Abstract Background: Small non-coding RNAs (smRNAs) are characteristically associated with Argonaut family proteins and function as the core of RNA-induced silencing complex in RNA interference. Recent studies have shown a subgroup of smRNAs that are derived from other types of known RNAs. In smRNA deep sequencing (smRNA-seq) data sets, they usually appear to be fragmented tRNAs, rRNAs, SnoRNA, etc. Until recently, they had been considered as by-products in cells. Here, we report the study of discovery and characterization of 5′-tRFHis-GTG, a novel tRNA-derived novel miRNA-like smRNA, in renal cell carcinoma (RCC) metastasis. Design: (1) Discovery of a group of novel smRNAs by deep sequencing analysis of Ago2 protein co-immunoprecipitated small RNA fragments in RCC cell lines; (2) Examination of 5′-tRFHis-GTG expression profiles in benign kidney tissue, localized and metastatic RCCs in a frozen tissue cohort (n=24) by smRNA-seq; (3) Validation of the differential expression of 5′-tRFHis-GTG in a 237-specimen FFPE tissue cohort of localized and metastatic RCCs by RT-PCR. (4) Exploration of the 5′-tRFHis-GTG biogenesis by knocking down the gene expression of miRNA processing proteins Drosha, Dicer and Exportin-5, Ago2 in RCC cell lines; (4) Characterization the function of 5′-tRFHis-GTG on cell proliferation, migration and invasion by knocking down 5′-tRFHis-GTG expression in the metastatic RCC cell line ACHN by MTS, wound healing and transwell/matrigel assays; (5) Oxidative stress test by examining the expression levels of 5′-tRFHis-GTG with/without adding H2O2. Results: (1) Among the group of Ago2-associated novel smRNAs, 5′-tRFHis-GTG is a 24-mer RNA fragment derived from the mature form of tRNA-His(GTG). (2) 5′-tRFHis-GTG has been found to be up-regulated in metastatic RCCs, comparing to the localized tumors in the 24-sample frozen tissue cohort. (3) Validation test using a separate FFPE tissue cohort of 237 clinical specimens confirmed the upregulated 5′-tRFHis-GTG expression in metastatic RCC tissues. (4) Knock-down of Drosha, Dicer, Exportin-5 and Ago2 gene expression caused a significant decrease of the 5′-tRFHis-GTG expression in the cell line. (3) Knocking down 5′-tRFHis-GTG by its LNA antisense inhibited the proliferation of RCC tumor cells (ACHN, 769P, Caki-1 and Caki-2) but not the benign renal cells (HEK293T and HK-2). (4) Knocking down 5′-tRFHis-GTG also suppressed the tumor cell migration and invasion in the metastatic RCC cells (ACHN). (5) Adding H2O2 significantly induced 5′-tRFHis-GTG expression in renal cell lines. Conclusions: 5′-tRFHis-GTG, a novel tRNA-derived miRNA-like smRNA that is probably induced by oxidative stress, is upregulated in metastatic RCCs and involved in metastatic tumor cell proliferation and migration. Citation Format: Yuping Li, Xiwei Wu, Hanlin Gao, Jennifer Jin, Sumanta Pal, Arthur Li, Chao Guo, Rebecca Nelson, Jihui Wang, Young Kim, Francis Wang, Jessica Wang, Wengang Chen, Shirong Wang, John Rossi, Huiqing Wu. 5′-tRFHis-GTG, a novel tRNA-derived novel miRNA-like small non-coding RNA, aberrantly expressed in metastatic renal cell carcinoma by promoting tumor cell proliferation, migration and invasion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-247. doi:10.1158/1538-7445.AM2013-LB-247