Long Noncoding RNA H19: A Key Player in Liver Diseases

Supported by a VA Merit Award (I01BX004033), a Research Career Scientist Award (IK6BX004477); VA ShEEP grants (1IS1 BX004777‐01 and 1IS1BX005517‐01), and the National Institutes of Health (R01 DK104893, R01DK‐057543, and 1R21 AA026629‐01). Potential conflict of interest: Nothing to report. The completion of human genome sequencing in the early 2000s identified that most of the human genome (~93%) is transcribed but that <2% of transcripts encode proteins. Most of the transcripts represent noncoding RNAs (ncRNAs). Long noncoding RNAs (lncRNAs) are ncRNAs with >200 nucleotides. LncRNAs are further classified into sense, antisense, bidirectional, intronic, and intergenic lncRNAs based on their topographic relation to the nearest protein‐coding gene.(1) A recent advance in next‐generation sequencing has identified thousands of lncRNA loci, and the number of lncRNAs which are linked to human diseases, including liver disease, is rapidly growing.(2) However, compared to protein‐coding genes and microRNAs (small ncRNAs with 20‐24 nucleotides), lncRNAs are poorly characterized, and their biological functions remain largely unknown. LncRNA H19 was the first lncRNA and the first imprinted gene identified in eukaryotes as a hepatic, fetally specific, nontranslatable mRNA in the late 1980s.(3) Its role in embryogenesis has been well characterized. The biological function of H19 as an RNA molecule remained a mystery until the identification of another lncRNA X‐inactive‐specific transcript in the early 1990s.(4) During the last three decades, H19 has been well characterized as a multitasking lncRNA. The aberrant expression of H19 has been linked to various human cancers, including gastric, liver, and pancreatic cancers.(5,6) Recent studies also reported that H19 is involved in chronic liver diseases such as nonalcoholic fatty liver disease (NAFLD) and cholestatic liver disease, which are major global health issues. The high mortality and morbidity associated with hepatocellular carcinoma (HCC) and cholangiocarcinoma, the end stages of most chronic liver diseases, have imposed huge financial burdens on individuals and health care systems.(7) Therefore, there is an unmet need to identify diagnostic biomarkers and therapeutic targets for chronic liver diseases. In this concise review, we discuss the current understanding of H19 in the pathogenesis of chronic liver diseases. Expression and Regulation of H19 and Functional Mechanisms The role of lncRNA H19 in the regulation of liver development has been well documented.(8) Genetic and molecular studies have shown that H19 is a parentally imprinted and maternally expressed gene, which is localized to chromosome 7 in mice and chromosome 11p15.5 in humans, downstream of another maternally imprinted and paternally expressed protein‐coding gene, insulin‐like growth factor 2 (Igf2).(9) The H19 gene contains five exons and four small introns and encodes an ~2.3‐kb fully capped, spliced, and polyadenylated transcript.(8) The expression of H19 is controlled by a promoter and an imprinting control region (ICR), also called a "differentially methylated domain" or a "differentially methylated region." H19 and IGF2 are expressed in the same tissues, and their reciprocal expression is controlled by the zinc‐finger protein CCCTC binding factor (CTCF), which binds to unmethylated maternal ICR and prevents the activation of Igf2 by downstream enhancers. It also has been reported that H19 is the precursor of microRNA 675 (miRNA675), which is embedded in exon 1 of H19. The excision process of miRNA675 from H19 is regulated by the RNA‐binding protein (RBP) human antigen R (HuR) (Fig. 1).(10) H19 is highly expressed during fetal development and down‐regulated after birth, except in skeletal muscle. Aberrant expression of H19 has been linked to various human diseases, especially tumorigenesis.