LncRNA NEAT1: Shedding light on mechanisms and opportunities in liver diseases

With advances in genome and transcriptome research technology, the function and mechanism of lncRNAs in physiological and pathological states have been gradually revealed. Nuclear Enriched Abundant Transcript 1 (NEAT1, a long non‐coding RNA), a vital component of paraspeckles, plays an indispensable role in the formation and integrity of paraspeckles. Throughout the research history, NEAT1 is mostly aberrantly upregulated in various cancers, and high expression of NEAT1 often contributes to poor prognosis of patients. Notably, the role and mechanism of NEAT1 in liver diseases have been increasingly reported. NEAT1 accelerates the progression of non‐alcoholic fatty liver disease (NAFLD), liver fibrosis and hepatocellular carcinoma, while exerting a protective role in the pathogenesis of acute‐on‐chronic liver failure by inhibiting the inflammatory response. In this review, we will elaborate on relevant studies on the different casting of NEAT1 in liver diseases, especially focusing on its regulatory mechanisms and new opportunities for alcoholic liver disease.

and function of nuclear bodies, whereas cytoplasmic lncRNAs play essential roles in modulating mRNA stability and translation, competing with endogenous RNAs and interfering with protein modification. [5][6][7] More importantly, lncRNAs have been reported to be impressive regulators in diverse aspects of biology, thus, playing critical biological functions in mammals, such as development and growth, immune responses and substance metabolism. [8][9][10] However, various emerging diseases show aberrant depletion, amplification or disruption of some crucial lncRNAs. 11 Accumulating evidence suggests that many lncRNAs are abnormally changed in liver diseases and might play an indispensable role as potential non-invasive therapeutic biomarkers. [12][13][14][15] Moreover, profound mechanistic studies have revealed that lncRNAs can regulate cellular metabolism, 16 inflammatory responses, 17 autophagy, 18,19 cell proliferation and apoptosis [20][21][22][23] in liver diseases by directly or indirectly modulating key protein-coding gene expression ( Figure 1). For example, lncRNA HULC (highly upregulated in liver cancer), MALAT1, H19, CASC9 (Cancer Susceptibility 9) and lncRNA EGFR contribute to the deterioration of hepatocellular carcinoma (HCC) by regulating lipid and glucose metabolisms, cell aggressiveness, autophagy and immune evasion, whereas lncRNA TSLNC8 ameliorates HCC progression by impairing the aggressiveness of cancer cells. 16,[24][25][26][27][28] Therein, lncRNA H19 is involved in several liver diseases, playing an important role in the regulation of hepatic lipid metabolism in non-alcoholic fatty liver disease (NAFLD) via interaction with polypyrimidine tract-binding protein 1 (PTPB1), 29 promoting the activation of hepatic stellate cells (HSCs) in cholestatic liver fibrosis via repressing ZEB1-mediated inhibition of EpCAM, 30 and aggravating cholestatic liver injury by inhibiting small heterodimer partner (SHP) expression in hepatocytes. 31 In addition, cholangiocyte-derived exosomal lncRNA H19 mediates vital cellcell communication between cholangiocytes with hepatocytes and HSCs. 31,32 Interestingly, LncRNA MEG3 plays promotive and suppressive roles in different liver diseases, which promote the apoptosis and steatosis of hepatocytes by targeting miR-let-7c-5p/NLRC5 in alcoholic liver disease (ALD), 33 disrupt the homeostasis of bile acids by facilitating SHP mRNA decay in cholestatic liver injury 34 while alleviate lipid over-deposition via regulation of the miR-21/LRP6 axis in NAFLD. 35 Importantly, serum lnc AK054921 and lnc AK128652 in patients can be viewed as potential biomarkers for predicting ALD progression. 36 However, some functions of lncRNA, such as H19 in HCC and GAS5

Key points
• NEAT1 is a structural lncRNA that is essential for the formation of paraspeckle.
• NEAT1 has two isoforms, NEAT1_1 and NEAT1_2, each plays an important role in hepatocellular carcinoma progression.
• Studies of NEAT1 in various liver diseases are summarized.
• NEAT1 may be a potential therapeutic biomarker for diagnosing and prognosing liver diseases.

