3.1.1. HULC (Highly Upregulated in Liver Cancer)

HULC is a 482 bp single spliced, oncogenic, canonically polyadenylated ncRNA, transcribed from chromosome 6p24.3. It localizes in the cytoplasm and co-purifies with the ribosomes of cancer cells [5, 13, 25]. It is present in the primates, although the genome of mouse and rat do not possess the HULC homolog. HULC was firstly identified as novel transcript associated with the molecular pathogenesis of HCC [25]. It is reported to be linked to anomalous lipid metabolism in HCC cells. HULC is upregulated in tumors and plasma of patients suffering from HCC and is a potential biomarker of HCC [13]. Downregulation of miR-372 is mediated by HULC because of its activity as an endogenous sponge. This down-modulation represses the translational inhibition of miR-372 gene. The transcriptional activity of HULC is highly dependent upon a cAMP response element-binding (CREB) site in the promoter region [6, 8, 14-16].

Long-term infection with hepatitis B virus (HBV) is one of the major contributors in HCC development. HBx, a protein produced by HBV, is found to be raised in the liver cells of patients suffering from HCC. This protein is found to be associated with genes regulating cellular growth [26, 27]. Du and collaborators reported the positive association between expression levels of HBV and HULC. Knockdown or overexpression of HBx is directly linked to a decrease or increase in the expression profile of HULC respectively [28].

Various other studies have also revealed the role of HULC in HCC development. Downregulation of p18 (a tumor suppressor gene, positioned near HULC) by HULC has led to the proliferation of hepatoma cells. HBx downregulates the expression of P18, which is overturned by HULC knockdown, incriminating a mechanism in which downregulation of p18 by HBX-mediated downregulation of HULC might lead to the proliferation of cancer cells [5, 8, 15, 25, 29]. HULC has been reported to upregulate the levels of SPHK1 (Sphingosine kinase 1), an enzyme that contributes to the survival, growth, differentiation, and proliferation of cells, and angiogenesis. HULC regulates a cascade of pathway leading to the upregulation of E2F1, a transcription factor that binds to SPHK1 promoter. MiR-107, a regulator of E2F, is sequestered by HULC leading to a series of events and eventually, tumor angiogenesis [25]. The metastasis and recurrence of HCC are regulated by epithelial-mesenchymal transition (EMT), which acts on ZEB1 and miR-200a-3p. HULC sequesters miR-200a-3p, corresponding to amplified levels of ZEB1, leading to the stabilization of ZEB1 [25]. Together, these studies reflect novel mechanisms by which HULC contributes to the pathophysiology of HCC.

3.1.2. HOTAIR (Homeobox [HOX] Transcript Antisense RNA)

HOTAIR is a 2158 nucleotide long antisense lncRNA, which is transcribed near the HOXC loci on chromosome 12q13.13 [5, 13, 16]. It is a polycomb group protein that represses the transcription of thousands of genes involved in stem cell pluripotency and various differentiation pathways during development [26]. Initially, it was identified to be upregulated in the breast epithelial tumors, inducing the metastasis of breast cancer [16]. It is reported to be a therapeutic biomarker of oesophageal squamous cell carcinoma. These lncRNAs have been reported to interact with two major

The upregulation of HOTAIR is also observed in the HCC tissues and is reported to be directly interrelated with recurrence and poor survival rate in cancer patients receiving liver transplants. The downregulation of HOTAIR also inhibits the tumor invasion in liver cancer cell lines. Reduction in the levels of matrix metalloproteinase-9 and vascular endothelial growth factor (VEGF) protein (necessary for motility and metastasis of cells) is observed upon reduction in the expression of HOTAIR [25]. Overexpression of HOTAIR in HBx-positive human liver tumors is reported to be regulated by Wnt signaling genes like Sox2, Oct4, Nanog and
EpCAM [13, 25, 27].

3.1.3. MALAT1 (Metastasis Associated Lung Adenocarcinoma Transcript)/ NEAT2 (Nuclear Rich Abundant Transcript 2)

It is a 7.5kb long lncRNA, originally expressed in primary non-small cell lung cancers. It is conserved in mammals and is expressed in many tissues [16-18]. The expression of this lncRNA is found to be dependent on the cell cycle and necessary for G1/S and mitotic progression. The literature reflects that Hippo/yes associated protein (YAP) and Wnt/TCF/β-catenin signaling pathway, generally, acts on MALAT1. Also, it is negatively controlled by serine and arginine rich splicing factor 1 (SRSF1). SRSF1 acts by either promoting the degradation of MALAT1 or by binding to YAP. This binding prevents the transcription of YAP and hence the activity of YAP on the promoter site of MALAT1 is inhibited [30]. Post-transcriptional changes in MALAT1 produce short, cytoplasmic RNA and a long transcript, localized in the nucleus. This influences the level of phosphorylated splicing-associated serine arginine (SR) proteins [31, 32].

