A Systematic Review of Long Noncoding RNAs in Hepatocellular Carcinoma: Molecular Mechanism and Clinical Implications

Hepatocellular carcinoma (HCC) has the second highest mortality rate worldwide among all cancers. Previous studies have revealed the significant involvement of long noncoding RNAs (lncRNAs) in numerous human cancers including HCC. Both oncogenic and tumor repressive lncRNAs have been identified and implicated in the complex process of hepatocarcinogenesis. They can be further explored as prospective diagnostic, prognostic, and therapeutic markers for HCC. An in-depth understanding of lncRNAs' mechanism in HCC is therefore required to fully explore their potential role. In the current review, we will concentrate on the underlying function, molecular mechanisms, and potential clinical implications of lncRNA in HCC.


Introduction
Among all cancers, hepatocellular carcinoma (HCC) has the second highest mortality rate worldwide [1]. The risk factors, including HBV or HCV infection, alcoholism, liver cirrhosis, and metabolic diseases, contribute to HCC [2]. The molecular mechanism of hepatocarcinogenesis is highly complex and involves an interplay between dysregulated cell cycle, apoptosis, tumor cell invasion, and metastasis [2]. Despite advances in diagnosis and therapy, the incidence and mortality of liver cancer continue to increase [3]. It is vital therefore to illustrate the molecular mechanism of HCC in order to improve diagnosis, treatment, and overall prognosis.
With the development of human genome sequencing technology, about 20000 protein-coding genes have been identified, which account for less than 2% of the entire genome [4]. In fact, greater than 90% of the human DNA would be converted into noncoding RNAs (ncRNAs), which, despite not being translated into proteins, are involved in several cellular functions [5,6]. The long ncRNAs (lncRNAs) with more than 200 nucleotides play significant roles in cell growth and differentiation, chromatin organization, and regulation of gene expression [7,8]. lncRNAs are classified into intronic, intergenic, sense, and antisense types based on their genomic location [9] and into signaling, decoy, guide, and scaffold lncRNAs on a functional basis [10]. Signaling lncRNAs mainly act as transcription factors or as intermediates in various signaling pathways [10], and decoy lncRNAs act as "molecular sponges" by binding to and sequestering transcription factors away from their target genes [10]. Guide lncRNAs can regulate gene expression through chromatin remodeling by recruiting chromatinmodifying enzymes [10]. Finally, the scaffold lncRNAs act as recruiting platforms for multiple proteins and form lncRNAribonucleoprotein (lncRNA-RNP) complexes, which subsequently regulate downstream signaling [10].

BioMed Research International
Numerous lncRNAs have been identified recently with the help of high-throughput sequencing and microarrays. Most of them are aberrantly expressed in tumors like HCC, breast cancer, lung cancer, colorectal cancer, and others [11]. lncRNAs are known to regulate cell proliferation, epithelialmesenchymal transition (EMT), angiogenesis, metastasis, autophagy, and so forth. Considering their cancer specific expression and detectable presence in clinical samples like blood and urine, lncRNAs are potential diagnostic markers for tumors. Therefore, a better understanding of HCC specific lncRNAs will greatly contribute to the diagnosis and treatment of HCC.
lncRNAs exhibit both tumor suppressive and oncogenic roles. In the present review, we will concentrate mainly on the functions, molecular mechanisms, and potential clinical implications of HCC-related lncRNAs that are abnormally expressed and therefore have critical roles in hepatocarcinogenesis.

HOTTIP.
"HOXA transcript at the distal tip" or HOTTIP is greatly expressed in HCC tumor tissues and cells [52] and is linked with a greater threat of metastasis and poor OS [52]. Studies have shown the effect of HOTTIP on HCC proliferation, metastasis, and glutamine metabolism (Table 1) [52][53][54].
HOTTIP downregulates miR-125b, miR-192, and miR-204 and enhances the cell growth and migration through the miR-192/-204-HOTTIP axis [53,54], while HOTTIP inhibition decreases growth of HCC cells [52,54]. In addition, HOXA13 and GLS1 are further revealed to be the likely target genes of miR-192/-204-HOTTIP axis, and overexpression of miR-192 and miR-204 is associated with increased survival in the patients [54]. Taken together, these findings imply the oncogenic role of HOTTIP in hepatocarcinogenesis through miRNA interaction.