(11) It also has been reported that H19 expression is up‐regulated by estrogen, c‐Myc, hypoxia, and oxidative stress.(12‐15) The hepatic H19 expression level is very low under normal physiological conditions, but it can be up‐regulated under pathological conditions.(16) The relative expression levels of H19 in different types of hepatic cells depend on the pathophysiological conditions. Despite the discrepancy found in previous studies, there is consensus that H19 impacts various hepatic cells as a multitasking regulator of gene expression. The mechanisms by which H19 regulates cellular functions include epigenetic regulation, sponge of miRs, production of miR‐675, and regulation of target gene expression through binding to RBPs.(17)FIG. 1: Regulation of H19 and Igf2 expression. (A) H19 is expressed from the maternal allele. The binding of CTCF to the unmethylated ICR prevents the downstream enhancer from interacting with the promoter region of Igf2 but allows the enhancer to interact with the H19 promoter. The H19 transcript contains five exons. In exon 1, there is the coding region for miR675. The production of pre‐miR675 is inhibited by RBP HuR. (B) In the parental allele, the ICR is methylated, which prevents the binding of CTCF and allows the enhancer to interact with the promoter region of Igf2. Abbreviation: DMR, DNA methylation region.LncRNA H19 in NAFLD The liver is the most important metabolic organ and plays many vital life functions. The incidence of NAFLD has rapidly increased over the last two decades due to the global pandemic of obesity. NAFLD can progress to nonalcoholic steatohepatitis (NASH) and is the second most common indication for liver transplantation and a major cause of HCC. Due to the complexity of disease pathology, no reliable diagnostic biomarkers and regulatory agency–approved drugs are available.(18) There is compelling evidence supporting the "multihit" over the "two‐hit" hypothesis of NAFLD pathogenesis.(19) NAFLD is not a single‐organ disease. The progression from simple steatosis to NASH is correlated with systemic and adipose tissue inflammation, dysbiosis, and disruption of gut barrier function. It has been reported that overexpression of H19 disrupts the intestinal barrier function through miR675.(20) A recent study further showed that H19 inhibited the function of Paneth and goblet cells by suppressing autophagy.(21) The role of H19 in NAFLD remained unexplored until the identification of its aberrant expression in the livers of patients with NASH.(22) By using H19−/− and adeno‐associated virus 8–mediated overexpression of H19 mouse models, Liu et al. reported that H19 promoted lipogenesis by facilitating polypyrimidine tract‐binding protein 1 (PTBP1), an RBP, to stabilize sterol regulatory element‐binding protein 1c mRNA and increase protein cleavage and nuclear translocation.(23) Recently, two groups reported that H19 expression is induced by free fatty acids in hepatocytes and high‐fat diet feeding in vivo.(24,25) Mechanistically, H19 promotes hepatic lipogenesis by down‐regulating miR130a, an inhibitor of peroxisome proliferator–activated receptor γ, or up‐regulating the transcription factor MLX‐interacting protein‐like (MLXIPL, also called "carbohydrate‐responsive element‐binding protein") and the phosphoinositide 3‐kinase (PI3K)/mammalian target of rapamycin (mTOR) pathways.