Epigenetic and transcriptional regulation of oncogenic programs by nuclear lncRNAs in HCC

In case of their functionality, lncRNAs that are enriched at their own loci of transcription should be part of the process of chromatin modification. The functions of lncRNAs in regulating the transcriptional activity of local or distant genes can be achieved through various methods, including but not limited to the down-regulation of the activity of epigenetic regulators, direct contact between lncRNAs and the DNA, or enhancing enhancer-promoter interaction.

Cytoplasmic lncRNAs as key modulators of post transcriptional processes

LncRNAs are not limited to regulating protein localization on chromatin. Most lncRNAs are exported to the cytoplasm and regulate two processes that take place in the cytoplasm, which significantly affect the process of protein translation turnover and translation of the mRNA.

LncRNAs are capable of controlling mRNA dynamics at the post-transcriptional level by (1) miRNA sponge, (2) mRNA contact, and (3) RBP recruitment. The sequence-specific interaction makes lncRNAs fine-tune the gene expression with a preference toward the HCC tumorigenesis through regulating the mRNA stability and miRNA availability. miRNAs have also been shown to bind the target mRNAs, which are suppressed by the lncRNA-mRNA complex, reversing the suppressive action of miRNAs on mRNA targets [20]. As an example, the lncRNA DANCR (differentiation antagonizing non-protein coding RNA) has been shown to bind the miRNA binding site of CTNNB1 (catenin beta 1) on 3′ UTR that amplifies the number of tumor cells with stemness characteristics [21].

Evidence compilation has suggested that lncRNAs are able to mediate protein stability by regulating ubiquitin or proteasome machinery. In HCC, some lncRNAs stabilize oncoproteins by exerting their oncogenic activities [22]. As an example, lncCSMD1-1 binds to and stabilizes MYC protein by inhibiting its ubiquitin-proteasome-mediated degradation, thereby promoting hepatocarcinogenesis [22]. In the same way, lncRNA PSTAR (p53-stabilizing and activating RNA) suppresses the tumorigenicity of HCC through increasing the SUMOylation of heterogeneous nuclear ribonucleoprotein K (hnRNP-K), which also intensifies the interaction between hnRNP-K and p53, resulting in transactivation of p53 [23]. Interestingly, there are lncRNAs that regulate protein translation. GMAN is a gastric cancer metastasis-associated lncRNA that was discovered to bind with eukaryotic translation initiation factor 4B (eIF4B) and stimulate its phosphorylation through inhibition of dephosphorylation of another protein mediator phosphatase 2A subunit B (PPP2R2A). The above changes in turn enhance anti-apoptotic protein expression, thus leading to tumorigenesis of HCC [24].

Research has failed to explain either the biological functions or molecular mechanisms of most lncRNAs because scientists have only partly characterized a small fraction of these molecules. The existing evidence shows that these molecules operate as vital components for regulating particular cellular events with a focus on protein-coding gene expression across epigenetic, transcriptional, and post-transcriptional levels (Figure 1).

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Figure 1. Summary of the molecular roles of lncRNA in liver cancer and its use in clinical therapies. Dysregulation of lncRNAs through epigenetic and genetic changes causes the lncRNAs to play the roles of either activators or suppressors in the process of initiating liver cancer. The dysregulated expression of lncRNAs can serve as a signal, guide, decoy, and scaffold and influence the expression of protein-coding genes, which consequently triggers complex interactions and regulations with either positive or negative effects on tumor proliferation and metastasis. The abnormal lncRNAs can be detected, targeted, and even used as indicators of the prognosis of liver cancer. LncRNA: long non-coding RNA.

The diversity of lncRNA structures results in a wide array of molecular functional roles in both normal and abnormal biology. A complex regulatory system that regulates the expression of lncRNAs has developed as a result of the magnitude of the non-coding transcriptome. The expression pattern of lncRNA molecules depends on developmental processes that make them both cell-type-specific and time-dependent. LncRNA expression in specific tissues helps preserve cell differentiation alongside cellular features. Many EZH2-associated lncRNAs have been demonstrated to exhibit tissue-specific binding capacity, implying a tissue-specific function for lncRNAs in chromatin remodelling and cellular imprinting [25]. Achieving global transcriptional control occurs through two methods: chromatin remodelling along with transcriptional repression/activation. Temporal control of lncRNA expression may influence differentiation and cell fate as global transcription regulators, either directly or epigenetically. LncRNAs utilize specific signals in their 3′ UTRs to regulate spatial positioning as well as temporal activities of their transcript location. Studies show this transcript placement pattern mainly occurs within nervous tissues because their cells have intricate structures [26]. The DNA-binding ability of lncRNAs allows them to recruit proteins, which initiate DNA looping that results in super-enhancer activation both within cis regulators and between trans regulators. The lncRNAs UCA1 and HCCL5 activate YAP gene super-enhancers through their association with epithelial-mesenchymal transition (EMT) transitions in ovarian cancer cells as well as HCC cells [27, 28]. The DNA-binding ability of lncRNAs enables them to attach to promoter areas where they potentially operate as either activating or repressive elements. LncRNAs can bring in transcription factors or DNMTs. The transcriptional machinery cannot make contact with DNA when lncRNAs bind to it in antisense orientation to block transcription. Molecular sponge functions of lncRNA transcripts enable them to trap both transcription factors and miRNAs. These transcripts function as endogenous competing endogenous RNAs (ceRNAs) that have the ability to either deplete proteins/enzymes or small RNA transcripts to block normal functionality. The transcripts can be used to transport their binding targets into subcellular areas where they prompt physiological responses. Messenger RNA stability changes through two distinct actions by lncRNAs: they either trap miRNAs or directly interact with them. When lncRNA binds RNA directly, it causes cells to move proteins or enhance stability while suppressing expression and changing processing outcomes. LncRNAs perform dual roles within the cell by orchestrating alternative splicing events in other transcripts and engaging in post-translational modifications (Figure 2). The intricate structures and substantial lengths of lncRNA transcripts render them ideal components for functioning as subunits of ribonucleoprotein (RNP) complexes. Within these complexes, lncRNAs provide structural support while also guiding them to their designated binding sites. The RNPs that lncRNAs create consist of polycomb repressive complex alongside histone deacetylases (HDACs) and histone acetyl transferases (HATs), as well as other components. The formation of heterochromatin and euchromatin, along with chromatin stability, receives essential contributions from lncRNAs. XIST is a paradigmatic chromatin-regulatory lncRNA that facilitates X-chromosome inactivation, and its female-specific expression is a paradigm example of sex-biased lncRNA expression [29]. Research shows that particular lncRNAs might create heritable effects through modifying epigenetic patterns [30].

