Circular RNAs in Cholestatic Liver Diseases

Primary sclerosing cholangitis (PSC) is a 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. A large fraction ( about 98%) of the transcribed mammalian genome is noncoding RNAs

(ncRNAs), including long noncoding RNAs (lncRNAs), circular RNA (circRNAs), and microRNA (miRNAs). However, the biological functions of ncRNAs remain largely unknown. As the first identified and well- characterized lncRNA, aberrant expression of lncRNA H19 has been associated with numerous human diseases,

including liver diseases. CircRNAs are a large emerging class of ncRNAs that are important regulators of various physiological and pathological processes. Recent advances in high-throughput RNA sequencing 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 and is the focus of this new application. We reported that aberrant expression of H19 is associated with the severity of cholestatic liver injury in Mdr2-/- mice. Our new preliminary data showed that 1) Key genes

involved in inflammation, fibrosis, and senescence, as well as sphingolipid metabolism, were upregulated in both bile duct ligation (BDL)-mice and Mdr2-/- mice; 2) The number of cholangiocytes, pro-inflammatory macrophages and activated stellate cells were significantly increased, while the number of Kupffer cells (KCs) was significantly

reduced in Mdr2-/- mice; 3) Deletion of H19 reduced cholestatic liver injury in both BDL and Mdr2-/- mice; 4) The deletion of RNA binding protein HuR (human antigen R) upregulated H1926. 5) 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) and mmu_circRNA_19966 (circAff3) are among the most significantly regulated circRNAs by H19; 7) Sequence analysis suggested that circRNA-Edil3 and circAff3 may serve as a "sponge" for miRNA-215-3p, miRNA-129-5p, miRNA-212-5p, and miRNA-185-3p; 5)

Previous studies have shown that these miRNAs inhibit HSC activation and suppress fibrogenic signaling pathways by targeting high mobility group box protein 1 (HMGB1), Edil3 and Aff3. Based on our published results and a large amount of exciting new preliminary data, we HYPOTHESIZE that H19-induced upregulation of

circRNAs plays a critical role in cholestatic liver fibrosis by sequestering miRNAs and HuR. Two specific aims are proposed to test our hypothesis. Aim 1 is to examine the role of H19-induced upregulation of circRNAs (circEdil3 & circAff3) in regulating cholangiocyte proliferation, senescence and hepatic

inflammation. Aim 2 is to identify the mechanisms by which H19-induced circRNAs promote cholestatic liver injury. Completion of our proposed studies will elucidate the potential cellular/molecular mechanisms involved in the progression of cholestatic liver diseases and identify novel diagnostic and therapeutic targets.