MVIH
. "Microvascular invasion in HCC" or MVIH is situated at chromosome 10 and was firstly identified by Yuan et al. in HCC [55]. High levels of MVIH in HCC were correlated with enhanced invasion and poor prognosis with decreased RFS and OS [55]. As shown in Table 1, MVIH plays important roles in proliferation, migration, apoptosis, metastasis, and angiogenesis in HCC [55][56][57].
MVIH exerts its proangiogenic action by inhibiting PGK1 secretion [55]. It also acts like a sponge for miR-199, and MVIH-mediated inhibition of miR-199 leads to increased proliferation and apoptosis inhibition in HCC cells [56]. Recently, MVIH was reported to control proliferation and migration of HCC cells via modulation of ARID1A-mediated regulation of CDKN1A [57]. Taken together, these findings underscore the oncogenic role of MVIH in HCC.
2.6. PVT1. Murine PVT1 was first identified in the liver where it accelerated proliferation and cell cycling and enhanced stem-cell-associated properties [58]. Human PVT1 is overexpressed in HCC tumor tissues and cell lines and is linked with advanced TNM stage and poor prognosis as well as RFS [58][59][60], and upregulation of PVT1 can also predict HCC recurrence [59]. As shown in Table 1, PVT1 plays an oncogenic role in multiple cellular processes like proliferation and invasion and increases the stemness of HCC cells [58,60,61].
Functionally, through interaction between PVT1 and NOP2, PVT1 enhances the expression of NOP2 via stabilizing NOP2, thus promoting proliferation, cell cycle, and stemness of HCC cells [58] (Table 1). In addition, PVT1 can also induce miR-214 inhibition via interaction with EZH2 to promote cell proliferation and invasion [60], forming a PVT1/EZH2/miR-214 axis (Table 1) [60]. The clearly oncogenic role of PVT1 indicates its potential use as a biomarker in diagnosing and predicting recurrence in HCC.
In addition to the lncRNAs mentioned above, several others are upregulated during hepatocarcinogenesis, including DANCR, HEIH, and Linc-ROR (Table 1).
MEG3 can be negatively regulated by UHRF1 via modulating DNA methylation, since its promoter region is highly methylated [73]. One mechanism of MEG3 mediated tumor suppression is the activation of p53 by increasing its stability and modulating the downstream genes [72,74] (Table 2). Using a novel delivery system, MEG3 was introduced into HCC cells and resulted in tumor growth inhibition via the p53 signaling, indicating a bona fide tumor suppressive role of MEG3 in HCC [74]. Furthermore, it acted as a molecular sponge for miR-664 and could inhibit cell proliferation by modulating miR-664-mediated regulation of ADH4 [76]  ( Table 2). Taken together, MEG3 is a tumor suppressor and might be considered a prospective diagnostic, predictive and therapeutic biomarker in HCC.

3.3.
Dreh. "Downregulated expression by HBx" or Dreh was first identified by lncRNA microarray on WT and HBxtransgenic mice [77]. It is low expressed in the tumor tissues of HBV-related HCC patients and corresponding cell lines [77,78]. Patients with decreased expression of Dreh showed poor survival [77]. As shown in Table 2, Dreh is linked with the proliferation and metastasis of HBV-related HCC.
A previous study revealed a negative correlation of Dreh expression with HBx and HBs [78]. Dreh is downregulated by HBx via downregulation of vimentin, which results in the suppression of HCC growth and migration [77,78] (Table 2), thus underscoring the tumor suppressive role of Dreh in HBV-related HCC.
3.4. LET. "Low expression in tumor" or LET is present in significantly low levels in HCC tumor tissues [79] and is linked with metastasis [79]. As shown in Table 2, LET influences the invasiveness and metastasis of HCC cells.
LET is downregulated by HDAC3 [79], and LET inhibition increases the stability of NF90, thus promoting hypoxiainduced invasion [79] (Table 2). This was successfully validated in an HCC clinical sample with abnormal histone acetylation, downregulation of LET, and upregulation of NF90. These findings suggest a tumor suppressive role of LET centered around regulating metastasis under hypoxia.
As shown in Table 2, along with the lncRNAs discussed above, several others have been indicated to influence hepatocarcinogenesis, such as ZNFX1-AS1, PTENP1, and XIST.  (Table 3) [15, 17]. In addition to plasma, serum and exosomes can also be used for lncRNA detection. For example, HEIH, an oncogenic lncRNA expressed highly in HCC tissues, was also found to be overexpressed in the serum and exosomes of patients with HCV-related HCC (Table 3). In addition to HULC and HEIH, many other lncRNAs could also serve as biomarkers of HCC which are shown in Table 3.

lncRNAs as Diagnostic Biomarkers and Drug Targets in HCC
Since various lncRNAs are abnormally expressed in HCC and affect many downstream genes and related signaling pathways through oncogenic or tumor suppressive action, restoring these lncRNAs to their normal expression level is a therapeutic option worth considering, especially as an alternative to the chemotherapeutic drugs which usually result in chemoresistance [80]. Pharmaceutical companies have recently shown a great interest in lncRNA-targeted therapy and have already taken actions [81,82]. lncRNAs could be upregulated by exogenous overexpression and directly targeted by their specific siRNAs or antisense oligonucleotides [83,84]. For example, the tumor suppressor MEG3 introduced into HCC tumor through a novel delivery system effectively induced apoptosis in HCC cells [74], presenting a potential lncRNA-targeted therapy with fewer side effects. Therefore, clarifying the specific mechanism of lncRNA action will greatly promote the advancement of lncRNAbased diagnosis and therapy for HCC.

Conflicts of Interest
The authors declare that they have no conflicts of interest.