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Figure 2. Mechanistic roles of lncRNAs. (A) Signal lncRNA. The primary role of a signal lncRNA is to act as a molecular signal to coordinate the expression of some genes to different stimuli, e.g., transcription factors or chromatin modifiers. (B) Guide lncRNA. Guide lncRNAs are associated with chromatin-modifying enzymes and are directed to the particular location on the genome in order to control gene expression. (C) Decoy lncRNA. Decoy lncRNAs attach to the miRNAs or the transcription factors in order to sequester the miRNA or transcription factor away from its target, affecting translation and transcription. (D) Scaffold lncRNA. LncRNAs are capable of serving as scaffolds to multi-component complexes, including ribonucleoprotein (RNP) complexes, to temporarily form and influence histone modifications. LncRNA: long non-coding RNA; miRNA: microRNA.

Invasion/Migration

Cancer cell migration and stromal invasion, while related to metastasis, significantly influence disease development and treatment. HCC is considered a whole-organ disease due to the highly invasive and migratory nature of tumor cells within the liver. These characteristics lead to extensive intrahepatic spread, making HCC a particularly aggressive form of cancer [31]. Surgical removal of highly infiltrative tumors proves complex and tends to result in tumor recurrence after surgery. Cancer cell invasion and migration potentials show an association with numerous lncRNAs, which demonstrate both promoting and inhibiting effects. The invasive and migratory behavior in cervical cancer cells increases when lncRNA RHPN1-AS1 is expressed because it regulates the miR-299-3p/FGF2 signaling pathway [32]. Papillary thyroid carcinoma susceptibility candidate 3 (PTCSC3) overexpression leads to inhibition of cervical cancer cell invasion and migration through capturing the miR-574-5p miRNA [33]. Breast cancer cells exhibit enhanced EMT and proliferation due to nuclear enriched abundant transcript 1 (NEAT1), but this noncoding RNA also enables endometrial cancer cells to migrate and invade through controlling miR-144-3p/EZH2 activity [34]. NEAT1 drives metastasis and invasion events in colon cancer cells by means of the miR-185-5p/IGF2 regulatory pathway [35]. Through different signaling pathways, these three lncRNAs, EZR-AS1, LINC00261, and LINC01082, exhibit suppressive effects on cell migration in colon cancer cells [36]. The MYLK-AS1 axis leads EGFR-AS1 to promote migration and invasion in HCC patients [37].

Angiogenesis

The rapid tumor cell multiplication leads to higher requirements for gas and nutrient exchange processes. Metastasis depends on tumor-associated blood vessels. Both angiogenesis promotion and endothelial cell differentiation capabilities are common in cancer cells [38]. HITT functions as a novel translation-level inhibitor of HIF-1α that is frequently downregulated in human cancers, thus promoting both angiogenesis and tumor growth through HIF-1α expression [39]. The repression of HIF-1α occurs when HITT functions together with EZH2 to establish epigenetic regulation [40]. Breast cancer metastasis and angiogenesis occur due to hypoxic conditions that induce transcription of the lncRNA RAB11B-AS1 [41]. Breast cancer angiogenesis occurs through IGF-1/ERK signaling regulation, which is controlled by the lncRNA NR2F1-AS1 [42]. The NF-κB-interacting lncRNA (NKILA) controls immune responses against cancer together with its ability to regulate blood vessel formation in breast cancer cells by modifying NF-κB/IL-6 signaling pathway activity [43]. The expression of mesoderm-specific transcript (MEST) becomes inhibited by LINC00284 through an NF-κB dependent pathway, which causes increased angiogenesis in ovarian cancer cells [44]. The DANCR facilitates tumor angiogenesis by regulating the miR-145/VEGF relationship in ovarian cancer [45]. The therapeutic outcomes for HCC patients can be improved through LINC01446-targeted therapy because it limits both tumor evolution and angiogenesis through the SRPK2/SRSF1/VEGFA165 pathway [46].

