Tsix inhibits Xist transcription by recruiting CTCF to the Xist promoter region, while JPx inhibits CTCF transcriptional repression of Xist by binding CTCC (CCCTC-binding factor); Khps1a participates in the T-DMR (tissue-dependent) region demethylation of Sphk1 by an unknown mechanism; Dum cis-recruits DNMT1, DNMT3A, DNMT3B, and etc. to the promoter region of the neighboring gene Dppa2. differentially methylated region) of Sphk1; Dum cis-recruits DNMT1, DNMT3A and DNMT3B to the promoter region of the neighboring gene Dppa2 and causes silencing of methylation in this region, thereby suppressing Dppa2 expression and leading to differentiation of skeletal muscle myogenic cells into myoblasts (Guttman et al., 2011; Bian et al., 2019; He et al., 2020).

Xist (X chromosome inactivation specific transcript) and RepA (transcript of the adenine repeat region at the 5′ end of the Xist gene) synergistically wrap the X chromosome and recruit PRC2 to establish H3K27me3 to cause X chromosome inactivation; Bxd (Bithoraxoid) binds to ubx-TRE (Ubx cis-regulatory trithorax response elements) and recruits ASH1 to activate Ubx transcription; HOTAIR regulates the expression of the HoxD gene cluster in trans by interacting with two histone modification complexes: catalytic PRC2 complex established by H3K27me3, and the LSD1-CoREST-REST complex catalyzing H3K4me2/3 erasure (lysine-specific demethylase1-RE1-Silencing Transcription factor corepressor 1-RE1 -Silencing Transcription factor complex); HOTTIP (HoxA transcript at the distal tip) recruits MLL (Mixed lineage) via WDR5 (WD repeat-containing protein 5) leukemia) to the 5′ region of the HoxA gene cluster, which catalyzes the establishment of H3K4me3 and activates the expression of genes such as Hoxa11 and Hoxa13 in cis; Mira can form a DNA/RNA heterodimer with its locus and recruit MLL1, a member of the TrxG complex, which catalyzes the establishment of H3K4me3 and activates adjacent genes Hoxa6 and Hoxa7 expression, leading to differentiation of mES to the germline; Evf2 acts as a coactivator of DLX2 at high concentrations, enhancing Dlx5/6 enhancer activity and activating Dlx5 and Dlx6 transcription, while at low concentrations it can cis-suppress Dlx6 transcription through its Dlx6 antisense property, by recruiting MECP2, and thus HDAC Dlx5; asOct4-pg5 can recruit histone methyltransferases such as EZH2 and G9a to bind to the promoter regions of Oct4 and Oct4-pg5, establishing repressive chromatin modifications such as H3K27me3 and H3K9me3, which in turn repress transcription of Oct4 and Oct4-pg5, and when as Oct4-pg5 is combined with PURA (purine rich element binding protein A) and NCL (nucleolin), the ability to recruit EZH2 and G9a is lost and the repressive function is lost; the nascent ANRIL (antisense noncoding RNA in the INK4 locus) binds to CBX7 and ANRIL binds to CBX7 and promotes heterochromatin formation, while the formed ANRIL-CBX7 complex unbinds CBX7 to H3K27me3, leaving transcriptional repression in a dynamic state of flux. ANRIL recruits PRC2, allowing the INK4β/INK4α/ARF gene cluster to establish repressive modifications such as H3K27me3. ANRIL binds to SUZ12 and cis-represses INK4β transcription; DBE-T cis-recruits Ash1L to the 4q35 region, catalyzing the establishment of activating chromatin modifications such as H3K36me2, which activates gene transcription in the 4q35 region, ultimately leading to FSHD (facioscapulohumeral muscular dystrophy) disease (Guttman et al., 2011; Kopp and Mendell, 2018; Riaz and Li, 2019).

Xite and DXPas34 regulate Tsix expression in cis with enhancer activity; transcription of SER3 gene biosynthesis-associated 3-phosphoglycerate dehydrogenase in the biosynthesis of serine is regulated by lncRNA SRG1 (SER3 regulatory gene 1); Pwr1 interferes with the transcription of Icr1; Icr1 interferes with the transcription of Fol11; DHFRtinc interferes with the transcription of DHFR; Airn interferes with the transcription of Igf2r; Gas5 inhibits the binding of activated GR to target genes (Zhang et al., 2014; Ma et al., 2016b).

Malat1 regulates variable shear of Cat1 pre-mRNA; Zeb2-anti is involved in variable shear of Zed2; PTENpg1 asRNA β promotes PTENpg1 exit from the nucleus; Neat1 promotes retention of mRNA with IRAlus structure in the 3′ UTR region within paraspeckles; BACE1-AS increases BACE1 stability; MDRL as ceRNA promotes pri-miR484 maturation (Bian et al., 2019; He et al., 2020).

It has been reported that lncRNAs are usually involved in the progression of human-related diseases by such ways as being deregulated (Guttman et al., 2011; He et al., 2020), and some studies have shown that lncRNAs are involved in the key process of liver fibrosis by acting as regulators of HSC activation (Wang et al., 2014; Bian et al., 2019). Even though we can observe an impressive amount of literature suggesting an important role of lncRNAs in the liver fibrosis process, it is undeniable that the detailed mechanisms of lncRNAs in liver fibrosis remain unclear until now. In this review, we aim to provide a review of the latest developments in lncRNAs research, elaborate on the interactions between lncRNAs and miRNAs, and further evaluate the potential of lncRNAs as new therapeutic targets in liver fibrosis.