F I G U R E 1
The known role and mechanism of lncRNA in diverse liver diseases, and representative lncRNAs are outlined. In different liver disease, lncRNAs regulate cell metabolism, inflammatory response, autophagy, cell proliferation and apoptosis by directly or indirectly modulating key protein-coding gene expression. ECM: extracellular matrix, EpCAM: epithelial cell adhesion molecule, NASH: non-alcoholic steatohepatitis. The upward arrow represents upregulation in liver disease, the downward arrow represents downregulation, the dotted arrow represents indeterminacy and the asterisk (*) represents the expression and function of GAS5 in liver fibrosis and H19 in HCC are controversial (growth arrest-specific transcript 5) in liver fibrosis, are controversial. In hepatocarcinogenesis, H19 was reported to be decreased in valuable models and exert tumour suppressor function, whereas large patient cohorts reported both up-and downregulation of H19 in HCC, which is probably resulted from a panel of studies using rather small sample sizes. [37][38][39] Studies have shown that GAS5 is downregulated in experimental liver fibrosis models and human cirrhosis, while another vital study indicated GAS5 is increased in plasma of liver fibrosis patients accompanying the progression of fibrosis degree except for that in cirrhotic livers. 22,40,41 These studies suggest that variations in some ln-cRNAs may be correlated with specific liver diseases, and large patient sample studies are more conducive to elucidating the role of lncRNA in certain diseases. However, the regulatory role of lncRNA nuclearenriched abundant transcript 1 (NEAT1) in diverse liver diseases is poorly understood.
In this study, we summarized the function and mechanism of NEAT1 in distinct liver diseases and proposed new potential regulatory opportunities for ALD.

| B I OG ENE S IS AND FE ATURE S OF N E AT1
NEAT1, a familial tumour syndrome multiple endocrine neoplasia type 1 locus (Chr 11q13.1) transcript, is a structural lncRNA that plays an essential role in the constitution and assembly of nuclear paraspeckles, a group of extremely dynamic nuclear subdomains. 11,42 A recent gene-wide analysis of lncRNA stability revealed that NEAT1 is highly unstable, but the instability does not limit the function of NEAT1.
Instead, the high turnover of NEAT1 may be beneficial to the extremely dynamic characteristics of paraspeckles. 43 NEAT1 has two variant isoforms, NEAT1_1 (3735bp) and NEAT1_2 (22741bp). The two isoforms share an identical promoter and 5′-end, and the different 3′-end make them two separate subtypes. As we know, paraspeckles are shaped in the form of a core-shell spheroidal structure, and the middle part of NEAT1_2 is organized to the core, which is encircled by NEAT1_1 and the 5′-and 3′-ends of NEAT1_2 44 ( Figure 2). Of note, NEAT1_2, and not NEAT1_1, is requisite for paraspeckle formation and potent for restoring the effect of NEAT1 knockdown. 45,46 Yamazaki et al proposed that NEAT1_2 has one modular domain structure, which plays an important role in the stabilization of NEAT1_2, switching of NEAT1 isoform and induction of paraspeckle assembly through phase separation. 46 Moreover, a recent genome editing study revealed that NEAT1_1 is not a major component of paraspeckles, and that the vast non-paraspeckle foci of NEAT1_1 is probably responsible for its paraspeckle-independent functions. 47 In brief, NEAT1 can act as a scaffold for paraspeckles, allowing paraspeckle proteins, including PSPC1, P54NRB/NONO and SFPQ/PSF, to form ordered, compact structures. It has been reported that MALAT1 localizes to nuclear speckles and NEAT1 overlaps with MALAT1 in many genomic sites. 48 A study by West et al revealed that NEAT1 and MALAT1 interact with proteins located in nuclear bodies and specifically target active chromatin sites, and they showed distinct binding patterns at these sites, indicating their independent but complementary functions. 49 The main features of NEAT1 are shown in Figure 2.
More importantly, progressive evidence suggests that NEAT1 not only contributes to vital physiological processes, such as immune responses, organogenesis and myogenesis, but also serves as a pivotal regulator in various pathological processes, such as lung cancer, gastric cancer, breast cancer and HCC. 50