Replicative immortality

The behavior of cancer cells includes continuous, uncontrolled cellular reproduction. The key characteristic of many cancer cells involves their ability to bypass replicative senescence as they develop replicative immortality by stabilizing their chromosomes, most commonly through extended telomeres [47]. The RNA molecule that plays the primary role in telomere maintenance is telomeric repeat-containing RNA (TERRA) [48]. Elongated telomeres are linked with high TERRA expression in the human placenta [49]. Increased expression of TERRA has been observed in multiple human cancer cell lines [50]. SENEBLOC inhibits cellular senescence through the stabilization of p53-MDM2 interaction, and inhibition of p21 transcription, and also by an independent p53-independent mechanism, which includes HDAC5 [51]. Linc-ASEN disables cell aging through p21 reduction, which permits unlimited cell cycle progression [52].

Genome instability and mutation

Cancer cells develop extremely high mutation rates when they undergo multiple divisions triggered by genomic instability. Genomic instability in cells causes the removal of tumor suppressors and the duplication of oncogenes. The loss of the PTEN gene marks an example of this phenomenon in HCC. LncRNA transcripts have been identified as genomic instability regulators. TERRA is a telomere-regulated lncRNA, which helps in maintaining telomeres and stability at chromosome ends, hence maintaining genomic integrity [53]. The lncRNA MANCR (LINC00704) demonstrates an elevated expression within highly mitotic cells in triple-negative breast cancer (TNBC) [54]. ncRNA activated by DNA damage (NORAD) is a long noncoding RNA that functions as a scaffold for the building of topoisomerase complexes essential for preserving genomic stability [55]. NORAD also fosters chromosomal integrity by sequestering destabilizing PUMILIO (PUF family RNA-binding) proteins [56]. When NORAD expression decreases in cancer cells, it may create genomic instability, which produces more copy number variants (CNVs) along with elevated mutational loads. CCAT2 has been shown to induce chromosomal instability by activating the BOP1–AURKB signaling axis [57].

Proliferation

Among the fundamental characteristics of cancer cells are sustained proliferation, together with growth suppressor evasion. Evidence shows that the lncRNA CASC11 (cancer susceptibility candidate 11) promotes bladder cancer cell proliferation by binding to miR-150 [58]. Studies have revealed that the lncRNA CCAT1, which was discovered in CRC, actively stimulates bladder cancer cell proliferation as well as enhances cell migration and invasion [59]. The lncRNA GClnc1 activates proto-oncogene MYC to promote both proliferation and invasion in bladder cancer cells [60]. The antisense lncRNA DLG1-AS1 enables cervical cancer cell proliferation by reducing miR-107, which allows ZHX1 levels to increase [61]. The research community has extensively studied the lncRNA transcript NEAT1 because it activates breast cancer proliferation and EMT pathways [62]. The lncRNA SNHG6 affects breast cancer cell proliferation and invasion through its impact on miR-26a/VASP signaling [63]. The breast cancer proliferation and migration rates increase due to lncRNA PSMG3-AS1-mediated titration of miR-143-3p [64]. Evidence shows that elevated TUG1 levels in HCC tissue enhance cell proliferation and inhibit apoptosis, which indicates its possible use as both a diagnostic tool and therapy target for this disease [10].

Immune evasion

Cancer cells employ two distinct immune evasion strategies: intrinsic mechanisms that suppress immune recognition and extrinsic mechanisms that reprogram normal immune cells to support tumor survival. The tumor creates inflammatory situations that promote tumor growth. Research shows that lncRNAs take part in programming cancer cells for immune-related processes. SATB2-AS1 is a tumor-suppressive lncRNA in colorectal cancer; the reduced expression of it is linked to an aggressive form of the disease, whereas its increased expression suppresses the progression of CRC by inhibiting the transcription of Snail through SATB2 and epithelial-mesenchymal transition [65]. LINK-A stands as a novel lncRNA that suppresses tumor antigenicity and immune-related tumor suppression by activating TRIM71 signaling pathways in TNBC cells [66]. NKILA has been shown to promote activation-induced cell death in cytotoxic T cells, resulting in immune evasion in breast and lung cancer [67]. During breast cancer development, SNHG1 regulates regulatory T cell (Treg) differentiation, which affects immune escape through the miR-448/IDO pathway [68]. Some tumors use recruitment tactics to bring tumor-associated macrophages (TAMs) to help tumors grow and survive. Breast cancer cells receive survival-promoting glycolysis stimulation through the HISLA lncRNA, which TAM cells release within extracellular vesicles [69]. The lncRNA DGCR5 functions as a tumor suppressor for HCC while influencing immune-related biological processes by employing three mechanisms that include sponge activity against miR-346 and the regulation of KLF14 and Wnt signaling pathway inhibition [70].