Panzitt et al. identified for the first time the highly up-regulated hepatocellular carcinoma LncRNA (HULC) of approximately 500 nucleotides containing two exons located on chromosome 6p24.3 as the most highly expressed lncRNA in human hepatocellular carcinoma, whose transcribed RNA does not have a considerable open reading frame nor does it produce any protein (Panzitt et al., 2007). The HULC promoter and its first exon are in a long terminal repeat sequence (LTR) retrotransposon-like sequence (Kapusta et al., 2013). The upregulation trend of HULC can be observed in all accessible studies on hepatocellular carcinoma (HCC) (Chen et al., 2017; Xin et al., 2018; Ghafouri-Fard et al., 2020b). Several literatures have reported that HULC is upregulated in cancer and is regulated as an oncogene lncRNA in tumorigenesis and progression (Kitagawa et al., 2013; Parasramka et al., 2016). Considering its high expression in HCC cells, previous studies have also shown the potential of HULC as a novel antitumor therapeutic agent (Klec et al., 2019). cAMP response element binding protein (CREB) is usually bound to and activated by the target promoter (Mayr and Montminy, 2001; Kong et al., 2016), and Wang et al. demonstrated the presence of CREB binding sites in the HULC promoter region and the ability to further activate the HULC promoter (Wang et al., 2010), which can affect the expression of HULC at the transcriptional level (Shen X. et al., 2019). Shen et al. revealed the role of lncRNA HULC in the progression of liver fibrosis in rats with nonalcoholic fatty liver disease (NFALD) and that inhibition of HULC suppressed steatosis. The degree of hepatic steatosis, inflammation, hepatocyte red lipid vesicles and apoptosis were also significantly reduced with the knockdown of HULC gene. Inhibition of HULC significantly reduced liver fibrosis scores and liver fibrosis indices (HA, LN, PC III, and IV-C) (Shen X. et al., 2019). Inhibition of HULC improved liver fibrosis and reduced hepatocyte apoptosis in NAFLD rats. In general, the above findings not only provide valuable candidate molecular markers for liver fibrosis and indicators of advanced liver fibrosis but also provide new insights into the role of lncRNA in the biology of cancer.

Nuclear paraspeckle assembly transcript 1 (NEAT1) was characterized as an unusual RNA polymerase II transcript that lacks introns and accompanied by non-canonical processing of the non-polyadenylated 3′-end by RNase P (Ding et al., 2019). NEAT1 was found to be upregulated in gastric adenocarcinoma and human laryngeal squamous cell carcinoma (Ma et al., 2016a; Wang et al., 2016), which suggested that it promotes tumor development by promoting cell proliferation and survival as well as inhibiting apoptosis (Choudhry et al., 2015). A similar situation has been observed in hepatocellular carcinoma (Guo et al., 2015). Yu et al. examined NEAT1 expression in cCl4-induced mice. qRT-PCR analysis showed increased expression of NEAT1 in CCl4-treated livers compared to control livers, and a significant increase in NEAT1 expression in HSCs was also observed during different weeks of CCl4 treatment (Yu et al., 2017b), Huang et al. screened the aberrantly expressed microRNAs in the CCl4-induced mouse liver fibrosis model by analyzing the GSE77271 microRNA microarray based on the Agilent-046065 mouse miRNA V19.0 platform. Neat1 simultaneously targeted miR-148a-3p and miR-22-3p, and showed the most significant increase in liver fibrosis mice that displayed the most marked increase in expression upregulation, and its expression in the CCl4 group exceeded 2-fold that of the control group (Huang et al., 2021), and inhibition of NEAT1 was observed to reverse isotropic liver fibrosis with concomitant reduction in α-SMA and type I collagen content, which was further confirmed by NEAT1 knockdown assays and NEAT1 overexpression assays (Yu et al., 2017b). A similar situation was confirmed in the alcoholic steatohepatitis (ASH) assay by Ye et al. (Ye et al., 2020). Inhibition of NEAT1 suppressed ethanol-stimulated elevated lipid metabolism and inhibited inflammatory responses in AML-12 cells. More importantly, inhibition of NEAT1 upregulated ethanol-induced hepatic function in ASH mice and inhibited lipid, inflammatory responses, hepatocyte apoptosis, and hepatic fibrosis, demonstrating that knockdown of NEAT1 inhibited hepatic fibrosis in ASH mice and thus slowed down the development of ASH (Ye et al., 2020). Related mechanistic studies suggest that Kruppel-like factor 6 (KLF6), as an important pro-fibrotic gene, is involved in the regulation of liver fibrosis by NEAT1 (Yu et al., 2017b), and that NEAT1 overexpression induces KLF6 mRNA and protein expression. However, it is of interest that KLF6 knockdown experiments showed NEAT1-induced proliferation of HSC, while KLF6 siRNA blocked NEAT1-induced α- SMA and type I collagen production, suggesting that NEAT1 could mediate HSC activation through KLF6 (Yu et al., 2017b). Huang et al. suggested that NEAT1 knockdown could inhibit the process of liver fibrosis and HSCs activation by regulating the expression of a cellular adhesion element 3 (Cyth3) associated with allosteric insulin signaling in mammals (Jux et al., 2019; Huang et al., 2021). And Ye et al. further identified that downregulation of NEAT1 could limit the inflammatory response and liver fibrosis in ASH mice by reducing suppressor of cytokine signaling 2 (SOCS2) (Ye et al., 2020), which is a feedback inhibitor of the growth hormone/insulin-like growth factor axis (Monti-Rocha et al., 2018).

