The pivotal role of EMT-related noncoding RNAs regulatory axes in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) remains a major health problem worldwide, being the leading cause of cancer-related deaths, with limited treatment options, especially in its advanced stages. Tumor resistance is closely associated with the activation of the EMT phenomenon and its reversal, being modulated by different molecules, including noncoding RNAs (ncRNAs). Noncoding RNAs have the potential to function as both tumor suppressors and oncogenic molecules, controlling the malignant potential of HCC cells. Basically, these molecules circulate in the tumor microenvironment, encapsulated in exosomes. Their impact on cell biology is more significant than originally expected, which makes related research rather complex. The temporal and spatial expression patterns, precise roles and mechanisms of specific ncRNAs encapsulated in exosomes remain primarily unknown in different stages of the disease. This review aims to highlight the recent advances in ncRNAs related to EMT and classifies the described mechanism as direct and indirect, for a better summarization. Moreover, we provide an overview of current research on the role of ncRNAs in several drug resistance-related pathways, including the emergence of resistance to sorafenib, doxorubicin, cisplatin and paclitaxel therapy. Nevertheless, we comprehensively discuss the underlying regulatory mechanisms of exosomal ncRNAs in EMT-HCC via intercellular communication pathways.


Introduction
Hepatocellular carcinoma (HCC) is a common lethal malignancy among patients with chronic liver disease, with approximately 800,000 deaths annually, according to the GLOBOCAN 2020 report (Sung et al., 2021).Several treatment options are available for therapeutic purposes, such as trans-arterial chemoembolization (TACE) with anthracyclines, cisplatin, and multikinase inhibitor, sorafenib (Pratama et al., 2019).However, these treatments become challenging to manage, due to the appearance of invasion, metastasis and recurrence, whose key molecular sign is EMT (Yan et al., 2018).
EMT (epithelial-mesenchymal transition) is a morphogenetic process in which epithelial cells get a mesenchymal phenotype.In early EMT, transcriptional factors (TFs) are activated to repress epithelial genes and activate the mesenchymal ones.These transcriptional changes trigger the following key events: cell-cell junction dissociations, apical-basal polarity loss, cytoskeleton architecture reorganization, the production of extracellular matrix (ECM) degradation enzymes, and cellular shape transformation.The activation of cellular pathways associates this process with proliferation, invasion, metastasis, and chemotherapy resistance (Yan et al., 2018;Dudas et al., 2020;Yang et al., 2020;Huang et al., 2022).Among these transformations, EMT is associated with numerous signaling pathways involved in inflammation, oncogenic and metabolic stress, hypoxia or apoptosis (Huang et al., 2022).
Moreover, many studies suggest that noncoding RNAs (ncRNAs), such as microRNAs (miRNAs), long-noncoding RNAs (lncRNAs) and circular RNAs (circRNAs), have been linked to both the EMT process activation and inhibition.Indeed, these types of RNAs have multiple roles in cancerous cells because one ncRNA transcript could target many molecules involved in different signaling pathways (Toden et al., 2021;Khanbabaei et al., 2022).
This review highlights ncRNAs' significant direct and indirect signaling pathways in the EMT process and how these mechanisms are involved in HCC progression and chemoresistance.Finally, we provide an update on developing exosome-based therapies against HCC and their molecular aspects in EMT (Figure 1).