Metastasis/Tumorigenesis

Multiple cancers develop their metastatic ability in specific groups of perivascular cells. Metastatic cells must transfer into lymphatic system before exiting to establish new tumors at sites removed from the primary tumor. The evidence suggests that lncRNAs provide metastatic abilities to these specialized cells. Research shows that the novel lncRNA XLOC_006390 promotes cervical cancer tumorigenesis and metastasis by acting as a competing endogenous RNA against miR-331-3p and miR-338-3p [71]. Under hypoxic conditions, the lncRNA BX111 gets activated to promote pancreatic cancer metastasis through EMT activation of ZEB1 transcription [72]. The lncRNA PTAR facilitates ovarian cancer metastasis through inhibition of miR-101-3p, which releases ZEB1 expression to promote EMT [73]. The functions of lncRNAs are not usually explicitly defined. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been characterized as imparting metastatic capability, especially in lung-to-brain metastasis via the regulation of EMT [74]. MALAT1 promotes metastatic progression through the suppression of pyroptosis and T cell-mediated destruction of incipient metastatic cells in breast cancer [75]. LINC01133 prevents breast cancer metastasis by regulating SOX4 expression through EZH2 activity [76]. LINC0273 promotes breast cancer metastasis by stabilizing hnRNPL, hence activating AGR2 transcription [77].

Resisting cell death

Replicative immortality represents a distinctive cancer cellular feature because it enables cells to escape from normal programmed death mechanisms. The regulation of apoptosis by lncRNAs includes both positive and negative effects, which have been documented in this cellular hallmark. In the same manner, lncRNA ENST00000422059 was demonstrated to stimulate the growth of breast cancer cells and suppress their apoptosis via miR-145-5p/KLF5 axis [78]. Similarly, another lncRNA PART1 controls apoptosis in prostate cancer cells through the regulation of Toll-like receptor signaling pathways [79]. LncRNA-ATB has been found to induce apoptosis in non-small cell lung cancer via miR-200a/β-catenin [80]. The absence of the lncRNA MEG3 in prostate cancer cells causes lower apoptosis levels because QKI-5 expression is not restrained by miR-9-5p [81]. Research indicates that LncKLHDC7B links to TNBC resistance against cell death along with unfavorable clinical expectations [82]. CCAT1 enhances the progression of tumor by sponging miR-181a-5p and modulating the autophagy involving ATG7 in HCC [83].

Since HCC has become more resistant to traditional treatment methods such as sorafenib, alternative treatment modalities, such as those that take advantage of a regulated cell death mechanism such as ferroptosis are urgently needed [84]. This is especially considering that inhibition of the retinoblastoma protein has been identified to increase the activity of sorafenib by aggravating ferroptosis in vitro and in vivo [85]. Ferroptosis, a specialized type of programmed cell death that involves accumulation of lipid peroxides and reactive oxygen species, which is iron-dependent, is a promising option to avoid apoptosis resistance that is common in most cancer types. In fact, it has been shown that ferroptosis can be used to suppress HCC progression and increase its sensitivity to sorafenib, and ferroptosis inhibition predictably accelerates the progression of HCC and promotes resistance to the drug [86]. Therefore, the complexity of regulatory mechanisms of ferroptosis, especially lncRNAs, is an essential barrier to the creation of effective treatment methods.

Altered metabolism

The therapeutic target of aberrant metabolism in cancer cells attracts researchers due to its long-standing attractiveness. Cancer cells exhibit the Warburg effect while producing energy from aerobic glycolysis for their cellular needs [87]. The research shows that LINC00504 stimulates aerobic glycolysis in ovarian cancer cells by depleting miR-1244 [88]. The lncRNA MALAT1 creates simultaneous glycolytic increases and suppresses gluconeogenesis in HCC by enhancing mTOR-mediated translation of transcription factor 7-like 2 (TCF7L2) [89]. EPB41L4A-AS1 functions as a Warburg effect repressor through its binding with HDAC2 and NPM1, which occurs within multiple cancer sites [90]. The lncRNA SNHG14 (small nucleolar RNA host gene 14) acts as an important promoter for cancer progression in HCC. The H3K27 acetylation of PABPC1 regulated by SNHG14 leads to increased cell proliferation as well as migration and angiogenesis in HCC tissues and cells [91]. The research shows that SNHG14 affects metabolic processes in HCC cells by controlling the activity of genes connected to glycolysis and other metabolic pathways. The therapeutic evaluation of SNHG14 or its subsequent targets represents a possible therapeutic approach to treat HCC.

Stemness and multipotency

Research indicates that CSCs function as the main agents behind tumor formation while simultaneously causing treatment resistance. CSCs present three essential capabilities, including self-renewal, multipotency, and survival capacity. CSC replication and differentiation produce a hierarchical cell arrangement within tumors, which enhances their aggressive nature and makes them hard to overcome. LncRNAs sustain both stemness properties and cellular fate programs with tumor microenvironment (TME) niches that support CSCs. MALAT1 has also been reported to mediate miR-375/YAP1 axis to promote cancer stem cell properties in HCC [92]. Research indicates that stemness-related transcription factor Oct4 operates in a feedback loop by triggering the expression of MALAT1 and NEAT1 [93]. The YAP transcription factor functions as an oncogene that enables multiple cancer programs including stemness [94]. The lncRNA B4GALT1-AS1 functions to bring YAP into the nucleus so it can boost its transcriptional output, which increases cancer stemness in colon cancer cells [95]. It has been reported that SNHG20 enhances the development of HCC and is linked to an unfavorable prognosis, partially by regulating the EZH2/E-cadherin axis [96].