It was first reported by Chaudhry in 2013 that a new full-length 2,176 bp oncogenic lncRNA, known as lncRNA small nucleolar rna host gene 7 (SNHG7), expressed in lymphoblastoid cell lines TK6 and WTK1 (Chaudhry, 2013), which is located on chromosome 9q34.3. Recent studies have shown a significant increase in its expression in tumor cells of digestive system, breast, and prostate (Wu F. et al., 2020; Wu X. et al., 2020), and further studies have demonstrated that SNHG7 is widely involved in the proliferation, invasion and migration of various tumor cells (Xia et al., 2020), including its regulation in the progression of HCC and liver fibrosis (Cui et al., 2017). Just as Xie et al. found increased expression of SNHG7 in primary HSC mice as well liver fibrosis, suggesting its regulation of HSC activation (Xie et al., 2021), and SNHG7 knockdown experiments showed decreased expression levels of α-SMA and Col. I (Xie et al., 2021), similarly SNHG7 inhibition was associated with reduced survival and proliferation rates in liver fibrosis mice. Current studies have identified several types of high confidence indicators of autophagy, such as the cytoplasmic form of LC3, a key protein in autophagosome formation (LC3-I), the active membrane-bound form of LC3 (LC3-II), and Beclin1 (Wirawan et al., 2012; Alirezaei et al., 2015; Dodson et al., 2017; Feng et al., 2017). Xie et al. revealed that knockdown of SNHG7 could reduce the decrease the expression level of Beclin1, LC3-II and LC3-I ratio, demonstrating the inhibitory effect of SNHG7 knockdown on HSC autophagy (Xie et al., 2021). DNMT3A induces a DNMT-regulated DNA ab initio methylation process, and DNA methylation/hydroxy methylation, a key step in HSC activation and liver fibrosis development, can be inhibited by activation of DNMT3A expression in HSCs (Garzon et al., 2009; Page et al., 2016). Several recent mechanistic studies suggest that SNHG7 knockdown is significantly associated with low expression levels of DNMT3A. These results confirm the relationship between SNHG7 and DNMT3A, which are novel regulators of HSC activation, autophagy, and proliferation in liver fibrosis (Xie et al., 2021). Yu et al. identified a positive correlation between SNHG7 levels and type I collagen mRNA levels in patients with cirrhosis (Yu et al., 2017b). In addition, SNHG7 showed a significant association in regulating activated HSCs proliferation and the cell cycle associated with increased G0/G1 phase cells and decreased S phase cells. SNHG7 knockdown experiments performed in activated HSCs inhibited type I collagen expression (Yu et al., 2017b), as well as collagen deposition and hydroxyproline due to carbon tetrachloride were similarly blocked by silencing of SNHG7 in vivo, suggesting that inhibition of liver fibrosis can be mediated by downregulation of SNHG7 (Yu et al., 2017b). Furthermore, Yu et al. demonstrated at the mechanistic level the role of SNHG7 in regulating the expression level of irregular fragment polarity protein 2 (DVL2) (Yu et al., 2017b; Nielsen et al., 2019), which was positively correlated with DVL2, the deletion of which also blocked its effect on HSCs activation (Yu et al., 2017b). In conclusion, all these data suggest that SNHG7 is an impressive possible therapeutic target and a potential diagnostic marker for liver fibrosis.

LncRNA H19 is expressed only by the maternal allele 11p15.5, which can encode 2.3 kb RNA and is transcribed by RNA polymerase II (Gabory et al., 2006), splicing and polyadenylation (Ghafouri-Fard et al., 2020a). It is exported from the nucleus to the cytoplasm, adjacent to the insulin-like growth factor 2 (IGF2) gene, and they are expressed from the maternal and paternal genetic chromosomes, respectively (Raveh et al., 2015; Wang J. et al., 2020). H19 RNA molecules have now been observed to be present in the cytoplasm at much higher levels than in the nucleus. H19 plays an essential role in biological processes such as apoptosis, angiogenesis, inflammation and cell death through regulatory RNA or ribosomal regulators (Yoshimura et al., 2018). This includes the regulation of proliferation, invasion, and metastasis processes in a variety of tumors of the digestive system (Zhou et al., 2017; Wei LQ. et al., 2019). Multiple complex mechanisms have been demonstrated in different cancers (Zhang D. M. et al., 2017; Zhou et al., 2017). Of interest is the upregulation of the level of intracellular transcripts (Zhu et al., 2019) and extracellular exosomes (Li X. et al., 2018), known as lncRNA-H19, observed in activated HSCs, which is thought to be associated with HSCs-activated metabolic processes like lipid (Liu et al., 2018) and cholesterol metabolism (Xiao et al., 2019). Previous research concluded that the level of fibrosis in the liver was positively correlated with the level of H19, and that H19 knockdown attenuated Bcl-2-induced liver injury (Zhang Y. et al., 2016), while conversely H19 overexpression significantly exacerbated the process of HSCs and EMT activation in hepatocytes (Zhu et al., 2019). Song et al. demonstrated the overexpression of H19 in bile duct ligation (BDL)-induced liver fibrosis with abnormal liver function parameters (Song et al., 2017), and identified a new downstream target gene of ZEB1, called EpCAM (Song et al., 2017), which promotes cholestatic liver fibrosis by interacting with the ZEB1 protein to prevent its binding to the EpCAM promoter and thus the inhibitory effect of ZEB1 (Song et al., 2017). Liu et al. reported that cholangiocyte-derived exosomal H19 promotes cholestatic liver injury in Mdr2^−/− mice and promotes HSCs transdifferentiation and activation, along with upregulation of fibrotic gene expression in HSCs-derived fibroblasts (Liu et al., 