Tan Lu Ping, Seinen Erwin, Duns Gerben, de Jong Debora, Sibon Ody C M, Poppema Sibrand, Kroesen Bart-Jan, Kok Klaas, van den Berg Anke
Department of Pathology and Laboratory Medicine, University Medical Center Groningen, University of Groningen, Hanzeplein 1, 9700 RB Groningen, The Netherlands.
Nucleic Acids Res. 2009 Nov;37(20):e137. doi: 10.1093/nar/gkp715. Epub 2009 Sep 4.
The study of human microRNAs is seriously hampered by the lack of proper tools allowing genome-wide identification of miRNA targets. We performed Ribonucleoprotein ImmunoPrecipitation-gene Chip (RIP-Chip) using antibodies against wild-type human Ago2 in untreated Hodgkin lymphoma (HL) cell lines. Ten to thirty percent of the gene transcripts from the genome were enriched in the Ago2-IP fraction of untreated cells, representing the HL miRNA-targetome. In silico analysis indicated that approximately 40% of these gene transcripts represent targets of the abundantly co-expressed miRNAs. To identify targets of miR-17/20/93/106, RIP-Chip with anti-miR-17/20/93/106 treated cells was performed and 1189 gene transcripts were identified. These genes were analyzed for miR-17/20/93/106 target sites in the 5'-UTRs, coding regions and 3'-UTRs. Fifty-one percent of them had miR-17/20/93/106 target sites in the 3'-UTR while 19% of them were predicted miR-17/20/93/106 targets by TargetScan. Luciferase reporter assay confirmed targeting of miR-17/20/93/106 to the 3'-UTRs of 8 out of 10 genes. In conclusion, we report a method which can establish the miRNA-targetome in untreated human cells and identify miRNA specific targets in a high throughput manner. This approach is applicable to identify miRNA targets in any human tissue sample or purified cell population in an unbiased and physiologically relevant manner.
由于缺乏能够在全基因组范围内鉴定miRNA靶标的合适工具，人类微小RNA（miRNA）的研究受到严重阻碍。我们在未经处理的霍奇金淋巴瘤（HL）细胞系中，使用针对野生型人类AGO2的抗体进行核糖核蛋白免疫沉淀-基因芯片（RIP-芯片）实验。未处理细胞的AGO2免疫沉淀（IP）组分中富集了基因组中10%至30%的基因转录本，这些转录本代表了HL的miRNA靶标组。计算机分析表明，这些基因转录本中约40%代表大量共表达miRNA的靶标。为了鉴定miR-17/20/93/106的靶标，我们对用抗miR-17/20/93/106处理的细胞进行了RIP-芯片实验，并鉴定出1189个基因转录本。对这些基因在5'-非翻译区（UTR）、编码区和3'-UTR中的miR-17/20/93/106靶位点进行了分析。其中51%在3'-UTR中有miR-17/20/93/106靶位点，而通过TargetScan预测，19%的基因是miR-17/20/93/106的靶标。荧光素酶报告基因检测证实了miR-17/20/93/106对10个基因中的8个基因的3'-UTR具有靶向作用。总之，我们报告了一种能够在未经处理的人类细胞中建立miRNA靶标组，并以高通量方式鉴定miRNA特异性靶标的方法。这种方法适用于以无偏倚且与生理相关的方式，鉴定任何人类组织样本或纯化细胞群体中的miRNA靶标。