EMT-related ncRNAs mechanisms of action
As mentioned above, noncoding RNAs (ncRNAs), including microRNAs, lncRNAs and circRNAs, have oncogenic and tumor suppressor roles and regulate essential processes involved in cancer progression.
MicroRNAs (miRNAs) are noncoding single-stranded RNAs of approximately 22 nucleotides transcribed in pri-miRNA by RNA Pol II (Bartel, 2004).As described in the canonical pathway, Ribonuclease III and double-stranded-RNA-binding protein, DGCR8, recognize this structure in the nucleus, generating a pre-miRNA of ~65 nucleotides.Pre-miRNA is exported to the cytoplasm by an Exportin 5 and Ran-GTP complex and recognized by RNase III Dicer, which forms a miRNA duplex.This mature form is incorporated into an RNA-induced silencing complex (RISC), directing RISC to complementary mRNA targets (Cai et al., 2004).In brief, miRNAs function as negative regulators of genes when binding to RNA 3′-untranslated region (3′-UTR) (Ha and Kim, 2014).Besides that, the interaction with coding sequences, gene promoters, and 5′-UTR has been proved (O'Brien et al., 2018).Because each miRNA can regulate multiple targets containing specific miRNA response elements (MREs) (Bassett et al., 2014) and play a crucial role in a variety of molecular processes, they have been studied in all cancer types (Esquela-Kerscher and Slack, 2006;Volinia et al., 2006;Nicoloso et al., 2009).In HCC, miRNAs modulate cell cycle, proliferation, apoptosis, epithelialmesenchymal transition and metastasis (Sidhu et al., 2015).Furthermore, our previous studies have shown that miRNAs are an important tool in the prognostic and diagnostic HCC (Mjelle et al., 2019;Sorop et al., 2020).
Long noncoding RNAs (lncRNAs) are transcripts of approximately 200 nucleotides, which usually RNA Pol II transcribes, but so do RNA Pol I and RNA Pol III (Statello et al., 2021;Mattick et al., 2023).Moreover, they have a wide diversity, with an average of 100,000 human lncRNAs (Mattick et al., 2023).At first, lncRNAs were defined as transcriptional "junk" or "noise."Still, in the past few years, more studies have shown the involvement of lncRNAs in different molecular pathways (Sun et al., 2017), indicating their interaction with DNA, RNA, or protein.The interaction mechanism could be: scaffold, decoy, guide, signal, or SINEUPs.Scaffold lncRNAs could act as archetype RNAs and are involved in the assembly of transcriptional regulators.The decoy mechanism implies acting as a competing endogenous RNA (ceRNA) or sponge of miRNAs, transcriptional factors, or RNAbinding proteins.In contrast, the guide mechanism involves the formation of a ribonucleoprotein complex, which targets a promoter or genomic loci (Rinn and Chang, 2012).Furthermore, lncRNAs could act as regulatory molecules (Nadhan et al., 2022) or SINEUPs containing SINE elements which enhance mRNAs translation (Toki et al., 2020).
Circular RNAs (circRNAs) are single-stranded RNAs with closed-loop structures and resistance to RNase R and exonucleases.They are generated from precursor RNA (pre-RNA) through back-splicing (Chen, 2016).This mechanism involves connecting a downstream donor site of a flanking downstream intron to an upstream acceptor site (Kristensen et al., 2019).Increasing research has revealed that circRNAs can sponge miRNAs, interact with proteins, interfere with transcription or splicing, or encode peptides (Zhang and Wang, 2021).
EMT plays a pivotal role in the early stage of metastasis (Bakir et al., 2020); thus, many studies have been conducted to determine the function of ncRNAs in this highly dynamic phenomenon.Therefore, this review underlines two types of mechanisms: direct and indirect.

