Galoian K, Guettouche T, Issac B, Navarro L, Temple H T
Tumour Biol. 2014 Jan;35(1):483-92. doi: 10.1007/s13277-013-1068-5.
The goal of this study was to compare and analyze differentially expressed miRNA and their targets in human chondrosarcoma JJ012 and chondrocytes C 28 cell lines (control) to elucidate deregulation of major signal transduction pathways involved in sarcomagenesis. Total RNA extraction was followed by analyzing RNA quality and integrity. Exiqon human miRNA panel of 743 unique miRNA assays and Illumina microarray HT-12 platform and quantitative reverse transcriptase–PCR verification of targets were performed. The results from human miRNA Exiqon arrays with biological triplicates indicated 28 significant miRNAs (P value ≤0.01). A total 3,045 target genes were derived from the miRWalk database for these 28 miRNAs with 587 common and 2,458 unique target genes. The results of our analyses of the significantly downregulated and upregulated miRNAs in chondrosarcoma cell line indicated the predominant dysregulation of NOTCH, insulin-like growth factor receptor (IGFR), and downstream rat sarcoma, and Src pathways, compared to control. Among the upregulated targets for upregulated miRNAs were the cluster of cancer testis antigen (CTA) genes, located on X chromosome, and their expression was correlated to IGFR pathway activity. Based on our observations, lost miRNA surveillance of NOTCH and IGFR pathways is involved in and leads to sarcomagenesis. We conclude that upregulation of CTA genes is due to hypomethylation that are controlled by epi-miRNAs. We do not preclude the possibility that the upregulated miRNAs, which target CTA genes located in adjacent regions in chromosome X, are epi-miRNAs that influence target gene expression by directly regulating epigenetic processes. Future endeavors will be directed towards understanding the posttranscriptional modifications that affect miRNA expression in sarcomas.
本研究的目的是比较和分析人软骨肉瘤JJ012和软骨细胞C 28细胞系（对照）中差异表达的miRNA及其靶标，以阐明肉瘤发生过程中主要信号转导通路的失调。提取总RNA后分析RNA质量和完整性。进行了Exiqon人类miRNA芯片，包含743种独特的miRNA检测，以及Illumina微阵列HT-12平台和靶标的定量逆转录PCR验证。来自具有生物学重复的人类miRNA Exiqon芯片的结果表明有28个显著的miRNA（P值≤0.01）。从miRWalk数据库中为这28个miRNA获得了总共3045个靶基因，其中有587个共同靶基因和2458个独特靶基因。我们对软骨肉瘤细胞系中显著下调和上调的miRNA的分析结果表明，与对照相比，NOTCH、胰岛素样生长因子受体（IGFR）以及下游大鼠肉瘤和Src通路存在主要失调。上调的miRNA的上调靶标中包括位于X染色体上的癌胚抗原（CTA）基因簇，其表达与IGFR通路活性相关。基于我们的观察，NOTCH和IGFR通路的miRNA监测缺失参与并导致肉瘤发生。我们得出结论，CTA基因的上调是由于表观遗传miRNA控制的低甲基化。我们不排除这样一种可能性，即靶向位于X染色体相邻区域的CTA基因的上调miRNA是通过直接调节表观遗传过程来影响靶基因表达的表观遗传miRNA。未来的研究将致力于了解影响肉瘤中miRNA表达的转录后修饰。