| CELLUL AR AND PHYS IOLOG IC AL ROLE S OF N E AT1
Given the pivotal role of paraspeckles in controlling gene expression during multiple cellular processes, such as differentiation, viral infection and stress responses, NEAT1 may play an indispensable role in certain cellular processes owing to its inalienable component of paraspeckles. 51  Hirose et al also reported that NEAT1 −/− fibroblasts were more sensitive to proteasome inhibition that triggered cell death, which revealed that paraspeckles or NEAT1 attenuated the cell death pathway. 52 In 2014, a study by Nakagawa et al identified NEAT1 as indispensable for the formation of corpus luteum and subsequent establishment of pregnancy under suboptimal condition in mice. 54 They found that NEAT1_2 was expressed during the development course of the corpus luteum, which further induced the formation of paraspeckles in luteal cells, as indicated by the enrichment of SFPQ. However, these phenomena disappeared in NEAT1knockout mice. 54 Another study indicated that genetic ablation of NEAT1 could lead to abnormal mammary gland morphogenesis and lactation defects. 55 Through RNA FISH using a NEAT1_2 probe, they found that NEAT1-contaning paraspeckles assembled in luminal epithelial cells of the mammary gland. 55 These studies suggest that NEAT1 is essential for mammalian breeding and feeding. It has been frequently reported that NEAT1 (two isoforms) is associated with inflammatory processes such as chemokine modulation, cytokine production and inflammasome activation. 56,57 Of note, Zhang et al reported that NEAT1 enhances the activation of NLRP3, NLRC4 and AIM2 inflammasomes as well as facilitates caspase-1 activation, cytokine production and pyroptotic cell death. Further mechanistic studies revealed that NEAT1 is transferred from paraspeckles upon various inflammasome-activating signals, and then promotes the assembly of inflammasomes in the cytoplasm. 58 Interestingly, a recent study by Wang et al found another important role of NEAT1 in mouse muscle development and regeneration.
They found that NEAT1 expression gradually increased during myogenic differentiation and muscle regeneration. A functional study showed that NEAT1 promotes myoblast proliferation in vitro and in vivo, while impeding the differentiation of myoblasts. In this mechanism, NEAT1 recruited EZH2 to the promoter of P21, subsequently inhibiting P21 expression and augmenting C2C12 cell proliferation. 59 In parallel, NEAT1 inhibits the expression of Myog, Myh4 and Tnni2, three muscle-specific genes, by recruiting EZH2 to promoters of the target genes, thus, inhibiting myogenic differentiation. 59 Above all, these studies emphasized that NEAT1 plays an important role not only in cellular processes but also in vital physiological processes.

| ROLE OF N E AT1 IN LIVER D IS E A S E S
The liver, a multifunctional organ, is essential for metabolism, immunity, digestion and detoxification. Importantly, the liver has a great capacity to repair itself after minor damages. 60 However, morbidity and mortality caused by liver diseases have sharply increased in recent decades. Fortunately, increasing studies have indicated that many lncRNAs are abnormally changed in liver diseases (Figure 1), and they may be regarded as promising biomarkers for treating liver diseases. NEAT1 was reported to be involved in various liver diseases ( Figure 3), and the concrete mechanisms are listed in Table 1.

| HCC
Recent epidemiology data showed liver cancer as the sixth-most commonly diagnosed cancer and the fourth leading cause of cancerrelated deaths, which results in a great burden for people worldwide. 61 HCC is the most common type of primary liver cancers. 62 Increasing evidence has indicated that the diversity of non-coding RNAs is significantly altered in HCC. 63 Encouragingly, lncRNAs are reported to be aberrantly dysregulated in HCC and viewed as a budding star in predicting and diagnosing HCC based on its non-invasive advantage. 63 74 Recently, some studies reported that inadequacy of ATGL could alleviate the growth and motility of some tumour cells. 75 92 Overall, NEAT1 aggravates the progression of HCC, and disturbance of NEAT1 may contribute to chemotherapy, radiotherapy and immunotherapy of HCC as well as interfere with the maintenance of liver CSC properties. The mechanisms are described in Figure 4. Furthermore, the detection of serum NEAT1 in HCC makes NEAT1 a promising therapeutic biomarker.

