Recent studies have revealed that feed-forward loops (FFLs) as regulatory motifs have synergistic roles in cellular systems and their disruption may cause diseases including cancer. FFLs may include two regulators such as transcription factors (TFs) and microRNAs (miRNAs). In this study, we extensively investigated TF and miRNA regulation pairs, their FFLs, and TF-miRNA mediated regulatory networks in two major types of testicular germ cell tumors (TGCT): seminoma (SE) and non-seminoma (NSE). Specifically, we identified differentially expressed mRNA genes and miRNAs in 103 tumors using the transcriptomic data from The Cancer Genome Atlas. Next, we determined significantly correlated TFgene/miRNA and miRNA-gene/TF pairs with regulation direction. Subsequently, we determined 288 and 664 dysregulated TF-miRNA-gene FFLs in SE and NSE, respectively. By constructing dysregulated FFL networks, we found that many hub nodes (12 out of 30 for SE and 8 out of 32 for NSE) in the top ranked FFLs could predict subtype-classification (Random Forest classifier, average accuracy ≥90%). These hub molecules were validated by an independent dataset. Our network analysis pinpointed several SE-specific dysregulated miRNAs (miR-200c-3p, miR-25-3p, and miR-302a-3p) and genes (EPHA2, JUN, KLF4, PLXDC2, RND3, SPI1, and TIMP3) and NSE-specific dysregulated miRNAs (miR-367-3p, miR-519d-3p, and miR-96-5p) and genes (NR2F1 and NR2F2). This study is the first systematic investigation of TF and miRNA regulation and their co-regulation in two major TGCT subtypes. Testicular germ cell tumors (TGCT) occur most frequently in men between ages of 20 and 40 1,2. Accordingly to histology, TGCT can be separated into two major types: seminoma (SE) and non-seminoma (NSE) 1-4 , and NSE has several subtypes. While the etiology of the two TGCT subtypes is well studied, their molecular profiles, signature genetic markers, and regulatory mechanisms have not been systematically investigated, unlike other common cancers. Such an investigation is much needed now to identify molecular signatures either common in two subtypes, or unique in subtype. The molecular signatures may be further useful for clinical implications, such as patient stratification and subtype-based or personalized treatment. Currently, there are several challenges in TGCT treatment. First, TGCT patients have a high risk of relapse with poor prognosis. Second, there are severe side effects for current chemotherapy and radiotherapy that lead to development of other pathologies. Third, since most of the patients are adolescent or young men, there is a heavy burden for the patients and families in the long run 2,3. During the last decade, a number of studies have been conducted to explore insights into the genetic, epigenetic, and molecular mechanisms of TGCT. For example, after collecting TGCT related genes from previous