(24,25) These studies identified H19 as an essential player in diet‐induced hepatic steatosis (Fig. 2). However, it remains unclear whether and how H19 is involved in NAFLD/NASH disease progression.FIG. 2: Potential mechanisms of H19‐induced hepatic lipid accumulation. High‐fat diet and free fatty acids induce up‐regulation of H19 in hepatocytes. (A) H19 inhibits miR130a expression, an inhibitor of peroxisome proliferator–activated receptor Ɣ, and results in activation of peroxisome proliferator–activated receptor Ɣ and hepatic lipogenesis. (B) H19 facilitates the RBP PTBP1 to stabilize the Srebp1c mRNA; promotes SREBP1c protein cleavage and nuclear translocation of the activated nuclear form, nSREBP1c; and results in the increase of transcription of lipogenic genes. (C) H19 induces activation of the PI3K/mTOR pathway and up‐regulates the lipogenic transcription factor MLXIPL, resulting in increased lipid accumulation. Abbreviations: Acc1, acetyl‐CoA carboxylase 1; Fasn, fatty acid synthase; FFA, free fatty acid; HFD, high‐fat diet; nSREBP1, nuclear form of SREBP1c; PPARƔ, peroxisome proliferator–activated receptor Ɣ; Scd1, stearoyl‐CoA desaturase 1; Srebp1c, sterol regulatory element‐binding protein 1c.LncRNA H19 in Cholestatic Liver Disease Cholestatic liver diseases, such as primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) in adults and biliary atresia (BA) and Alagille syndrome in children, are a significant cause of morbidity and mortality and liver transplant. "Cholestasis" is defined as the impairment of bile flow due to disruption of bile acid formation or excretion or obstruction of bile ducts.(26) Bile acids are exclusively formed in hepatocytes and play critical roles in nutrient absorption through intrahepatic circulation. More importantly, bile acids function as signaling molecules in regulating lipid and glucose metabolism.(27) Disruption of intrahepatic bile acid circulation or accumulation of bile acids in the liver can cause hepatocyte injury, cholangiocyte proliferation, ductal reaction, activation of hepatic stellate cells (HSCs), and inflammation. Although cholestatic liver diseases are relatively rare compared to NAFLD, the incidence and prevalence are increasing. The available therapeutic agents are limited to ursodeoxycholic acid and obeticholic acid for PBC, which are largely nonspecific and often ineffective. There is an urgent need to identify diagnostic biomarkers and therapeutic targets for cholestatic liver diseases. LncRNAs are increasingly recognized as promising potential therapeutic targets for cholestatic liver diseases.(28) Zhang et al. identified H19 as a key player in bile duct ligation (BDL)–induced cholestatic liver injury.(22) Several studies have reported up‐regulation of H19 in different hepatic cells, cholestatic mouse models, and human patients with PSC, PBC, and BA.(29‐39) H19 not only functions as a sponge of miRNAs but also activates different signaling pathways involved in the activation of macrophages, cholangiocytes, and HSCs. Table 1 summarizes the key findings of the most recent studies which assessed the roles of H19 in different hepatic cells and cholestatic animal models. Table 1 - Potential targets of lncRNA H19 in cholestatic liver fibrosis Animal Models or Human Samples In Vitro Models Targets Effects Reference or Year C57BL/6 and H19−/− mice, HepG2, Huh7, Hep3B, H69, Mz‐Cha‐1, CCLP‐1, HuCCT1, SG231; mouse Hepa1, MLC, and MSC ZEB1 BDL‐induced H19 suppressed ZEB1 expression, which resulted in the derepression of EpCAM by ZEB1 and cholestatic liver fibrosis 32 C57BL/6 and H19−/− mice, EpCAM 2017 2‐week BDL SOX9 C57 male mice Primary mouse hepatocytes, AML12 cell line Sox9 Sox9‐mediated up‐regulation of H19 is responsible for CCl4‐induced liver fibrosis 5 3‐week and 4‐week CCl4 2017 Sprague‐Dawley male rats, 12‐week CCl4 model HSC‐T6 cell line DNMT1 DNMT1‐mediated epigenetic regulation of H19 and H19‐mediated activation; ERK1/2 promoted HSC activation and liver fibrosis 38 ERK 2018 Mdr2−/−, H19−/− (Δ exon1/+) Primary mouse hepatocytes, cholangiocytes, and Kupffer cells FXR/SHP Bile acid/estrogen‐induced H19 expression in cholangiocytes is responsible for gender disparity of cholestatic liver injury in Mdr2−/− mice by down‐regulation of SHP and activation of S1PR2 and ERK1/2 signaling pathways 16, 29 PSC liver samples (n = 16), MLC cell