| NAFLD
Non-alcoholic fatty liver disease, a chronic liver metabolic disorder, is prevalent worldwide and is associated with human health.
However, there is a lack of detection of NEAT1 in the serum and liver tissues of human NAFLD, and whether NEAT1 is correlated with disease risk, severity and prognosis of NAFLD remains unknown.
Moreover, which of the two variant isoforms plays a profound role remains to be clarified. Therefore, further studies are needed to examine circulating NEAT1 levels and validate the potential function as well as the mechanism of NEAT1 in NAFLD.

| Liver fibrosis
It is widely acknowledged that the activation of HSCs is a pivotal event in liver fibrosis. 102  intensifying autophagy in Ad-IGFBPrP1-treated mice. 105 The known roles of NEAT1 in liver fibrosis are shown in Figure 5B. Intriguingly, a recent review proposed that NEAT1 might regulate liver fibrosis by influencing P53 owing to the intimate regulatory relationship between NEAT1 and P53 in tumorigenesis. 106 This suggests another underlying mechanism of NEAT1 in liver fibrosis. Therefore, NEAT1 may be a potential biomarker for predicting liver fibrosis, and further studies are needed to investigate the deep mechanism of NEAT1_1 or NEAT1_2 in the pathogenesis of liver fibrosis.

| Liver injury
Sepsis, a systemic inflammatory syndrome, tends to stimulate inflammatory cascades and promotes ROS production, ultimately leading to multiple organ dysfunction. 107   In conclusion, NEAT1 plays a crucial role in the above-mentioned liver injury types ( Figure 5C) and may be a promising therapeutic biomarker. However, it is not known whether NEAT1_1 or NEAT1_2 exert a greater role in regulating liver damage and whether serum NEAT1 levels could predict the progression of liver injury remains to be clarified.

| Viral hepatitis
Recently, growing evidence has emphasized that NEAT1 is involved in virus infection and innate immune response, either acting as antiviral or promoting viral effect. 114

| CON CLUS I ON AND FUTURE PROS PEC TS
As an architectural lncRNA, NEAT1 is critical for the formation, integrity and assembly of paraspeckles. Nevertheless, scientists have unveiled the potential regulatory role of NEAT1 in various physiological and pathological processes in recent years. Of note, substantial research has been performed to explore the enigmatic role of NEAT1 in liver diseases, ranging from viral hepatitis to HCC Notably, knockdown of some mitochondrial proteins or treatment of mitochondrial stressors both result in the abnormal expression of NEAT1 via ATF2 and altered morphology as well as the amount of paraspeckles. 53 Given the important function of mitochondria in different liver diseases, whether NEAT1 exerts its role by regulating mitochondrial proteins is far from comprehension.
Recently, exosome-derived lncRNAs and mechanisms of intercellular lncRNA shuttling via exosomes have gradually been revealed in different liver diseases, which can assist in signal communication between cells. 31,118,119 Notably, NEAT1 was also identified as an exosomal lncRNA, inhibition of which could influence inflammatory responses in inflammatory bowel disease (IBD) through exosome-mediated polarization of macrophages. 120 Moreover, NEAT1 was enriched in large extracellular vesicles secreted by hypoxic cardiomyocytes, which were taken up by fibroblasts, causing the expression of profibrotic genes. 121 However, it has not been reported whether NEAT1 exists in exosomes secreted by liver cells and whether exosome NEAT1 has a certain effect on mediating cellular communications in liver diseases, regardless of origin from the liver or other distant organs. Given that circulating NEAT1 may become a potential biomarker in several liver diseases, it is necessary for future investigators to detect NEAT1 expression in plasma exosomes.
In addition, it is unclear whether NEAT1 is involved in the process of ALD. Further mechanistic studies are urgently needed to achieve a comprehensive understanding of NEAT1 in various liver diseases.

| FINAN CIAL SUPP ORT S TATEMENT
This review work was supported by grants from the National Natural

ACK N OWLED G EM ENTS
The authors appreciate the study participants and experts who helped correct the grammatical errors in our manuscript.

CO N FLI C T S O F I NTE R E S T
All authors listed in this review declare no conflicts of interest.