Profiling and functional analysis of microRNAs in follicular granulosa cells and their clinical application

我们通过高通量测序对人颗粒来源细胞系(例如KGN)中的microRNA(MiRNAs)进行了大规模的分析。在这些细胞系中，我们发现miR-21占AGO2(命名为EIFC2)结合的miRNAs的80%以上，这表明它作为一种功能性miRNA在人GCs中发挥着核心作用。我们还在KGN细胞中克隆了AGO2结合的mRNAs，并确认COL4A1 mRNA是KGN细胞中可能的miR-21靶点，这一点得到了随后的实验验证。在利用人工合成的miRNA前体或短干扰RNAs的体外功能研究中，我们还发现这些短双链RNAs(23和/或24核苷酸dsRNAs)容易诱导人颗粒细胞肿瘤来源的细胞凋亡，但对其他类型的细胞不诱导凋亡。此外，我们还发现，短dsRNAs通过dsRNA激活的蛋白激酶在转录水平上高水平诱导维甲酸诱导基因-I。

We performed large-scale profiling of microRNA (miRNAs) in human granulosa-derived cell lines (e.g., KGN) by high-throughput sequencing. In these cell lines, we found that miR-21 accounted for more than 80% of AGO2 (designated EIFC2) -bound miRNAs, suggesting that it played a central role as a functional miRNA in human GCs. We also performed cDNA cloning of AGO2-bound mRNAs in KGN cells and identified COL4A1 mRNA as a possible miR-21 target in KGN cells, which was corroborated by subsequent experimental validation. In proceeding in vitro functional studies using synthetic mimics of miRNA precursors or short-interference RNAs, we also found that these short double-strand RNAs (23- and/or 24-nucleotide dsRNAs) readily induced apoptosis in human granulosa cell tumor-derived cells but not in other cell types. Furthermore, we revealed that the short dsRNAs transcriptionally hyper-induce retinoic acid-inducible gene-I through dsRNA-activated protein kinase.