LncRNA H19 in Cholestatic Liver Diseases

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Primary sclerosing cholangitis (PSC) is the major cholestatic liver disease with high morbidity and mortality. Inflammation and fibrotic injury of the bile ducts due to impairment of bile formation or flow represent the major characteristics of PSC. The majority of the mammalian genome are non-protein-coding sequences. Less than 2% of the human genome is made up of protein-coding genes. A large fraction of the transcribed mammalian genome is noncoding RNAs (ncRNAs), including long noncoding RNAs (lncRNAs), circular RNA (circRNAs), and microRNA (miRNAs), but only a few of them have been structurally annotated. The biological functions of ncRNAs remain largely unknown. Recent advances in high-throughput RNA sequencing (RNAseq) and circRNA- specific bioinformatics algorithms have identified thousands of circRNAs with tissue-specific expression patterns. However, the relevance and function of circRNAs in disease, especially in liver diseases, remain to be determined. Our previous studies reported that the conjugated primary bile acid (CBA), taurocholic acid (TCA), activated sphingosine-1phosphate receptor 2 (S1PR2) and induced H19 expression in cholangiocytes. TCA- induced upregulation of H19 is a major promoter of cholestatic liver injury by activating inflammatory macrophages and hepatic stellate cells (HSCs). Our new preliminary data using Arraystar circRNAseq identified 33 overlapping circRNAs, which were significantly upregulated in Mdr2-/- mice and downregulated in Mdr2-/-/H19- /- mice. Among them, mmu_circRNA_26644 (circRNA-Edil3), which is encoded by epidermal growth factor-like repeats and discoidin I-like domains 3 (Edil3) gene, is the most significantly downregulated circRNA in Mdr2-/- /H19-/- mice. Sequence analysis suggested that circRNA-Edil3 may serve as a "sponge" for miRNA-215-3p, miRNA-129-5p and miRNA-3075-3p. TargetScan analysis suggested that High Mobility Group Box Protein 1 (HMGB1), AF4/FMR2 Family Member 3 (Aff3) and Edil3 are potential targets of miRNA-215-3p, miRNA-129-5p and miRNA-3075. Based on our published results and exciting new preliminary data, we HYPOTHESIZE that H19-induced upregulation of circRNA-Edil3 plays a critical role in cholestatic liver injury by sequestering miRNAs. Two specific aims are proposed to test our hypothesis. Aim 1. To examine the role of H19-induced circRNA-Edil3 in regulating cholangiocyte proliferation, senescence and hepatic inflammation using cholestatic liver injury mouse models and human PSC patient liver and serum samples. Aim 2: To identify the mechanisms by which H19-induced circRNA-Edil3 promotes cholestatic liver injury. We will use multiple newly-established approaches and animal models to test our hypothesis. Completion of these specific aims should elucidate the potential cellular/molecular mechanisms involved in lncRNA H19-mediated progression of cholestatic liver diseases and identify novel diagnostic and therapeutic targets. Since the effects of cholestasis are profound and widespread, leading to worsening liver diseases and systemic illness, the subject matter of this proposal is timely, important, and has direct clinical application.