MALAT1 is found to be overexpressed in various cancers including breast cancer, liver cancer, ovarian cancer, and bladder carcinoma and is a potential therapeutic target. The upregulation of MALAT1 is seen in neoplastic samples obtained from patients suffering from liver cancer. A high level of MALAT1 in patients has been observed as a signature of tumor recurrence after liver transplantation. This relativity implies that this lncRNA may show important clinical implications in the treatment of hepatocellular cancer [30]. Further, reduced expression of MALAT1 inhibits the motility and migration of cells and increases apoptosis in liver carcinoma and bladder cancer [7, 30].

For malignant cells to become invasive, epithelial-mesenchymal transition (EMT) is necessary. Downregulation of MALAT1 alters the expression profile of various genes like ZEB1, ZEB2, E-cadherin, Slug, which are associated with EMT. An increase in the levels of E-cadherin and a decrease in the levels of Slug, ZEB1, and ZEB2 have been widely reported [30, 33].

3.1.4. HEIH (High Expression in HCC)

HEIH is a 1.7-kb SP1-regulated long lncRNAs, located in the 5q34.3 locus. It is differentially expressed in HCC and closely associated with HCC recurrence. It is considered as a prognostic factor for HCC [5, 14, 15]. HEIH networks with enhancer of zeste homolog 2 (EZH2) and regulates EZH2 target genes including cell-cycle–regulatory genes p15, p16, p21, and p57. HEIH knockdown leads to reduction in the proliferation of cells and suppression of tumor growth. In short, HCC recurrence has a close association with HEIH and HBV infection [2, 8, 13, 18, 26, 33, 34]. Hence, the role of this dyad is worth reading further to detect the possibility of HCC recurrence.

3.1.5. HOTTIP (HOXA transcript at the distal tip)

It is a 7.9kb sized lncRNA, located at 7p15.2 and originated from the distal tip of the HOXA13 gene [14, 19]. It is the most upregulated variant in human HCC, even at an early stage. The oncogenic effect of HOTTIP is conferred by upregulation of several HOXA genes via interaction of WD repeat-containing protein 5 (WDR5) [an adapter protein with mixed-lineage leukemia (MLL) complexes]. This complex along with HOTTIP, mediates the trimethylation of H3K4 and HOXA, facilitating gene transcription [26, 15]. In the HOX cluster, chromatin interactions are chiefly regulated by HOTTIP. As observed in an orthotopic implantation nude murine model, knocking down HOTTIP abrogates lung metastasis by significantly inhibiting the cell proliferation and migration. HOTTIP also regulates chemoresistance by activation of β-catenin signaling. The knockdown of HOTTIP is able to suppress chemoresistance and mesenchymal characteristics [35].

3.1.6. H19

H19 is a 2.3kb long ncRNA located at 11p15.5 and is solely expressed from the maternal allele. The upregulation of H19 is widely seen in HBV-associated HCC. Its involvement is highly recorded in genomic imprinting during growth and development [5, 14]. Authors have reported the role of two imprinted genes located on the same gene locus: Insulin-like growth factor 2 (IGF2) and H19. Biallelic expression of H19 and IGF2 are the critical players in the epigenetic mechanism employed behind the development and progression of tumors [13, 15]. The expression of this lncRNA is mediated by c-Myc. It binds to E-boxes, positioned near the imprinting control region, facilitating acetylation of histone and translationally initiating H19 gene in HCC cells [15, 34]. In in vivo models of HCC, knockdown of H19 significantly abolishe anchorage-independent growth after hypoxia recovery. The possible mechanism behind the same is the diminished expression of p57kip2 [36]. Furthermore, the expression of P-glycoprotein and multidrug resistance 1 (MDR1)-allied drug resistance in HCC cells has a direct association with the expression of the H-19 gene [8]. H19 also activates miR-200 by linking with the protein complex hnRNP U/PCAF/RNAPol II, and subsequently, suppressing the progression and metastasis of HCC [37].

3.1.7. MEG3 (Maternally Expressed gene 3)

MEG3 is a paternally imprinted single-copy gene which comprises of 10 exons [33]. 12 isoforms of MEG3 have been detected and each of them can be distinguished by their differential blend of 4-7 exons in the transcript. The original isoform of MEG3 is a 1.6kb chromatin-associated polyadenylated transcript, located in the nucleus [16, 38].

MEG3 lncRNA is 34919bp sized, paternally imprinted and transcribed from chromosome 14q32.2 [5, 13]. It is normally expressed in human tissues but has its highest expression in the brain and pituitary gland. It has been revealed that ectopic expression inhibits the proliferation of a variety of cancer cell lines by majorly acting on tumor suppressor, p53. MEG3 is found to be involved in the accumulation of tumor suppressor p53 and regulation of TGF-b pathway genes involved in cell invasion, immune regulation, etc. It also interacts with PRC2 to repress murine double minute 2 homolog (MDM2), which contributes to p53 accumulation [13]. It has been observed that the activation of p53 depends upon the secondary structure of the MEG3 RNA, instead of its primary sequence. A study designed by Zhang and team provided proof for the preservation of the original secondary structure. They reported that replacement in some particular regions of MEG3 with unrelated sequence does not affect p53 activation [38]. From this, a conclusion can be drawn that in the absence of sequence conservation, the potential of these unique RNA species will still be preserved.

