2. Identified as promising candidates, the miR-17 miRNA family was targeted for knockdown with a miRNA sponge. Additionally, individual family members miR-17, miR-20b and miR-106a were knocked down using Anti-miR™ inhibitors. Subsequently, FOXL2 expression was analysed using RT-qPCR and Western blotting.
The profiling of COV434 and KGN cells revealed unique miRNA signatures, with COV434 expressing miR-17 family miRNAs whilst KGN expressed members of the let-7 miRNA gene family. Luciferase assays confirmed miRNA binding to FOXL2's 3'UTR. Reduction of miR-17 family miRNAs increased FOXL2 mRNA expression, however luciferase assays performed in combination with the sponge suggested this is an indirect effect. As no changes in protein were observed, we propose another miRNA is repressing the translation of FOXL2 mRNA.
Through miRNA profiling we have begun to unravel the profiles of GCTs, showing that juvenile and adult derived-cell lines are biologically distinct. By expanding on this discovery we may further elucidate the miRNA-mRNA pathways involved in GCT initiation and progression with potential for novel therapeutics for these cancers.
尽管颗粒细胞瘤（GCT）具有明显的生物学特征，但它们的治疗方法与其他卵巢肿瘤相似。转录因子叉头框 L2（FOXL2）主要在颗粒细胞中表达，在幼年型 GCT 中几乎不存在。本研究旨在探讨 miRNA 作为抑制幼年型 GCT 中 FOXL2 表达的一种机制。
使用 Affymetrix miRNA GeneChip 阵列对 COV434 和 KGN 两种 GCT 细胞系的 miRNA 丰度进行了分析。荧光素酶测定法用于证实 miRNA 与 FOXL2 的 3'UTR 结合。作为有前途的候选物，miR-17 miRNA 家族被靶向使用 miRNA 海绵进行敲低。此外，使用 Anti-miR™抑制剂分别敲低家族成员 miR-17、miR-20b 和 miR-106a。随后，使用 RT-qPCR 和 Western blotting 分析 FOXL2 的表达。
COV434 和 KGN 细胞的分析揭示了独特的 miRNA 特征，COV434 表达 miR-17 家族 miRNA，而 KGN 表达 let-7 miRNA 基因家族的成员。荧光素酶测定法证实了 miRNA 与 FOXL2 的 3'UTR 结合。miR-17 家族 miRNA 的减少增加了 FOXL2 mRNA 的表达，但荧光素酶测定法与海绵结合进行的实验表明这是一种间接效应。由于未观察到蛋白的变化，我们提出另一种 miRNA 正在抑制 FOXL2 mRNA 的翻译。
通过 miRNA 分析，我们已经开始揭示 GCT 的特征，表明幼年和成年来源的细胞系在生物学上是不同的。通过进一步扩展这一发现，我们可能会进一步阐明参与 GCT 起始和进展的 miRNA-mRNA 途径，为这些癌症提供新的治疗方法。