DNA damage response

Standard cancer treatments through radiation therapy or chemotherapy drugs struggle against cancer cells because these cells keep their DNA repair systems in overdrive. The transcriptional programs linked to DDR are controlled by specific lncRNAs. Homologous recombination and other DDR pathways are actively expressed in multiple myeloma (MM) cells when NEAT1 becomes activated [97]. The structural role of NEAT1 in forming paraspeckles plays a potential part in both DDR regulation and activity [98]. The DNA repair mechanism of TNBC depends on LINP1, which functions as a structural part of the IGFBP-3/NONO/SFPQ complex [99]. Alternative NHEJ in MM occurs through MALAT1, which binds PARP1 and LIG3 as well [100]. The sorafenib resistance mechanism in HCC depends on MALAT1, which regulates miR-140-5p/Aurora-A signaling, thus providing potential outlooks for patient prognosis and therapy development when using sorafenib [101]. The research about lncRNAs and drug resistance has been the focus of attention in recent years. Many lncRNAs have been demonstrated to be linked with drug resistance [102]. LncRNAs are important in chemotherapy resistance by controlling major cellular processes like drug efflux, DNA repair, apoptosis, and cell survival [103]. As illustrations, lncRNAs such as the H19 and MALAT1 have been reported to increase drug efflux-related resistance by increasing transporters including the MDR1/ABCB1 (P-glycoprotein) and MRP1, leading to decreased intracellular accumulation of drugs and decreased chemosensitivity [102]. Furthermore, the HCC-related lncRNA HANR has been reported to cause the resistance of HCC cells to doxorubicin through its interaction with GSKIP and subsequent alteration of GSK3β phosphorylation, which is a factor that leads to chemoresistance [104].

Although ICIs have a promising future, a significant percentage of patients show primary or acquired resistance, which restricts their effectiveness, and this indicates the necessity of further investigation of the mechanisms of resistance [105]. LncRNAs have the capability to control different cellular activities that are essential in immune evasion and tumor growth such as the regulation of the expression of immune checkpoint proteins and the complex signal transduction networks that regulate immune cell activities [106]. Moreover, some lncRNAs including lncRNA-MIAT (myocardial infarction associated transcript) and lncRNA-MIR155HG have been directly associated with the expression of immune checkpoints (PD-1, PD-L1, and CTLA-4), hence enabling immune evasion in HCC [103]. This is a regulatory capacity that is also applicable to tumor-infiltrating lymphocytes, and promotes T-cell exhaustion and dysfunction, which in turn impairs the anti-tumor immune response and promotes an immunosuppressive microenvironment favorable to ICI resistance [107].

Recruitment of stromal cells

The TME depends on lncRNAs to progress disease and invade tissues by using these molecules to change tumor-stroma relations and develop new blood vessels and hide from immunity and dismantle extracellular matrix structures [108]. Proliferating fibroblast cells in CRC tumors transfer the H19 ncRNA through exosome transmissions to generate cells with increased resistance to chemotherapy and stem-like properties [109]. Stromal lncRNA lnc-CAF, which is upregulated in the formation of CAF, enhances the growth of OSCC by maintaining IL-33 signaling [110]. Hypoxia-induced HIF-1alpha transcriptionally increases lncRNA HAS2-AS1 in OSCC that facilitates EMT and invasiveness through the stabilization of HAS2 [111]. The TGF-β signaling regulator lncRNA-ATB is believed to increase the risk of HCC metastasis and represents a promising therapeutic candidate to prevent disease spread [112]. To be precise, the MALAT1 overexpression in the HCC cells facilitates the angiogenic activity and creates an immunosuppressive environment. This is achieved when MALAT1 interacts with miR-140 to inhibit the activity of miR-140 and, therefore, enhances the production of VEGF-A, which assists the progression of HCC by promoting angiogenesis and the polarization of macrophages towards the M2 immunosuppressive lineage [113]. In addition to macrophages, lncRNAs have important functions in regulating T-cell functions in HCC, which affects the development of the disease and immune evasion. Indicatively, lncRNA epidermal growth factor receptor (lnc-EGFR) is profusely expressed in Tregs in the HCC. Lnc-EGFR binds to EGFR, prevents its ubiquitination through c-CBL, and enhances downstream signaling through AP-1/NFAT1 to induce differentiation of Treg cells and immune evasion [114]. The MIAT is another lncRNA whose expression is elevated in multiple cell types that are related to the disease, tumor cells, FoxP3+ Tregs, PD-1+ CD8+ T cells, and GZMK+ CD8+ T cells. Moreover, the expression of MIAT is related to the response of patients to sorafenib. The expression of this lncRNA is also correlated significantly with the expression of PD-L1, which is an immune evasion protein expressed by cancer cells [115]. The immune environment in HCC is a complicated interaction of different types of cells, which are coordinated and controlled by lncRNAs. In a study, Jiang and Li [116] have shown that this is indicated by the polarization of macrophages, which is a vital cellular constituent in the environment of HCC interplay.

