Abstract LB-247: 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.

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′-tRF His-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′-tRF His-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′-tRF His-GTG in a 237-specimen FFPE tissue cohort of localized and metastatic RCCs by RT-PCR. (4) Exploration of the 5′-tRF His-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′-tRF His-GTG on cell proliferation, migration and invasion by knocking down 5′-tRF His-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′-tRF His-GTG with/without adding H 2 O 2 . Results: (1) Among the group of Ago2-associated novel smRNAs, 5′-tRF His-GTG is a 24-mer RNA fragment derived from the mature form of tRNA-His(GTG). (2) 5′-tRF His-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′-tRF His-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′-tRF His-GTG expression in the cell line. (3) Knocking down 5′-tRF His-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′-tRF His-GTG also suppressed the tumor cell migration and invasion in the metastatic RCC cells (ACHN). (5) Adding H2O2 significantly induced 5′-tRF His-GTG expression in renal cell lines. Conclusions: 5′-tRF His-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′-tRF His-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