line S1PR2/ERK1/2 2017, 2018 8‐week CCL4 mouse model C57/BL6 male mice Primary mouse HSCs, human LX2 and L02 cell lines miR148a, USP4 H19 promoted hepatic fibrosis by activating HSCs through sponging miR148a and up‐regulating USP4, which enhanced the TGF‐β‐mediated activation of SMAD in HSCs 37 8‐week CCl4 mouse model TGF‐β/SMAD 2018 C57/BL6 Mdr2−/−, H19−/− Primary mouse hepatocytes, HSCs, cholangiocytes, and Kupffer cells Cyclin D1/p21 Cholangiocyte‐derived exosomal H19 is preferentially taken up by HSCs, and Kupffer cells significantly promoted the activation of HSCs and macrophages in liver fibrotic progression 30, 31 (Δ Exon1/+) and DKO mice; MLC, H69, and LX2 cell lines CCL2/CCR2 2019, 2020 2‐week BDL model C57/BL6 male mice BDL for 2, 4, and 6 weeks JS‐1 murine HSC cell line PI3K/AKT/mTOR H19 promoted autophagy by interacting with the PI3K/AKT/mTOR pathway, which was responsible for IGFBPrP1‐induced activation of HSC and liver fibrosis 39 2019 Human BA patient liver samples (n = 57) MLC cell line S1PR2 H19 expression level is correlated to disease severity in patients with BA; H19 promotes cholangiocyte proliferation and fibrotic liver injury by regulating S1PR2 and let‐7/HMGA2–mediated pathways 35 Mdr2−/−, H19−/− and DKO HUCCT1 Let‐7/HMGA2 2019 2‐week BDL model ICR male mice Primary mouse HSCs and human LX2 cell lines ADH3/ALDH1 H19 induced HSC activation by up‐regulation of ADH3/ALDH1 and retinoic acid signaling pathways and by activation of AMPKα through facilitating the formation of AMPKα/LKB1 complex 33,34 8‐week CCl4 mouse model RARα/RXRβ 2020 AMPKα/LKB1 C57/BL6j H19−/− male mice Human Huh7, mouse Hepa1, MSC, and MLC cell lines PTBP1 and Let‐7 H19 suppressed the expression of PTBP1, which inhibited the biogenesis of Let‐7 but enhanced the bioavailability to their targets 36 1‐week BDL model 2020 Abbreviations: ADH3, alcohol dehydrogenase 3; ALDH1, aldehyde dehydrogenase 1; AMPK, AMP‐activated protein kinase; CCL2, chemokine (C‐C motif) ligand 2; CCR2, chemokine (C‐C motif) receptor 2; DKO, double knockout; DNMT1, DNA (cytosine 5)‐methyltransferase 1; EpCAM, epithelial cell adhesion molecule; ERK, extracellular signal–regulated kinase; FXR, farnesoid X receptor; HMGA2, high‐mobility group AT‐hook 2; IGFBPrP1, IGF binding protein receptor protein 1; Let‐7, lethal 7; LKB1, liver kinase B1; Mdr2, multidrug resistance protein 2; RAR, retinoid A receptor; RXR, retinoid X receptor; SMAD, mothers against decapentaplegic; SOX9, SRY (sex determining region Y) box 9; S1PR2, sphingosine‐1‐phosphate receptor 2; USP4, ubiquitin‐specific protease 4; ZEB1, zinc finger E‐box‐binding homeobox 1. LncRNA H19 in HCC HCC is the most common primary liver cancer and is often diagnosed at late stages due to the lack of diagnostic and prognostic biomarkers. Liver transplantation remains the only therapeutic option. Aberrant up‐regulation of H19 in tumorigenesis has been well established in different types of human cancers.(11) However, the role of H19 in HCC is more complicated and remains controversial. Most studies with human HCC samples were limited by small sample size and variations in patient populations and tissue sampling. The major findings in the past three decades related to H19 in HCC, and the potential reasons for the conflicting results are summarized and discussed in an excellent recent review.(40) Studies with cultured HCC cell lines, in vivo animal models, and human HCC samples indicate that H19 can be an oncogene or tumor suppressor by regulating different miRNAs, RBPs, and diverse signaling pathways.