Conversely, the non-coding developmental regulatory lncRNA, fetal-lethal ncRNA [FOXF1 adjacent non-coding developmental regulatory RNA (FENDRR)], is a miR-423-5p sponge that inhibits the immune-suppressive activities of Tregs. An overexpressed FENDRR competitively binds miR-423-5p and increases growth arrest and DNA-damage-inducible beta protein (GADD45B), which have negative relationships with the number of Treg-cells and leads to decreasing immunosuppressive cytokines TGF-β1 and IL-10, resulting in tumor-cell apoptosis [117]. Besides, lncRNAs Tims and lncNNT-AS1 are linked with less tumor CD4 and CD8 T cell infiltration, which affects the clinical outcomes and immunotherapy response [118].

Coding potential of lncRNAs

Recent findings have radically changed the old perception according to which lncRNAs do not have the ability to code any protein and have shown that a significant fraction of lncRNAs contain non-canonical open reading frames that encode functional microproteins or small peptides. It is emerging that these microproteins coded by lncRNAs are key regulatory factors in tumorigenesis because they can directly regulate important oncogenic signaling pathways. In HCC, Zhao et al. [151] systematically discovered hundreds of translatable lncRNAs and established that the lncRNA ASH1L-AS1 encodes a microprotein, APPLE, which consists of 90 amino acids and is strongly expressed in HCC tissues and is linked to poor prognosis. Mechanistically, APPLE enhances HCC development by maintaining MAPK/ERK signaling through direct interaction with ERK1/2 and preventing PP1/PP2A-mediated ERK dephosphorylation, which is a non-canonical pathway activation mechanism that does not rely on classical RAS mutations [151]. These results demonstrate that lncRNAs may have oncogenic actions that are mediated by both RNA-mediated regulatory processes and through the products of their translation, microproteins, and so the translational potential of lncRNAs should be included in future analysis of HCC biology and therapeutic targeting.

It is increasingly evident that lncRNAs play key post-transcriptional regulatory roles, including but not limited to alternative splicing, and thus play a role in cancer pathogenesis and progression, such as in HCC. Malakar et al. [152] have shown that the lncRNA MALAT1 enhances the development of hepatocellular carcinoma by increasing the activity of the splicing factor, SRSF1, which leads to oncogenic alternative splicing and tumor growth. Mechanistically, MALAT1 has an oncogenic effect through the upregulation of SRSF1 and alteration of alternative splicing of cancer-related transcripts, which is accompanied by the activation of Wnt signaling and the output of the mTOR pathway, which triggers the development and progression of HCC [152]. These results have suggested alternative splicing as a major post-transcriptional mechanism by which lncRNAs promote hepatocarcinogenesis, and the crucial role of integrating splicing-based lncRNA mechanisms into models of HCC pathogenesis.

LncRNAs as biomarkers

Beylerli et al. [153] emphasized the fact that tumor cells secrete lncRNAs into human biological fluids, creating stable circulating lncRNAs, insensitive to RNA degradation. These lncRNAs have been detected in cancer patients as being expressed aberrantly [153]. In this way, lncRNAs are becoming an effective alternative to the conventional biomarkers (AFP, etc.) to diagnose HCC and provide a prognosis. The effectiveness of AFP as an early predictor of HCC has been a controversial subject since it raises the question of its sensitivity and specificity [154]. LncRNAs provide greater sensitivity and specificity, which may overcome limitations of conventional markers, e.g., AFP [155]. The resultant effect is an increasing need to identify new diagnostic markers that can potentially substitute AFP, and lncRNAs may be one of the possible alternatives. Thus, the expression level of these lncRNAs may be an important indicator of progression and prognosis of the diseases.

As the trend moves towards minimally invasive and non-invasive methods of diagnostic technology, circulating lncRNAs in serum are currently under intense research. As an example, overall survival, progression-free survival, tumor size, TNM stage, levels of C-reactive proteins, T stage, and portal vein thrombosis were all correlated with high serum levels of lncRNA-ATB, which suggests that they might be useful as serum biomarkers in patients with HCC [156].

Prominently, the lncRNAs like MVIH, X91348, and HOTTIP have demonstrated potential as prognostic markers in HCC. MVIH has been identified as a high level of microvascular invasion which is known to be an independent risk factor of recurrence-free survival and overall survival in HCC patients [157]. Similarly, lncRNA X91348 is significantly downregulated in HCC relative to healthy subjects, and reduced X91348 expression is associated with unfavorable overall survival [158].

In addition, a number of studies revealed lncRNAs, including UCA1 and WRAP53, as the potential biomarkers in the diagnosis of HCC when combined with AFP [159]. LINC00152, RP11-160H22.5, and XLOC014172 were also found to be new biomarkers of HCC by another group. A combination of these lncRNAs with the traditional marker AFP was observed to increase the accuracy of diagnosis of HCC, which suggests that they can be used to enhance the diagnosis of HCC [160].

In the environment of chemotherapy resistance, some lncRNAs, including CAHM, were recognized as main predictive biomarkers. By applying machine learning algorithms, CAHM was described as a central lncRNA, whose expression is upregulated in sorafenib-resistant cell lines, which makes it a promising biomarker for chemotherapy resistance in the future [161].

