fMiRNA-192 and miRNA-204 Directly Suppress lncRNA HOTTIP and Interrupt GLS1-Mediated Glutaminolysis in Hepatocellular Carcinoma

Accumulated evidence demonstrated that long non-coding RNAs (lncRNAs) play a pivotal role in tumorigenesis. However, it is still largely unknown how these lncRNAs were regulated by small ncRNAs, such as microRNAs (miRNAs), at the post-transcriptional level. We here use lncRNAHOTTIP as an example to study howmiRNAs impact lncRNAs expression and its biological significance in hepatocellular carcinoma (HCC). LncRNAHOTTIP is a vital oncogene in HCC, one of the deadliest cancers worldwide. In the current study, we identified miR-192 and miR-204 as twomicroRNAs (miRNAs) suppressing HOTTIP expression via the Argonaute 2 (AGO2)-mediated RNA interference (RNAi) pathway in HCC. Interaction betweenmiR-192 or miR-204 and HOTTIP were further confirmed using dual luciferase reporter gene assays. Consistent with this notion, a significant negative correlation between these miRNAs and HOTTIP exists in HCC tissue specimens. Interestingly, the dysregulation of the three ncRNAswas associated with overall survival of HCC patients. In addition, the posttranscriptional silencing of HOTTIP by miR-192, miR-204 or HOTTIP siRNAs could significantly suppress viability of HCC cells. On the contrary, antagonizing endogenous miR-192 or miR-204 led to increased HOTTIP expression and stimulated cell proliferation. In vivomouse xenograft model also support the tumor suppressor role of both miRNAs. Besides the known targets (multiple 5’ end HOX A genes, i.e.HOXA13), glutaminase (GLS1) was identified as a potential downstream target of the miR-192/-204-HOTTIP axis in HCC. Considering glutaminolysis as a crucial hallmark of cancer cells and significantly inhibited cell viability after silencingGLS1, we speculate that the miR-192/-204-HOTTIP axis may interrupt HCC glutaminolysis through GLS1 inhibition. These results elucidate that the miR-192/-204-HOTTIP axis might be an important molecular pathway during hepatic cell tumorigenesis. Our data in clinical HCC samples highlight miR-192, miR204 and HOTTIP with prognostic and potentially therapeutic implications. PLOS Genetics | DOI:10.1371/journal.pgen.1005726 December 28, 2015 1 / 19 OPEN ACCESS Citation: Ge Y, Yan X, Jin Y, Yang X, Yu X, Zhou L, et al. (2015) fMiRNA-192 and miRNA-204 Directly Suppress lncRNA HOTTIP and Interrupt GLS1Mediated Glutaminolysis in Hepatocellular Carcinoma. PLoS Genet 11(12): e1005726. doi:10.1371/journal.pgen.1005726 Editor: Luigi Terracciano, University of Basel, Università del Molise, SWITZERLAND Received: May 19, 2015 Accepted: November 13, 2015 Published: December 28, 2015 Copyright: © 2015 Ge et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All microarray files are available from the National Center for Biotechnology Institute Gene Expression Omnibus (GEO) repository database (accession number GSE60912). All other relevant data are within the paper and its Supporting Information files. Funding: The study was supported by grants from the National High-Tech Research and Development Program of China (2015AA020950); National Natural Science Foundation of China (31271382, 91229126 and 81201586); the Fundamental Research Funds for the Central Universities (YS1407); the open