3.1.8. DANCR (Differentiation Antagonizing Non-protein Coding RNA)

The stemness feature of the liver cancer cells has been greatly amplified in the presence of DANCR lncRNA. These lncRNAs are reported responsible for an increased intra- and extra-hepatic tumor development. It primarily interacts with cadherin-associated protein catenin beta 1, CTNNB1 mRNA at its 30 untranslated regions (UTRs). This leads to the blockage of the binding site of many miRs (miR- 214, miR-320a, and miR-199a) competitively. This also allowed this gene to upregulate, resulting in the activation of the Wnt pathway. This further results in increased spheroid formation and stem cell like-feature of the affected cells. On the other hand, knockdown of DANCR in vivo improves the survival rate in mice by decreasing tumor cell survival and increasing tumor shrinkage [26, 30, 39].

3.1.9. UCA1 (Urothelial Carcinoma-Associated 1)

The lncRNA urothelial carcinoma-associated 1 (UCA1) is 7375bp in size and located on chromosome 19p13.12. It was originally duplicated from the human bladder cell line and is overexpressed in embryonic tissues, bladder cancers, liver cancers, and other cancers [22-24]. Studies have shown that UCA1 acts as an endogenous sponge that attaches to miR-216b and downregulates its expression. This mitigates the repression of FGFR in the cancer cells, increasing its level in the HCC cells. Downregulation of UCA1 suppresses ERK ½ and p-ERK ½, inhibiting the cell proliferation in both in vitro and in vivo models of HCC. This argument suggests the positive correlation of UCA1 and HCC initiation and progression through the regulation of FGFR1/ERK signaling pathways [40]. Also, researchers have reported that UCA1 activates NF-κB/STAT3 signaling in cancer cells. It is observed in an experimental mouse model that the transformation of hepatocyte-like-stem cells is induced by UCA1 [41]. The proliferation of HCC cells is also linked to the expression of UCA1 via CDKN1B regulation [42]. Drug resistance is also a major factor associated with the expression of UCA1 [43].

3.1.10. Linc00974

Linc00974 is another lncRNA that is 4890bp in size and is located on chromosome 17q21.31. It is significantly upregulated in HCC [25]. A strong linkage between Linc00974 and KRT19 (biomarkers of HCC) and HCC development has been reported by Tang and coworkers. Hypomethylation of the promoter of Linc00974 upregulates this lncRNA. Linc00974 acts as an endogenous sponge, repressing the levels of KRT19. Deletion of Linc0097 increases apoptosis and arrests cell cycle in HCC cell lines. This further inhibits cell multiplication and invasion. Suppression of lncRNA, mediated by miR, amplifies the levels of KRT19. Recent literature reflects a strong connection between KRT19, NOTCH and TGF cascades [21]. Therefore, it can be deduced that Linc00974 and KRT19 may be innovative manifestations for the prediction of tumor initiation and metastasis. Linc00974 may also be utilized in the management of HCC in the future.

3.1.11. ZFAS1(ZNFX1 Antisense RNA 1)

ZFAS1 (ZNFX1 antisense RNA 1) is a spliced and polyadenylated lncRNA transcribed from the end of ZNFX1. It is derived from chromosome 20q13.13 and is implicated in different types of cancer including gastric cancer, colorectal cancer, and hepatocellular cancer, among others [13]. It has been reported to interact with CDK1 and cyclin B to control p53-dependent cell-cycle regulation [44]. In addition, it promotes cell proliferation by employing EZH2 and LSD1/ CoREST to the promoters of genes including KLF2 (Kruppel like factor 2) and NKD2 (naked cuticle 2) to regulate their expression [45]. It also acts as a sponge for tumor suppressor miR-150. The knockdown of ZFAS1 represses cell proliferation, migration, and colony formation [15]. Similarly, ZNFX1 sequesters miR-150, which directly targets the EMT regulator, ZEB1. Levels of ZEB1 are affected by the expression of ZEB1-AS1 lncRNA. This lncRNA has a bidirectional promoter and shares it with ZEB1. It is a positive regulator of ZEB1 expression and EMT [33].

3.1.12. SIRT1 (sirtuin/silent Mating type Information Regulation 2 Homolog 1)

SIRT1 gene has been found to play an imperative role in cell proliferation and apoptosis. Overexpression of SIRT-1 has been witnessed in various HCC samples. SIRT1- antisense RNA (SIRT1-AS) lncRNA shows its action by covering the binding site of miR-29c on 31-UTR via repression in the translational inhibition of SIRT1 mRNA. Binding of SIRT1-AS to 31-UTR region of SIRT1 prevents the binding of mir-29c and hence, stabilization SIRT1 mRNA is observed. This increased the level of SIRT1, subsequently increasing cell survival. This complete process is possible because of the 622C mutation in SIRT1-AS. In fact, increased expression of this mutant suppresses the proliferation of HCC cells [35]. This may offer a newer target for gene therapy.