In short, lncRNAs have become the potential biomarkers of HCC, and the importance of lncRNAs in diagnostic and prognostic precision medicine is clear. Despite the fact that PIVKA-II is a well-known biomarker of HCC, and lncRNAs have been independently identified as promising non-invasive biomarkers, there is little direct comparison between PIVKA-II and lncRNA biomarkers [162]. Large multicentric studies are required to confirm the clinical importance of lncRNAs. Also, it is essential to standardize approaches to detect lncRNAs that are circulating to achieve the same results. Nevertheless, the prospects of studying lncRNAs as biomarkers in HCC indicate that such studies can improve the diagnosis and prognosis of this malignancy, as well as targetable treatment.

Aberrant lncRNA expression and liver transplants outcome in HCC

LT is a highly demanded intervention to treat patients with HCC and cirrhosis, yet the emergence of HCC after the surgery is a critical issue that affects patient survival. LncRNAs have emerged as important regulators of diverse cellular functions such as tumorigenesis, immune regulation, and tissue repair, which are important in determining the success of LT in HCC patients. The role of lncRNAs in the development of HCC has been emphasized in a number of studies, with molecules such as HOTAIR and MALAT1 contributing to the proliferation, migration, and invasion of tumor cells [163]. The abnormal expression of the following lncRNAs can be a contributor to the aggressive progression of HCC, thus enhancing the chances of the tumor relapse after transplantation. Moreover, NEAT1 and other lncRNAs have been associated with sensitizing immune cell activation and the immunological reaction, which is essential in graft rejection or tolerance [164]. Unregulated lncRNAs in patients with HCC can upset the immune homeostasis, which can cause transplant rejection or post-surgical complications. Other than immune modulation, lncRNAs are also essential to liver regeneration, which is necessary to promote graft survival following LT. Research indicates that lncRNAs such as lncRNA-HEIH have a role in the proliferation of hepatocytes and changes in their expression may result in the failure of liver recovery after transplantation [165]. As illustrated by Lai et al. [166], MALAT1 is a lncRNA and is overexpressed in HCC and is linked to a high probability of tumor recurrence after LT. This research indicates that high MALAT1 expression is an independent prognostic factor for low disease-free survival, especially in patients who surpass the Milan criteria. In addition, the in vitro silencing of MALAT1 decreased cell viability, motility, and invasiveness and increased apoptosis, indicating that it could serve as a therapeutic target in the treatment of HCC patients to improve their LT [166]. Preclinical models showed promising outcomes of lncRNA-targeting in reducing metastasis and improving survival, and thus, lncRNA-targeted therapy may be considered to add to the overall success of LT in HCC patients.

Many researchers analyze and categorize hidden disease markers that are hard to identify. Diagnostic, prognostic, and theragnostic serve as the three primary types of biomarkers, which do not necessarily require exclusive distinctions from each other. Biomarkers with diagnostic functions help distinguish between normal tissues and cancer tissues. The expression levels of prognostic indicators demonstrate an independent relationship with disease prognosis in addition to disease progression. The detectable expression alterations of theragnostic biomarkers show forecasting capability regarding therapeutic response. Certain cancer-related lncRNAs are detectable in serum, even in circulating exosomes, and thus lncRNAs can be used as noninvasive biomarkers [167]. The diagnostic value of MEG3 tumor suppressor has been reported in the serum of CRC patients [168]. Studies reveal that CASC9 demonstrates elevated expression levels in HCC patients versus normal healthy subjects, along with prognostic potential for tumor metastasis outcomes [169]. Multiple examples of lncRNAs are activated due to radiation therapy [170]. The cellular induction of LINP1 occurs after radiotherapy in cervical cancer cells to enable DNA repair [171]. HOTAIR, BRM, and ICR serum lncRNA have demonstrated the potential of being prognostic biomarkers of HCC. High serum concentrations of these lncRNAs were linked to undesirable clinicopathological characteristics and unfavorable prognosis, and a combination of their evaluation could help to identify HCC at an early stage. Diagnostic performance was also compared in the mentioned study in combination with AFP and the panel of AFP and HOTAIR, BRM and ICR exhibited the best diagnostic accuracy [172]. The molecule MALAT1 presents an opportunity for both prognostic assessment and therapeutic interventions among HCC patients receiving sorafenib treatment [101]. The expression levels of lncRNA-GAS5 together with the promoter region rs145204276 polymorphism serve as prognostic biomarkers that predict postoperative pain experienced by patients who undergo hepatectomy for HCC [173].

LncRNAs as therapeutic targets

So far, the overall prognosis of HCC is poor, and at least partially it is explained by the absence of a therapeutic target. The targeted drug most frequently used in the treatment of HCC is sorafenib, which targets RTKs, but resistance to sorafenib is commonly observed with the treatment of HCC [174]. The important functions of lncRNAs in HCC render them attractive drug targets for new treatment regimens. Moreover, the lncRNA-targeting strategies possess certain benefits in comparison with protein-targeting strategies base-pairing principle is far simpler to use than designing a certain protein-binding inhibitor. ASOs and RNAi are the primary forms of these lncRNA-targeting and have both been demonstrated to exhibit positive anti-cancer effects against HCC through lncRNA targeting. For example, RNAi-mediated knockdown of CASC9 has been shown to suppress HCC growth [175]. In a nutshell, ASOs are short single-stranded DNAs that bind to a target lncRNA and make a DNA-RNA complex, which can be cleaved by RNaseH. RNAi, on the other hand, is short, double-stranded RNAs, which must be loaded into AGO2 protein to form an RNA-induced silencing complex (RISC) and subsequently, bind to target lncRNA to produce RNA-RNA complex and silence lncRNAs [176]. In addition to the knockdown of oncogenic lncRNAs, the delivery of tumor suppressive lncRNAs can also be used as an option. As an example, lncRNA PRAL can be a tumor suppressor by stabilizing p53, and its delivery by adenovirus vector has been demonstrated to significantly prevent HCC growth in mice bearing tumor significantly, which indicates its possible clinical use in the treatment of HCC [175]. It is important to mention that the latest pipeline of HCC-targeted ASOs and RNAi is extensive, and both of them have already been used to treat HBV. In short, ASOs and siRNAs are conjugated using chemical groups (e.g., N-acetylgalactosamine, GalNAc) or packaged in delivery vehicles (e.g., lipid nanoparticles); thus, an optimized pharmacokinetic profile is obtained [177]. The commercial experiences of HBV treatment with ASOs and RNAi give a significant foundation for lncRNA interference and the treatment of HCC.