2019). Wang et al. discovered that H19 can promote RARα and RXRβ mRNA and protein synthesis (Wang ZM. et al., 2020), and its reduced expression reversed the extent of HSCs activation induced due to increased retinoic acid signaling. Meanwhile, it should be mentioned that H19 knockdown-mediated HSCs inactivation was inhibited by the activation of retinoic acid signaling. Furthermore, they demonstrated that H19 enhancement was positively associated with a synergistic increase in retinoic acid metabolism during HSCs activation (Wang ZM. et al., 2020). More significantly, they confirmed that inhibition of ethanol dehydrogenase III (ADH3) completely abolished the effect of H19-mediated retinoic acid signaling, and that dihydroartemisinin (DHA), a natural inhibitor of H19, reduced both H19 and ADH3 expression and thus inhibited HSC activation (Wang ZM. et al., 2020). Taken together, these results reveal some of the molecular mechanisms underlying the increase in retinoic acid signaling during HSCs activation and suggest that the lncRNA-H19/ADH3 pathway is a potential target for the treatment of liver fibrosis. Similarly, H19 expression levels were increased in CCl₄-induced fibrotic liver thereby activating HSCs (Wang Z. et al., 2020). Further studies revealed the role of hypoxia-inducible factor-1α (HIF-1α) in promoting H19 expression by binding to the H19 promoter at two hypoxia response element (HRE) sites located at 492–499 and 515–522 bp (Wang Z. et al., 2020). H19 knockdown experiments also resulted in significant inhibition of HSC activation and attenuated liver fibrosis, suggesting that lncRNAH19 may be a potential target for antifibrotic therapeutic approaches. Moreover, the H19 silencing assay reduced the degree of lipid oxidation and the H19 knockdown assay restored the levels of lipid droplets, triglycerides, cholesteryl esters and retinyl esters in HSCs without changes in lipid uptake and synthesis (Wang Z. et al., 2020). In conclusion, as described above, the results highlight the role of H19 in the proliferation, activation and metabolism of lipid droplets in HSCs and reveal its feasibility as a new molecular target to attenuate liver fibrosis.

Ji et al. characterized a metastasis-associated lung adenocarcinoma transcript (MALAT1) transcribed by RNA polymerase II located on human chromosome 11q13 and mouse chromosome 19qA (Zhang et al., 2012; Wilusz, 2016), which is widely known for its properties in predicting early NSCLC metastasis and survival (Ji et al., 2003), the major transcript of MALAT1 is approximately mid-8 kb in humans and 6.7 kb in mice (Wilusz et al., 2008). MALAT1-associated small cytoplasmic RNA (mascRNA) is a larger fragment of approximately 6.7 kb and a smaller fragment of 61 nucleotides produced by the action of ribonuclease P and ribonuclease Z on MALAT1 (Brown et al., 2014), while the larger fragment or mature transcript is highly stable due to a unique triple-helix structure at the 3′ end that protects it from nucleic acid exonucleases (Zhang et al., 2012). The highly conserved and widespread expression of MALAT1 in mammalian tissues and cancers implies its functional importance; MALAT-1 dysregulation in a variety of cancers has been extensively studied. In most cases, it functions as a promoting role in the development of different types of tumors (Kim et al., 2018; Feng et al., 2019). MALAT1 upregulation is closely associated with the development of cancers such as lung (Wei S. et al., 2019), glioblastoma (Voce et al., 2019), esophageal squamous cell carcinoma (Chen M. et al., 2018), renal cell carcinoma (Zhang H. et al., 2019), colorectal cancer (Zhang H. et al., 2019; Xie et al., 2019), osteosarcoma (Chen Y. et al., 2018), multiple myeloma (Amodio et al., 2018), gastric cancer (Zhang YF. et al., 2020), gallbladder cancer (Lin et al., 2019), and other cancers (Tian and Xu, 2015), as well as other clinicopathological features including tumor location, tumor size, differentiation and tumor stage (Goyal et al., 2021). Numerous studies have shown that as a biomarker for tumor diagnosis and prognosis, the abnormal expression of MALAT1 in tumor tissues and/or body fluids is highly indicative (Leti et al., 2017; Peng et al., 2018). In a physical and functional interaction study with the liver fibrosis process, Yu et al. found that MALAT1 expression was significantly upregulated in fibrotic liver tissues and simultaneously activated HSCs (Yu et al., 2015a), while silencing MALAT1 suppressed the mRNA levels of α-SMA and Col.I and downregulated the protein levels of α-SMA and collagen type I in HSC respectively (Yu et al., 2015a). Sirius red staining of collagen in mouse liver tissue resulted in the observation that mice transduced by silencing MALAT1 showed a 54% downregulation of collagen accumulation compared to CCl4-treated mice (Yu et al., 2015a), reflecting the role of MALAT1 in accelerating the progression of liver fibrosis in vivo. Dai et al. used arsenite treatment of L-02 cells as well as co-culture of LX-2 cells and found that MALAT1 expression levels increased as well as co-culture promoting activation of LX-2 cells (Dai et al., 2019). They further discovered that MALAT1 levels were increased in exosomes of arsenite-treated L-02 cells and LX-2 cells exposed to exosomes from arsenite-treated L-02 cells (Dai et al., 2019), and these exosomes also promoted LX-2 cell activation; blocking MALAT1 expression simultaneously inhibited these changes, thus suggesting a mechanism by which MALAT1 induces LX-2 cell activation via exosomes. Silent information regulator 1 (SIRT1), as a member of the mammalian sirtuin family of proteins (SIRT1-SIRT7) (Dai et al., 2019), is homologous to the yeast Sir2 protein, and SIRT1 is involved in a variety