(41‐46) The most recent studies related to H19 in HCC are summarized in Table 2. The functions of lncRNAs are linked to their intracellular localization. More mechanistic and comprehensive studies are needed to define the role of H19 in the progression of HCC. Table 2 - Potential targets of lncRNA H19 in HCC Animal Models or Human Samples In Vitro Models Targets Potential Mechanisms Reference or Year C57/BL6J mice transplanted with TICs from DEN‐treated Tgfbr2fl/fl mice by splenic injection followed by i.p. injection of CCl4 for 3 weeks and tail vein injection of Ad‐Cre. TICs isolated from B6.129S6‐Tgfbr2fl/fl mice (male, 14‐day‐old) injected with DEN (25 mg/kg) TGFβ/ Tgfbr2‐Sox2 TGFβ regulated H19 expression through suppression of SOX2; inactivation of Tgfbr2 in TICs simultaneously increased SOX2 and H19 levels, which are responsible for HCC development and progression 43, 2019 Human HBV patient liver tissues and matched normal tissue (n = 20) Human L02 cell line miR675/PPARα HBVx protein–induced H19/miRNA675 is responsible for HBV‐associated hepatitis and liver injury by activating PPARα and Akt/mTOR signaling pathways 42, 2019 Akt/mTOR 16‐month‐old female and 17.7‐month‐old male C57/BL6 Mdr2−/−and Mdr2−/−/H19−/− DKO mice Primary cells from mouse nontumor liver tissues H19 is pro‐oncogenic in inflammation‐mediated HCC Single‐cell transcriptome analysis of nontumor tissue of an 18‐month‐old female Mdr2−/− mouse indicated that H19 was mainly expressed in hepatocytes, endothelial cells, and macrophages; 41, 2020 Human HCC liver tissues (n = 242) with matched nontumor tissues (n = 298) H19 expression level in HCC tumor inversely correlated to the patient's survival Human HCC liver tissues Huh7, Hep3B, SNU‐449, and SNU‐387 cell lines miR675 High H19 expression is negatively related to sorafenib sensitivity by up‐regulation of miR675 in HCC cells 45, 2020 Human HCC liver tissues and matched noncancerous liver tissues (n = 55) HepG2 cell line G3BP1 NSUN2‐mediated m5C‐modified H19 promotes HCC by recruiting G3BP1 oncoprotein, which leads to MYC accumulation 44, 2020 Myc Human HCC liver tissues (n = 64) and TCGA cohort (n = 393) Hep‐G2, Hep2B2, THP‐1, SK‐OV‐3, and NCI‐H520 miRNA‐193b TAM‐derived H19 promotes tumor cell migration and invasion and immune cell infiltration by hijacking miR‐193b as a sponge and activating MAPK1 46, 2020 MAPK1 axis Abbreviations: Ad‐Cre, adenoviral cyclization recombination; DEN, diethylnitrosamine; DKO, double knockout; G3BP1, Ras GTPase‐activating protein‐binding protein 1; MAPK1, mitogen‐activated protein kinase 1; Mdr2, multidrug resistance protein 2; NSUN2, NOP2/Sun domain family member 2; PPARα, peroxisome proliferator–activated receptor alpha; SOX2, SRY (sex determining region Y) box 2; TAM, tumor‐associated macrophage; TCGA, The Cancer Genome Atlas; Tgfbr2, TGF‐β receptor 2; TIC, tumor‐initiating cell. Conclusion and Future Perspectives LncRNA H19 has gained increasing attention due to its broad spectrum of physiological and pathological functions. The ease of detection of lncRNAs in serum and other bodily fluids makes them attractive biomarkers. Although H19 was discovered more than three decades ago, its potential roles in liver diseases remain largely obscure. Recent studies have solidified the idea that H19 represents a diagnostic and prognostic biomarker for various liver diseases. The fundamental role of H19 in promoting hepatic lipogenesis, inflammation, and epithelial–mesenchymal transition makes this lncRNA a promising therapeutic target in liver diseases. Recent advances in technologies for gene profiling and editing, as well as nanotechnology for RNA delivery, have rapidly moved the lncRNA research field forward. Considering the complexity of various liver diseases, it is essential to understand the comprehensive and systemic effects of H19 in different physiological and pathological settings. More mechanistic and translational studies with tissue‐specific and cell type–specific H19−/− animal models are needed in order to validate H19 as a therapeutic target for liver diseases. Author Contributions Y.W. and H.Z. were responsible for the original idea and writing. P.B.H. was responsible for manuscript review.