NRSA for Allen Yu: Probing the Role of Highly Expressed IncRNA in Hepatocellular Carcinoma

ABSTRACT Hepatocellular carcinoma (HCC), the most common type of liver malignancy, is one of the most lethal forms of cancer. HCC is not diagnosed until late stages and has a poor 5-year survival rate of less than 14%. Excluding liver transplantation, the current standard of care for HCC is treatment with sorafenib, a multi-kinase inhibitor that targets Raf, receptor tyrosine kinases, and platelet-derived growth factor receptor, which extends median survival time from 7.9 months to 10.7 months. This modest gain emphasizes the urgent need to identify new and effective therapeutic targets for HCC. Genome wide analyses such as the ENCODE (ENCyclopedia Of DNA Elements) project have revealed that most of the genome is transcribed, even though less than 2% of the genome encodes for proteins. Thousands of transcripts greater than 200 nucleotides in length, called long non- coding RNAs (lncRNAs), are expressed in a tissue-specific manner and undergo changes in expression level during cellular differentiation and in cancers. LncRNAs have been implicated in numerous molecular functions including modulating transcriptional patterns, regulating protein activities, serving structural or organizational roles, altering RNA processing events, and serving as precursors to small RNAs. One lncRNA (lnc05) is of particular interest in that it is enriched in mouse hepatocellular carcinoma cell lines and it is conserved in human. In preliminary studies, I have found that lnc05 is up-regulated in HCC cells compared to normal mouse hepatocytes as well as human HCC and cirrhotic liver tissue samples. Knock-out of lnc05 in HepA1-6 cells increases the doubling time by 23%, and modulates the fraction of cells in S and G2 phases. Moreover, knock- down of lnc05 expression using anti-sense oligonucleotides (ASOs) decreases proliferation, as assessed by expression levels of Ki67, a proliferation marker, and colony formation assays. The objective of the proposed studies is to characterize lnc05 and its potential role in HCC. I propose to conduct a thorough molecular characterization of this novel lncRNA including determining its size, isoform diversity, and sub-cellular localization, as well as perform loss-of and gain-of-function studies using approaches such as knock-out (CRISPR/Cas9), knock-down using ASOs, and overexpression of the transcript. In addition, identification of interacting partners by pull-down and mass spectrometry will expand our understanding of its function. I will also employ mouse models of HCC that closely mimic the biogenesis and genetics of human HCC to determine the in vivo impact of this lncRNA on proliferation and growth. Finally, I will test our ASOs on human HCC cell lines to determine the impact of lnc05 depletion on growth. Together, these studies will result in comprehensive insights into the function of lnc05, as well as test the effect of knocking down lnc05 using ASOs in the context of mouse models of HCC and human tumor cells.