Despite progress in diagnostics and treatment of hepatocellular carcinoma (HCC), its prognosis remains poor. In this study, we globally assessed long non-coding RNAs (lncRNA) for contributions to HCC using publically available microarray data, in vitro and in vivo assays. Here we report that ZFAS1, encoding a lncRNA that is frequently amplified in HCC, is associated with intrahepatic and extrahepatic metastasis and poor prognosis of HCC. ZFAS1 functions as an oncogene in HCC progression by binding miR-150 and abrogating its tumor suppressive function in this setting. miR-150 repressed HCC cell invasion by inhibiting ZEB1 and the matrix metalloproteinases MMP14 and MMP16. Conversely, ZFAS1 activated ZEB1, MMP14 and MMP16 expression, inhibiting these effects of miR-150. Our results establish a function for ZFAS1 in metastatic progression and suggest its candidacy as a new prognostic biomarker and target for clinical management of HCC.

microRNAs (miRNA) are well suited to the task of regulating gene expression networks, because any given miRNA has the capacity to target dozens, if not hundreds, of genes. The simultaneous targeting of multiple genes within a pathway may enable miRNAs to more strongly regulate the pathway, or to achieve more subtle control through the targeting of distinct subnetworks of genes. Therefore, as our capacity to discover miRNA targets en masse increases, so must our consideration of the complex networks in which these genes participate. We highlight recent studies in which the comprehensive identification of targets has been used to elucidate miRNA-regulated gene networks in cancer, focusing especially upon miRNAs such as members of the miR-200 family that regulate epithelial–mesenchymal transition (EMT), a reversible phenotypic switch whereby epithelial cells take on the more invasive properties of their mesenchymal counterparts. These studies have expanded our understanding of the roles of miRNAs in EMT, which were already known to form important regulatory loops with key transcription factors to regulate the epithelial or mesenchymal properties of cells. Cancer Res; 75(13); 1–6. ©2015 AACR.

Analysis of clinical trial specimens such as formalin-fixed paraffin-embedded (FFPE) tissue for molecular mechanisms of disease progression or drug response is often challenging and limited to a few markers at a time. This has led to the increasing importance of highly multiplexed assays that enable profiling of many biomarkers within a single assay. Methods for gene expression analysis have undergone major advances in biomedical research, but obtaining a robust dataset from low-quality RNA samples, such as those isolated from FFPE tissue, remains a challenge. Here, we provide a detailed evaluation of the NanoString Technologies nCounter platform, which provides a direct digital readout of up to 800 mRNA targets simultaneously. We tested this system by examining a broad set of human clinical tissues for a range of technical variables, including sensitivity and limit of detection to varying RNA quantity and quality, reagent performance over time, variability between instruments, the impact of the number of fields of view sampled, and differences between probe sequence locations and overlapping genes across CodeSets. This study demonstrates that Nanostring offers several key advantages, including sensitivity, reproducibility, technical robustness, and utility for clinical application. Cancer Res; 75(13); 1–7. ©2015 AACR.

microRNA (miRNA) dysregulation is involved in the development and progression of various human cancers, including hepatocellular carcinoma (HCC). However, how to identify the miRNAs targeting specific mRNA in cells is a significant challenge because of the interaction complexity and the limited knowledge of rules governing these processes. Some miRNAs are not predictable by current computer algorithms available. Here, using p21 mRNA as target, we established a new method, called miRNA in vivo precipitation (miRIP), to identify which kind of miRNAs can actually bind to the specific mRNA in cells. Several unpredictable miRNAs that bound p21 mRNA in HepG2 and PC-3 cells were identified by the miRIP method. Among these miRNAs identified by miRIP, miR-92a was found and confirmed to interact robustly with p21 mRNA, both in HepG2 and PC-3 cells. miR-92a was found to be remarkably increased in HCC tissues, and higher expression of miR-92a significantly correlated with lower expression of p21, which is related to poor survival of HCC patients. Moreover, inhibition of miR-92a could significantly suppress HCC growth in vitro and in vivo by upregulating p21. Together, miR-92a, which is identified by miRIP, is functionally shown to be associated with HCC growth as an oncogenic miRNA by inhibiting expression of targeting gene p21. In addition, several unpredictable miRNAs that target STAT3 mRNA were also identified by the miRIP method in HepG2 cells. Our results demonstrated that the miRIP approach can effectively identify the unpredictable but intracellular existing miRNAs that target specific mRNA in vivo. Cancer Res; 75(14); 1–11. ©2015 AACR.

microRNAs (miRNAs) dysregulation is involved in the development and progression of various human cancers, including hepatocellular carcinoma (HCC). However, how to identify the miRNAs targeting specific mRNA in cells is a significant challenge because of the interaction complexity and the limited knowledge of rules governing these processes. Some miRNAs are not predictable by current computer algorithms available. Here, using p21 mRNA as target, we established a new method, named as miRNA in vivo precipitation (miRIP), to identify which kind of miRNAs can actually bind to the specific mRNA in cells. Several unpredictable miRNAs which bound p21 mRNA in HepG2 and PC-3 cells were identified by miRIP method. Among these miRNAs identified by miRIP, miR-92a was found and confirmed to interact robustly with p21 mRNA both in HepG2 and PC-3 cells. miR-92a was found to be remarkably increased in HCC tissues, and higher expression of miR-92a significantly correlated with lower expression of p21 which is related with poor survival of HCC patients. Moreover, inhibition of miR-92a could significantly suppress HCC growth in vitro and in vivo by up-regulating p21. Together, miR-92a, which is identified by miRIP, is functionally shown to be associated with HCC growth as an oncogenic miRNA by inhibiting expression of targeting gene p21. In addition, several unpredictable miRNAs which target STAT3 mRNA were also identified by miRIP method in HepG2 cells. Our results demonstrated that miRIP approach can effectively identify the unpredictable but intracellular existing miRNAs which target specific mRNA in vivo.