of biological processes and exhibits multiple physiological functions through the deacetylation of many non-histone proteins (Houtkooper et al., 2012). Wu et al. verified the important role of SIRT1 in hepatic stellate cell activation and reversal and its overexpression counteracting TGF-β1-induced LX-2 cell activation (Wu et al., 2015), suggesting its potential as an alternative for the treatment of liver fibrosis. Further studies found that the evolutionarily highly conserved MALAT1 has a strong tendency to interact with SIRT1 (discriminatory power 100%) (Wu et al., 2015), which was verified in CCL4-treated mice and LX-2 cells exposed to TGF-β1, considering that the expression level of MALAT1 mRNA was significantly upregulated and accompanied by negative changes in SIRT1 protein (Wu et al., 2015). MALAT1 silencing assay yielded results that eliminated the TGF-β1-induced upregulation of myofibroblast markers and the downregulation of SIRT1 protein. These phenomena suggest a role for MALAT1 in mediating the expression as well as the function of SIRT1 in regulating liver fibrosis. In conclusion, these findings highlight the role of MALAT1 in liver fibrosis and suggest a mechanism for fibrosis development (Wu et al., 2015). However, future efforts should be devoted to elucidating other regulatory mechanisms and clinical implications of MALAT1 in liver fibrosis.

HOXA transcript at the distal tip (HOTTIP) is a functionally characterized lncRNA (Li et al., 2016). Wang et al. demonstrated that the HOTTIP gene is located at chromosomal locus 7p15.2 and encodes a 4665 bp transcript (Wang et al., 2011). Its function is to directly interact with the Trithorax protein WDR5 and induce open DNA chromatin conformation, target the WDR5/MLL complex and drive histone H3 lysine 4 trimethylation for transcriptional regulation of the 50-terminal HOXA locus gene (Wang et al., 2011). This suggests that HOTTIP is not only involved in developmental processes but also enhances the effect of this lncRNA as a cancer-associated lncRNA considering its role as a signaling transmitter from higher-order chromosome conformation to chromatin coding (Wang et al., 2011). Overall survival (OS), distant metastasis (DM), lymph node metastasis (LNM), and tumor staging of human tumors have been extensively studied and determined to be closely associated with HOTTIP expression, suggesting that HOTTIP expression may influence the prognosis and metastasis of several human cancers (Broerse and Crassini, 1984; Quagliata et al., 2014). The most representative case is the high HOTTIP expression in human HCC specimens (Quagliata et al., 2014) and its close correlation with clinical progression and disease outcome (Tsang et al., 2015). It is worth mentioning that HOTTIP has long been shown to be dysregulated in the early stages of hepatocellular carcinoma formation, and recent studies suggest a positive correlation between its expression and liver fibrosis progression (Yang et al., 2019; He et al., 2020). Zheng et al. verified the specific expression status of HOTTIP in liver fibrotic tissue and primary quiescent HSC (Zheng et al., 2019), and their qRT-PCR results showed a 22.6-fold increase in HOTTIP expression on day 10 compared to day 2 increased 22.6-fold, similar to the results from the group of oil-treated mice compared to the group of CCl₄-treated mice suggesting a significant upregulation of HOTTIP expression in HSCs however this phenomenon was not observed in hepatocytes (Zheng et al., 2019). Furthermore, the mRNA and protein levels of α-SMA and Col. I was also found to be reduced by hot-end silencing but Edu incorporation assay demonstrated that hot-end downregulation inhibited the proliferation of activated HSCs. The above results suggest that HOTTIP downregulation could inhibit HSCs activation and proliferation. Related mechanistic studies suggest that HOTTIP is a target of miR-150 and is also recruited to Ago2-associated miRNPs (Zheng et al., 2019), possibly acting through miR-150 association. In addition, bioinformatics analysis and luciferase analysis of a series of experiments also confirmed the role of serum response factor (SRF) as a target of miR-150. They further demonstrated that the inhibition of HSCs activation was caused by an increase in SRF mRNA expression due to HOTTIP overexpression (Zheng et al., 2019). Li et al. revealed that HOTTIP expression was significantly upregulated in fibrotic and cirrhotic liver samples, with the highest in cirrhotic samples (Li Z. et al., 2018), and this was also found in liver fibrotic tissue, primary HSC and activated LX-2 cells. Inhibition of HOTTIP at the mRNA and protein levels was effective in reducing the expression of α- SMA and Col. I. They found that downregulation of HOTTIP attenuated CCl₄-induced liver fibrosis in mice. In contrast, the relative survival of HSC in LX-2 cells and the mRNA and protein levels of α-SMA and Col. I were significantly reduced by HOTTIP knockdown (Li Z. et al., 2018). Li et al. proposed a possible mechanism to promote HSC activation, i.e., negative regulation of HOTTIP mediated by miR-148a, considering that TGFBR1 and TGFBR2 were identified as miR-148a novel targets in HSCs. TGFBR1 and TGFBR2 levels were increased by high levels of HOTTIP, which led to the progression of liver fibrosis (Li Z. et al., 2018). These results highlight the potential of the HOTTIP/miR-148a/TGFBR1/TGFBR2 axis as a potential marker and target in patients with liver fibrosis. In conclusion, HOTTIP promotes HSCs cell proliferation and activation suggesting its possible role as a fibrogenic gene in liver fibrosis and plays a key role as a prognostic marker and novel therapeutic target. However, these still need to be investigated further.