LncRNA-targeted therapy also offers novel concepts for treating the disease. Unlike traditional treatments such as chemotherapy, which affect both cancer cells and normal ones, targeted therapies are designed to target and interfere with the very specific disease-causing parts, all without causing as much damage to normal cells as possible. Clinical trials have begun with research in H19-based treatment methods. A plasmid expressing diphtheria toxin can suppress tumor growth, and this has been confirmed in bladder cancer under the dual control of H19 and IGF2-P4 and is likely to be involved in HCC that expresses high levels of H19 [178]. Another possible therapeutic intervention is the interference of related regulatory pathways by exogenous lncRNA. In earlier research, scientists loaded MS2 virus-like particles (VLPs) with GE11 polypeptide and were able to form a MEG3 carrier, which was directed to EGFR-positive hepatocarcinoma cells using the clathrin-mediated endocytosis [179].

In the recent past, immunotherapy has been a hot topic in the cancer therapy field. With significant roles in immunoregulation, lncRNAs have the potential for immense application in immunotherapy [180]. Indicatively, lncEGFR has the capacity to specifically bind EGFR and stabilize EGFR expression in the inhibition of its interaction with c-CBL and subsequent ubiquitination, which leads to differentiation of Tregs, inhibition of cytotoxic T cells, and disease progression in HCC [114]. Thus, lncRNA targeting and regulation have great potential to help immunotherapy and should be investigated in the future.

Within recent years, an increasing amount of interest has focused on the development of lncRNA-targeting nanoparticle-based therapies for HCC. The HCC nanoparticle therapyʼs most popular lncRNA is MEG3, which is a tumor suppressor and regulates p53 target gene expression. Nevertheless, lncRNA MEG3 exhibits a relatively low or no expression in human HCC. Thus, MEG3 has been used to inhibit the growth, migration, and invasion of HCC cells. Ren et al. [181] examined the importance of using polymeric nanoparticles in the delivery of select lncRNAs to HCC cells. They used a polymer termed pullulan, which has an efficient cell-targeting capacity (targeting liver cells). The purpose of this approach was to provide plasmids with the lncRNA MEG3 and the p53 gene. The delivery of these plasmids was done by co-delivery through pullulan-based ethanolamine-modified poly(glycidyl methacrylate) (PuPGEA). This approach was successful in improving the lncMEG3 and p53 levels in the HCC cells, following which the HCC cell proliferation, migration, invasion, and tumor growth were suppressed further. The paper has shown that these polymeric nanoparticles can be used as dual-targeted theranostic therapy of the HCC [181].

Wider knowledge about lncRNA processes will develop new therapeutic intervention targets. The standard chemotherapeutics often generate insufficient treatment results while patients develop resistance to these treatments. Early-stage HCC diagnosis allows curative treatments to be effective, but advanced HCC receives limited therapeutic options. The use of therapeutic methods against ncRNAs or through ncRNA molecules shows promise to improve HCC treatment.

Standardization of lncRNA measurements in clinical samples

Among the available variety of methodologies to detect lncRNAs, quantitative polymerase chain reaction (qPCR), RNA sequencing, and microarrays are commonly used, and each of them has specific strengths and limitations in terms of work with clinical samples such as blood or tumor tissue. Comprehensive studies of ncRNA transcripts are enabled by next-generation sequencing and microarrays, but NGS has higher sensitivity and does not require prior knowledge of target sequences [182]. qRT-PCR, on the other hand, is characterized by its sensitivity, reproducibility, and quantification capabilities of trace ncRNAs, which are especially useful in confirming the results of the larger screening techniques such as microarray or RNA sequencing [183]. Moreover, lncRNAs and other ncRNAs, including miRNAs and circular RNAs (circRNAs), are characterized by great stability and degradation resistance, which means that they can be detected by regular laboratory methods [184]. Nevertheless, these approaches to cell-free RNA (cfRNA) in liquid biopsies, particularly for long RNA, have drawbacks such as low reproducibility, technological and biological variability, and the absence of standard procedures [185]. In order to overcome these challenges, more advanced sequencing methods, including those that can forego poly-selection in library preparation or employ single-molecule long-read sequencing, are being developed to enhance comprehensive disease profiling and accurate identification of lncRNAs and their isoforms in complex biological samples [186].