miRNA/mRNA axes
Numerous miRNA/mRNA axes have been found to be involved in the EMT process (Table 2).
For instance, Shen et al. (2021) have found that miRNA-10a-5p is downregulated in HCC tissues and decreases EMT in HCC cells by targeting spindle and kinetochore-associated complex subunit 1 (SKA1).SKA1 is upregulated in tumors, promoting cancer progression, and has a prognostic value in HCC (Chen et al., Note: downregulated expression (↓), upregulated expression (↑), inhibition of cellular process (−), enhance of cellular process (+).
Circ_0003288 is an oncogenic RNA that enhances EMT by increasing programmed death-ligand 1 (PD-L1) and Akt pathways via miR-145 sponging (Xu et al., 2021).Circ_0091579 has been demonstrated to pin HCC patients and its downregulation inhibits EMT and promotes apoptosis in vitro.Also, miR-136-5p is a direct target of circ_0091579 and its overexpression suppresses the malignant potential of HCC cells via regulating tripartite motif containing 27 (TRIM27) expression (Mao et al., 2022).
Moreover, the Toll interacting protein (TOLLIP)-derived circRNA (circTOLLIP) is also found to be involved in the EMT of HCC.CircTOLLIP is upregulated in HCC via eukaryotic translation initiation factor 4A3 (EIF4A3), an RNA-binding protein.This circRNA acts as a ceRNA for miR-516a-5p, thus upregulating PBX3 and exhibiting pro-tumor roles in vitro and in vivo (Liu et al., 2022).
3 The role of ncRNA/mRNA axes in HCC drug resistance As discussed above, EMT is associated with chemotherapy resistance by avoiding cell death mechanisms (De Las Rivas et al., 2021).Therefore, a growing number of studies have supported the importance of EMT-related ncRNAs in molecular pathways of different therapies (He et al., 2022).
Besides Sorafenib, TACE with doxorubicin and cisplatin is used in HCC advanced patients (Lu et al., 2017;Couri and Pillai, 2019).Doxorubicin (Adriamycin, DOX) is an anthracycline drug used as an antineoplastic agent.The most known mechanism of action involves the interaction with topoisomerase IIα (TOP2A) (Tewey et al., 1984) and the activation of apoptosis (Roos and Kaina, 2013).Anthracycline drug resistance is caused by the incapability of DOX to accumulate in the nucleus (Cox and Weinman, 2016).For instance, Zhang et al. (2021) reported that overexpression of linc-ROR (long intergenic non-protein coding RNA (linc)-regulator of reprogramming) increases DOX resistance in HCC cell lines by TWIST upregulation.Also, circFoxo3 has higher expression in adriamycin-resistant patients.It has been shown that circFoxo3 via miR-199a enhances ABCC1 expression, a known protein involved in drug resistance.Moreover, the downregulation of miR-199a promoted EMT signaling in HCC cells and reversed circFoxo3 inhibition effects (Huang et al., 2020).Li et al. (2020) identified that circ_0003998 downregulation facilitated DOX-sensitivity by E2F Transcription Factor 3 (E2F3) regulation.They further identified circ_0003998 as a sponge of miR-218-5p and Eukaryotic initiation factor 5A2 (EIF5A2) as a direct target of miR (Li et al., 2020).Moreover, EIF5A2 is involved in genistein resistance, an essential anti-tumoral phytoestrogen that promotes apoptosis (Sarkar and Li, 2002) and inhibits EMT and stemness.MiR-1275 is a tumor suppressor that can bind 3′-UTR EIF5A2 as a protein that upregulated PI3K/ Akt and EMT pathways.MiR-1275 was expressed at a higher level by genistein treatment (Yang et al., 2022).Furthermore, it has been shown that miR-140-5p is involved in drug resistance in HCC cells.In brief, miR-140-5p improves DOX sensitivity through PIN1 depletion (Gao et al., 2021) and catalpol sensitivity through EMT suppression (Wu et al., 2021).
Cisplatin is a chemotherapeutic that inhibits transcription and replication, inducing apoptosis and necrosis in HCC cells (Ishikawa, 2009).It has been shown that miR-9 increases cisplatin sensitivity in vitro and in vivo by targeting EIF5A2 and EMT process.Besides that, EIF5A2 depletion decreases vimentin expression and increases E-cadherin in HCC cell lines (Bao et al., 2020).Another ncRNA involved in cisplatin sensitivity is miR-138 by its direct target, enhancer of zeste homolog 2 (EZH2).This miRNA upregulates EMT markers; therefore, the miR-138/EZH2/EMT axis could regulate cisplatin resistance (Zeng et al., 2021), also involved in radiosensitivity.Bai et al. (2022) show that miR-138 is downregulated in HCC tissue and its expression is indirectly correlated with EZH2 expression, which is a direct target of miR-138-5p.By RNA-seq, they observed that miR-138-5p upregulation inhibits HIF-1α and EMT (Bai et al., 2022).Moreover, Lu et al. (2022) reported that miR-138-5p is negatively regulated by circ-TLK1.
These investigations show the complex and dual role of ncRNAs in EMT.The exact mechanism by which every ncRNA is involved in the HCC will be difficult to decode because of its functions in many hepatocellular processes.One way to start is by classifying the miRNAs based on their direct or indirect impact on the EMT process.Undoubtedly, future studies are necessary to report new miRNAs associated with HCC-EMT and to map their function in this process, which can lead to the development of novel therapies.
Therefore, to translate ncRNAs in a therapeutic situation, tools must be developed to analyze these ncRNA axes functionally and to devise therapy strategies, so as to overcome off-target and toxicity consequences.