The 7,598 nucleotide lncRNA sequence localized on chromosome 22q12.2, also known as taurine upregulated gene 1 (TUG1), was initially identified in a genomic screen of taurine-treated mouse retinal cells (Young et al., 2005; Zhang et al., 2013). Functional studies in mice further demonstrated that knockdown of TUG1 inhibits retinal developmental processes (Khalil et al., 2009). Khalil et al. demonstrated by whole genome RNA immunoprecipitation analysis that approximately 20% of lncRNAs (including TUG1) with methyltransferase activity promote demethylation and trimethylation of lysine residue 27 of histone 3 (H3K27me3) in the target gene and inhibit its expression by binding to polyclonal repressor complex 2 (PRC2), which inhibits its expression (van Kruijsbergen et al., 2015). Besides other PRC2-associated lncRNAs involved in tumorigenesis and progression, TUG1 regulates the biological behavior and molecular mechanisms of different cancer cells, including cell proliferation, invasion, apoptosis, differentiation, migration, drug resistance, radiation resistance, angiogenesis, mitochondrial bioenergetics, epithelial-mesenchymal transition (EMT), and regulation of blood-tumor barrier permeability among other different cancer cell (Niland et al., 2012; Katsushima et al., 2016; Cai et al., 2017; Chiu et al., 2018). TUG1 is closely associated with the mediation of radio resistance and angiogenesis in hepatoblastoma (Dong et al., 2016). TUG1 has also been extensively studied in liver diseases such as cirrhosis and liver fibrosis. Zhang et al. demonstrated that TUG1 is highly expressed in liver sinusoidal endothelial cells (LSEC), and the results of TUG1 knockdown experiments revealed inhibition of the extent of expression of autophagy and EMT-related genes (Zhang R. et al., 2020). In contrast, knockdown of TUG1 eliminated the most significant increase in autophagy-related genes in LPS-treated LSEC under starvation. The increase in ATG5 expression while inhibition of ATG5 attenuated autophagy and EMT (Zhang R. et al., 2020). Han et al. demonstrated that TUG1 was overexpressed in liver samples from patients with CCl4 and BDL-induced liver fibrosis in vivo as well as cirrhosis and activated HSCs while promoting a degree of expression of SMA, Col1a1, Mmp2/9/10, and Timp1. The possibility that TUG1 accelerates the progression of liver fibrosis by promoting the expression of these pro-fibrotic genes through downregulation of miR-29b is mechanistically argued (Han et al., 2018). Collectively, these studies revealed the mechanisms of TUG1 play a crucial role in liver fibrosis, suggesting its ability to monitor human liver fibrosis and its potential to be a candidate biomarker for new therapeutic strategies.

LincRNA-p21 (long intergenic non-coding RNA p21) localized at human chromosome 6p21.2 situated approximately 15 kb upstream of the cell cycle regulatory gene p21/Cdkn1a and approximately 3.0 kb in length has been described as an inducer of p53-dependent apoptosis in mouse embryonic fibroblasts (Huarte et al., 2010). lincRNA-p21 is available in two types both containing an exon and an Alu isoforms with a reverse repeat element (Yoon et al., 2012a; Yoon et al., 2012b; Wilusz and Wilusz, 2012). Coordinates the degree of autoregulation and expression of its target transcripts by interacting with RNA-binding proteins, miRNA, and mRNA targets (Yoon et al., 2012a). As a transcriptional target of p53 it is involved in the p53 pathway, downregulating many p53 target genes and triggering the apoptotic process by physically interacting with the p53 repressor complex (Fatica and Bozzoni, 2014). lincRNA-p21 has also been reported to regulate gene expression by directing protein binding partners in chromatin localization and thus directly binding to target mRNAs to act as a translational repressor and thus by activating p21 in cis participate in the regulation of the G1/S checkpoint (Dimitrova et al., 2014). It is also noteworthy that it can feedback regulate p53 activity by regulating the interaction of p53, p300, and MDM2 (Wu et al., 2014; Tang et al., 2015), thus participating in different tumorigenesis including hepatocellular carcinoma (Jia et al., 2016). In terms of tumor invasion lincRNA-p21 overexpression can be inhibited by Notch pathway (Wang et al., 2017). Besides it plays a key regulatory role in DNA damage response, apoptosis, and cell proliferation among other different processes (Ozgur et al., 2013). Zheng et al. observed in animal experiments that lincRNA-p21 expression was downregulated in liver fibrosis (Zheng et al., 2015). lincRNA-p21 was negatively correlated with disease progression and HSCs activation status, while in vitro and in vivo distribution inhibited HSCs activation and reduced liver fibrosis progression. Notably the reversibility of the inhibitory effect of lincRNA-p21 was confirmed by the removal of lincRNA-p21 leading to classical morphological changes associated with HSCs activation. lincRNA-p21 was found by Zhang et al. to inhibit the cell cycle and proliferation of primary HSCs by enhancing p21 (Zheng et al., 2015), while Tu et al. found a significant increase in hepatocyte lincRNA-p21 expression during hepatic fibrosis (Tu et al., 2017). These suggest that lincRNA-p21 contributes to a positive role in hepatocyte apoptosis and inhibition of hepatocyte growth in fibrotic livers. Knockdown of hepatocyte lincRNA-p21 attenuated CCl₄-induced hepatocyte apoptosis thereby reducing CCl₄-induced inflammatory cell infiltration and secretion levels of pro-inflammatory and pro-fibrotic cytokines in the fibrotic liver. Mechanistic studies have shown that inhibition of miR-30 impairs the effect of lincRNA-p21 in the development of liver fibrosis (Tu et al., 2017). lincRNA-p21/miR-30 axis has been highlighted as a potential marker and target for patients with liver fibrosis. Yang et al. found that