ORIGINAL PAPER Journal of Pathology J Pathol 2010; 222: 310–319 Published online 20 August 2010 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/path.2759 Systematic evaluation of the miRNA-ome and its downstream effects on mRNA expression identifies gastric cancer progression Oleg Tchernitsa, 1,2 Atsuko Kasajima, 1 Reinhold Sch¨ afer, 1 Ralf-J¨ urgen Kuban, 2 Ute Ungeth¨ um, 2 Balazs Gy ¨ orffy, 1,3 Ulf Neumann, 4 Eva Simon, 5 Wilko Weichert, 1 Matthias PA Ebert 6 and Christoph R ¨ ocken 1,5 * 1 Institute of Pathology, Charit´ e University Hospital, Berlin, Germany 2 Laboratory for Functional Genetics, Charit´ e University Hospital, Berlin, Germany 3 Joint Research Laboratory of the Hungarian Academy of Sciences and the Semmelweis University, Semmelweis University 1st Department of Pediatrics, Hungary 4 Department of General-, Visceral- and Transplant Surgery, Charit´ e University Hospital, Berlin, Germany 5 Institute of Pathology, Christian-Albrechts-University, Kiel, Germany 6 Department of Medicine II, Klinikum rechts der Isar, Technical University Munich, Germany *Correspondence to: Professor Christoph R¨ ocken, Institute of Pathology, Christian-Albrechts-University, Arnold-Heller-Strasse 3, D-24105 Kiel, Germany e-mail: christoph.roecken@uk-sh.de Abstract We investigated the differential expression of Dicer and Drosha, as well as that of microRNA (miRNA), in adjacent normal and tumour samples of patients with gastric cancer. The expression of Dicer and Drosha was studied by immunohistochemistry in 332 gastric cancers and correlated with clinico-pathological patient characteristics. Differential expression of miRNAs was studied using the Invitrogen NCode  Multi-Species miRNA Microarray Probe Set containing 857 mammalian probes in a test set of six primary gastric cancers (three with and three without lymph node metastases). Differential expression was validated by RT-PCR on an independent validation set of 20 patients with gastric cancer. Dicer and Drosha were differentially expressed in non-neoplastic and neoplastic gastric tissue. The expression of Drosha correlated with local tumour growth and was a significant independent prognosticator of patient survival. Twenty miRNAs were up- and two down-regulated in gastric carcinoma compared with non-neoplastic tissue. Six of these miRNAs separated node-positive from node-negative gastric cancers, ie miR-103, miR-21, miR-145, miR-106b, miR-146a, and miR-148a. Five miRNAs expressed differentially in node-positive cancers had conserved binding sites for mRNAs differentially expressed in the same set of tumour samples. Gastric cancer shows a complex derangement of the miRNA-ome, including Dicer and Drosha. These changes correlate independently with patient prognosis and probably influence local tumour growth and nodal spread. Copyright  2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Keywords: gastric cancer; lymph node metastases; miRNA; prognosis; Dicer; Drosha Received 14 June 2010; Revised 18 July 2010; Accepted 21 July 2010 No conflicts of interest were declared. Introduction Gastric cancer is the fourth most common malig- nancy and the second leading cause of cancer-related death worldwide [1,2]. Multiple genetic alterations are involved in the pathology and pathogenesis of gastric cancer and include the activation and overexpression of oncogenes and the inactivation of tumour suppressor genes. Recently it has become apparent that apart from genetic and epigenetic changes in classical oncogenes and tumour suppressor genes, small 18–25 nucleotides long non-coding microRNAs (miRNAs) regulate major cellular processes involved in tumour biology, such as cell proliferation, differentiation, and apoptosis. miR- NAs bind through sequence-specific base pairing to the 3 ′ -untranslated region (3 ′ -UTR) of the mRNA leading to its translational repression or degradation, thereby negatively regulating protein expression. At least 533 different miRNAs have been identified and it is believed that the human genome encodes about 1000 miRNAs [3]. The translation of 20–30% of all human genes is regulated by miRNAs. Each miRNA targets on average 200 gene products and multiple miR- NAs can bind to the 3 ′ -UTR of a single mRNA [3,4]. miRNAs are frequently located at fragile chromosomal sites, common break points, and regions of amplifi- cation or loss of heterozygosity [3,4], and may play significant roles in oncogenesis. It has been shown that miRNAs themselves may function as oncogenes and tumour suppressor genes. Wu et al [5] demonstrated genetic variations of miRNA genes including single nucleotide polymorphisms and mutations associated Copyright  2010 Pathological Society of Great Britain and Ireland. J Pathol 2010; 222: 310–319 Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk www.thejournalofpathology.com