The Role Lnc‐RHL in Hepatic Cell Lineage Decisions and Proliferation in HepaRG Cells

Long noncoding RNAs (lncRNAs) are functional transcripts with lengths of over 200 nucleotides that do not encode proteins. LncRNAs have received wide attention as key regulators of stem cell proliferation and differentiation, but knowledge of the specific role of lncRNAs in hepatocyte differentiation is still limited. Recently, our group identified and characterized a novel lncRNA, lnc‐RHL (regulator of hepatic lineage), which plays a critical role in the differentiation of bipotent hepatoblasts to hepatocytes and cholangiocytes. Lnc‐RHL maps to human chromosome 11q23.3 in an apolipoprotein (APO) gene cluster and consists of a 670‐base pair polyadenylated lncRNA with two exons and an intron. We knocked down lnc‐RHL expression with a doxycycline (Dox) inducible shRNA lentiviral vector in HepaRG cells to investigate the role of lnc‐RHL. Interestingly, knockdown of lnc‐RHL inhibited differentiation to hepatocytes but not to cholangiocytes in HepaRG cells. We observed a significant reduction in the number and size of hepatocyte colonies upon Dox induction. Moreover, knockdown of lnc‐RHL downregulated the mRNA levels of hepatocyte markers such as HNF4a and albumin, and many APO genes including APOA1, APOC3, and APOA5 but upregulated mRNA levels of the cholangiocyte marker, cytokeratin 7 (CK7). We also performed RNA‐sequencing analysis and ingenuity pathway analysis (IPA) to further investigate functions of lnc‐RHL and the top impacted canonical pathways due to loss of lncRHL during HepaRG cell differentiation. IPA results showed that several cell cycle‐ and proliferation‐associated canonical pathways were significantly regulated. In addition, a key proliferation‐associated transcription factor, FOXM1 was markedly upregulated after the loss of lnc‐RHL. In summary, this study explores the role and expression of lnc‐RHL in HepaRG cell differentiation and shows that it is required for the proper production of hepatocytes in these cells.