lincRNA-p21 overexpression promotes hepatocyte apoptosis, but its results can be blocked by thymosin β4 (Tβ4) blocked, and additionally Tβ4 reversed lincRNA-p21- induced cleavage of caspase-3 and caspase-9 levels (Tu et al., 2017). LincRNA-p21 overexpression increases the levels of fibrosis-associated proteins (type I collagen, α- SMA, and TIMP-1) and induces hydroxyproline and ALT production leading to pathological damage of liver tissue and progression of fibrosis. The potential utility of lincRNA-p21 in predicting cirrhosis is supported by the results of downregulation of serum lincRNA-p21 levels in cirrhotic patients (Yang L. et al., 2020). Yu et al. reported a decrease in serum lincRNA-p21 levels in patients with chronic hepatitis B that negatively correlated with the stage of liver fibrosis, thus revealing its diagnostic value (Yu et al., 2017c). There was also a negative correlation between serum lincRNA-p21 levels and markers of liver fibrosis (including α-SMA and Col. I) but not in markers of viral replication, liver inflammatory activity and liver function. The primary HSC culture results suggested that the deletion of lincRNA-p21 expression was associated with promoter methylation, and these conditions implied the potential of serum lincRNA-p21 as a potential biomarker of liver fibrosis in patients with chronic hepatitis B/cirrhosis. Promoter methylation may be involved in the downregulation of lincRNA-p21 in liver fibrosis (Yu et al., 2017c). Collectively, these findings demonstrate the ability of lincRNA-p21 to act as a mediator of HSCs activation and proliferation, suggesting its potential as a new therapeutic target for liver fibrosis.

The maternally expressed gene 3 (MEG3), located within the human chromosome 14q32.3 DLK1-MEG3 locus (Wylie et al., 2000), is 35 kb in size consisting of 10 exons (Zhou et al., 2012) and encodes an approximately 1.6 kb long non-coding RNA as a contained 10 exons (Zhang et al., 2010). Selectively spliced transcripts of Gtl2 (gene trap site 2 (Gtl2) is the mouse homolog of human MEG3) extend to contain intron-encoded C/D box SNORNAs and miRNAs, suggesting that Gtl2 may function as a host gene for these small RNAs (Cavaille et al., 2002; Lin et al., 2003; Tierling et al., 2006). MEG3 can be observed in unimprinted embryonic cells to silence genes involved in neurogenesis by regulating the chromatin targeting of multicomb proteins and plays an important role in neuronal development (Mercer et al., 2008; Kaneko et al., 2014; Mondal et al., 2015). Recent studies have suggested that MEG3 may act as a tumor suppressor considering the extent to which its loss of expression in several cancers is associated with inhibition of cell proliferation (Ghafouri-Fard and Taheri, 2019). Yu et al. showed that the process of liver fibrosis is accompanied by a decrease in MEG3 in vivo and in vitro and that restoration of MEG3 expression inhibits liver fibrosis while reducing α-SMA and type I collagen production (Yu et al., 2018). MEG3 overexpression inhibits HSC activation through EMT and is associated with E-calcium activation. The Hedgehog (Hh) pathway is one of the pathways involved in HSC activation by MEG3 as an EMT process. Smoothing (SMO) plays an important role in the Hh pathway. Bioinformatics analysis, RNA immunoprecipitation and deletion mapping results suggest that the interaction between MEG3 and SMO is involved in EMT repression caused by MEG3 overexpression (Yu et al., 2018). Gene expression in the DLK1-MEG3 region is controlled by two differentially methylated regions (DMRs) consisting of multiple methylated CpG sites located approximately 13 kb upstream of the MEG3 transcription start site intergenic DMR (IG-DMR) and overlaps with a 1.5-kb upstream promoter in the post-fertilization-derived secondary (MEG3-DMR) (Murphy et al., 2003), indicating the important role that DNA methylation plays in silencing the MGE3 gene (Anwar et al., 2012). The most widely studied epigenetic modification, DNA methylation and its relevance to the pathogenesis of liver fibrosis have been well established experimentally (Benetatos et al., 2008; Li et al., 2010), and previous studies have suggested a role for DNA methylation in the deletion of MEG3 expression in tumors (Zhao et al., 2005; Benetatos et al., 2008). He et al. revealed that MEG3 levels were significantly reduced in CCl4-induced liver fibrosis in mice and humans, while MSP was significantly reduced in CCl4-treated mouse liver tissue and human liver fibrosis tissue and TGF-β1-treated LX-2 cells where MEG3 promoter methylation was observed (He et al., 2014). The effect of 5-azadC to block MEG3 methylation could be achieved by the methylation inhibitor 5-azadC significantly eliminating TGF-β1-induced aberrant MEG3 hypermethylation and restoring MEG3 in TGF-β1-treated LX-2 cells thereby inhibiting HSC activation and proliferation expression illustration (He et al., 2014). The inhibition of activation and the degree of proliferation of LX-2 cells and the reversal of methylation of the MEG3 promoter were both closely associated with the deletion of DNMT1 thereby restoring MEG3 expression. While 5-azadC treatment or knockdown of DNMT1 downregulated mRNA and protein production of α-SMA and Col. I in TGF-β1-treated LX-2 cells, overexpression of MEG3 was detected in TGF-β1-treated LX-2 cells (He et al., 2014), which significantly activated p53 protein levels and induced a Bax/Bcl-2 ratio accompanied by a significant increase in cytoplasmic cytochrome c significantly increased. These suggest that the p53-dependent mitochondrial apoptotic pathway is partially involved in the MEG3-induced apoptosis process (He et al., 2014). In conclusion, these findings demonstrate that MEG3 may play an important role in stellate cell activation and liver fibrosis progression and presents as a new potential treatment target for liver fibrosis.