EMT-associated exosomal ncRNAs in HCC
Exosomes can be found in all human body fluids (blood, urine, saliva, ascites, cerebrospinal and synovial fluids) (Jiang et al., 2022).They are extracellular 30-100 nm vesicles (EVs) having a lipid bilayer; they are generated from the luminal membranes of multivesicular bodies (MVBs) and released into the extracellular matrix after MVBs fusion with the cell membrane (Kim et al., 2020).The primary physiological role of exosomes is to mediate cell-cell communication by transferring bioactive molecules, such as proteins or nucleic acids (Chen et al., 2021), thus being one of the most studied tools for the interchange of substances between tumor cells and the tumor microenvironment (Jiang et al., 2022).
In the last decade, more studies have highlighted the regulatory effects of different bioactive molecules delivered by exosomes, such as ncRNAs, in the EMT process in various types of cancers, including HCC.Interestingly, they can promote or suppress the EMT phenomena in HCC cells.
According to RNAseq investigation, exosomal miR-92a-3p expression level increases in two established high-metastatic HCC cell lines (97 hm and Huhm).Besides, treatment with high-metastatic HCC-derived exosomal miR-92a-3p facilitates the aggressiveness of HCC cells via PTEN inhibition and Akt/ Snail signaling activation, promoting EMT (Yang et al., 2020).Similarly, high levels of miR-4800-3p were found in Huh7 cellderived exosomes.Thus, Lin et al. (2022) demonstrated that exosomal miR-4800-3p heightened the progression of HCC by regulating the Hippo signaling pathway and targeting STK25 in both in vitro and in vivo experiments.Moreover, the treatment of low metastatic HCC cells with exosomal miR-4800-3p downregulates the expression of E-cadherin and ZO-1 and increases the expression of N-cadherin, activating the EMT process (Lin et al., 2022).
Interestingly, M2 macrophages can influence tumor development by secreting various cytokines and exosomes that can be loaded with specific miRNAs.For instance, miR-660-5ploaded M2 exosomes augmented EMT and enhanced the tumorigenic ability in HCC cells through downregulating Kruppel-like factor 3 (KLF3) expression (Tian et al., 2021).
Human umbilical cord mesenchymal stem cells (hucMSCs) have low immunogenicity and high proliferation and differentiation potential.Additionally, the treatment of HCC cells with hucMSC-Exo upregulates miR-451a.This miRNA inhibits a disintegrin and metalloprotease 10 (ADAM10), thus reducing EMT and aggressive phenotypes of HCC (Xu et al., 2021).
Similarly, miR-374c-5p was found to be downregulated in the EMT model and transferred by exosomes derived from bone marrow mesenchymal stem cells (BMSC) suppresses EMT via targeting LIM domain kinase 1 (LIMK1) and inhibiting Wnt/β-catenin and TGF-β1 axes in HCC cells (Ding et al., 2023).Yao et al. (2022) identified that lncRNA THEMIS2-211 is upregulated in plasma-derived exosomes from HCC patients.Knockdown of THEMIS2-211 increases E-cadherin and decreases N-cadherin and vimentin in HCC cells.Mechanistically, they showed that THEMIS2-211 is an oncogene that promotes proliferation, migration, invasion, and EMT by sponging miR-940 and increasing SPOCK1 expression (Yao et al., 2022).
In summary, these studies prove that exosomes act as ncRNAs cargo for tumor cells and have distinct regulatory effects on the EMT process in HCC and various underlying processes.Although exosomes are promising therapy in cancer, improvement of their purification, and additional studies on the interaction and mechanisms with other types of cells remain the main problems to be solved in their uses.

Conclusion and future perspectives
The development of transcriptomics approaches in the last decade has highlighted the essential roles of ncRNAs in cancer (Slack and Chinnaiyan, 2019;Winkle et al., 2021).The formation of ncRNA axes starts to become an essential tool in various cellular mechanisms, and its role in the progression of HCC is decisive (Wong et al., 2018).Furthermore, it will be crucial to comprehend how ncRNA axes regulate migration, proliferation, and EMT in HCC cells, so as to generate cuttingedge therapeutic medications based on ncRNAs, to prevent and manage HCC.
Taking together these observations, we find that defining ncRNA pathways in direct and indirect mechanisms could map a precise road to a therapeutic target as close to a clinical necessity as possible.The EMT-related miRNAs' direct mechanism of action could be a promissive path in developing new therapies against metastasis.However, more research is needed to understand how these miRNA axes work and to determine which transcripts are valuable targets.Undoubtedly, since a single miRNA could have several targets and can affect more therapeutic drugs, its use as a new therapy in cancer requires an in-depth study of the mechanisms involved.

FIGURE 2
FIGURE 2Molecular actions of EMT-related ncRNA axes in HCC drug resistance.Multiple regulatory components either increase or decrease sensitivity to sorafenib, paclitaxel, genistein, cisplatin, doxorubicin, or catalpol, affecting HCC progression.The signaling pathways of every drug are represented by different colors, as seen above (created with biorender.comaccessed on July 2023).

TABLE 1
Summary of ncRNAs direct signaling pathways and their action on HCC tumor cell processes.

TABLE 2
Summary of miRNAs signaling pathways and their action on HCC tumor cell processes.

TABLE 3
Summary of lncRNAs signaling pathways and their influence in HCC tumor cells processes.

TABLE 4
Summary of circRNAs signaling pathways and their influence in HCC tumor cells processes.
In the Table