Situated at 1q25 and composed of 12 exons, GAS5 was originally identified from a subtractive cDNA library, named according to the increased level of expression found in mammalian cells at growth arrest (Sun et al., 2017). Its exons are selectively spliced to produce two possible mature lncRNAs: GAS5a and GAS5b (Li J. et al., 2018) and 11 introns responsible for encoding 10 cassettes of C/D small nucleolar RNA (snoRNA) (Ni et al., 2019). Sequence similarity to the hormone receptor element of the glucocorticoid receptor (GR) in terms of function inhibits the effect of GR on its target gene expression (Zhong et al., 2020). Considering other regions of sequence similarity suggests a role for this lncRNA in regulating the function of other hormones such as androgen, progesterone, and salt corticosteroid receptors (Dong P. et al., 2019; Yang X. et al., 2020). Additionally, plasma GAS5 is involved during diabetes and coronary heart disease. Yu et al. showed that GAS5 could directly bind to miR-222 in mouse, rat and human fibrotic liver samples as well as in activated HSC but its overexpression inhibited the activation of primary HSC in vitro while attenuating collagen accumulation levels in fibrotic liver tissues in vivo, but this was not observed in response to GAS5 is predominantly localized in the cytoplasm (Yu et al., 2015b) accompanied by a higher copy number than miR-222 and is noted to increase p27 protein levels by binding to miR-222, thereby acting as a suppressor in HSC activation and proliferation (Yu et al., 2015b). Han et al. revealed that GAS5 expression was strongly correlated with liver fibrosis in patients with nonalcoholic fatty liver disease (NAFLD) (Han et al., 2020), and plasma GAS5 expression was significantly higher in patients with advanced stages than in non-advanced stages (Han et al., 2020). The progression of fibrosis was linearly correlated with plasma GAS5 expression, which also suggests the potential of plasma GAS5 as a noninvasive marker of liver fibrosis in patients with NAFLD (Dong Z. et al., 2019). Dong et al. investigated CCl4-induced in vivo assays in model rats and TGF-β1-induced in vitro assays in HSC and found that miR-23a expression was significantly increased while compared with miR-23a Compared with the NC group (Dong Z. et al., 2019), miR-23a inhibitor did not affect the expression levels of E-calmodulin, α-SMA and type I collagen in normal rats while up-regulating the expression levels of E-calmodulin and down-regulating the expression levels of α-SMA and type I collagen in model rats, suggesting that miR-23a plays a critical regulatory role in the development of liver fibrosis (Dong Z. et al., 2019). Further co-transfection revealed that the relative luciferase activity of pGL3-GAS5-wt was inhibited by miR-23a mimics while the luciferase activity of miR-23a NC and pGL3- GAS5-mut was unchanged (Dong Z. et al., 2019). RNA pull-down analysis suggested that approximately 5% of GAS5 bound to miR-23a compared to 100% of GAS5 in total RNA, and these results suggest that miR-23a could pull down GAS5 in liver tissue and HSC. lncRNA GAS5 silencing resulted in increased expression levels of miR-23a while addition of exogenous miR-23a resulted in downregulation of lncRNA GAS5 expression levels, this evidence suggested the ability of lncRNA GAS5 to bind directly to miR-23a. Thus, the ability of lncRNA GAS5 to act as a sponge platform for miR-23a and competitively reduce the expression level of miR-23a to inhibit liver fibrosis can be confirmed. Additionally, it is essential to mention the fact that TCM has been selected as an alternative therapy for liver fibrosis in view of the ineffectiveness and frequent occurrence of adverse side effects of synthetic drugs currently used to treat liver diseases, including liver fibrosis (Lam et al., 2016). Dahuang Zhezhuo Pill (DHZCP) as a typical Chinese medicine can inhibit the proliferation of vascular smooth muscle cells or further development of liver fibrosis in vivo by inhibiting the MAPK pathway (Zhang et al., 2009; Cai et al., 2010). Gong et al. identified that the proliferation of HSC was significantly inhibited after overexpression of GAS5 and DHZCP reversed the relative mRNA expression of GAS5, which suggest that DHZCP can mitigate liver fibrosis by enhancing the GAS5 expression (Gong et al., 2018).