Non‐coding RNA‐associated competitive endogenous RNA regulatory networks: Novel diagnostic and therapeutic opportunities for hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC), as the most prevalent liver malignancy, is annually diagnosed in more than half a million people worldwide. HCC is strongly associated with hepatitis B and C viral infections as well as alcohol abuse. Obesity and nonalcoholic fatty liver disease (NAFLD) also significantly enhance the risk of liver cancer. Despite recent improvements in therapeutic approaches, patients diagnosed in advanced stages show poor prognosis. Accumulating evidence provides support for the regulatory role of non‐coding RNAs (ncRNAs) in cancer. There are a variety of reports indicating the regulatory role of microRNAs (miRNAs) in different stages of HCC. Long non‐coding RNAs (LncRNAs) exert their effects by sponging miRNAs and controlling the expression of miRNA‐targeted genes. Circular RNAs (circRNAs) perform their biological functions by acting as transcriptional regulators, miRNA sponges and protein templates. Diverse studies have illustrated that dysregulation of competing endogenous RNA networks (ceRNETs) is remarkably correlated with HCC‐causing diseases such as chronic viral infections, nonalcoholic steatohepatitis and liver fibrosis/cirrhosis. The aim of the current article was to provide an overview of the role and molecular mechanisms underlying the function of ceRNETs that modulate the characteristics of HCC such as uncontrolled cell proliferation, resistance to cell death, metabolic reprogramming, immune escape, angiogenesis and metastasis. The current knowledge highlights the potential of these regulatory RNA molecules as novel diagnostic biomarkers and therapeutic targets in HCC.


| Hepatocellular carcinoma
Hepatocellular carcinoma (HCC) accounts for an aggressive primary form of liver cancer. Annually, over 500,000 new cases of HCC are diagnosed across the world and its incidence continues to rise. 1,2 It is an epithelial tumour that originates from stem cells or mature hepatocytes characterized by chemotherapy resistance and poor prognosis. 3 A multitude of genetic and epigenetic changes contributes to the multi-step malignant transformation of liver tissue. 4 Chronic viral infections of hepatitis B (HBV) and C (HCV), alcoholism and cirrhosis are recognized as the most important risk factors for HCC. 5 The risk of disease development is also increased in chronic medical conditions such as diabetes mellitus and obesity. As the liver tissue plays a crucial role in glucose metabolism, diabetes mellitus can lead to a variety of liver-associated disorders including chronic hepatitis, fatty liver, liver failure and cirrhosis. 6 Orthotopic liver transplantation (OLT) and surgical resection are known as the most effective approaches for HCC treatment. However, a high rate of metastasis/ recurrence (~50%-70%) has been observed within five years postoperation. 7 Although sorafenib and regorafenib are used as the firstand second-line systemic chemotherapy for HCC, concerns about drug resistance, which leads to a high mortality rate, are rising. Over the recent decades, a large body of evidence has been obtained about the role played by genes crucial for cellular processes, such as cell cycle control, cell growth, apoptosis and migration in HCC development. [8][9][10] This highlights the necessity of unravelling the mechanisms underlying HCC progression as well as finding efficient molecular biomarkers.

| Non-coding RNAs
Recent advances in transcriptome sequencing have revealed that less than 3% of human genome encodes exons, while almost 97% of genome is transcribed into non-coding RNAs (ncRNAs) including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs). 11,12 NcRNAs are RNA molecules that do not code for proteins and play key roles in DNA replication, RNA splicing, translation and epigenetic regulation. Based on transcript length, ncRNAs are divided into two major categories: those shorter than ~200 nucleotides are known as short ncRNAs (miRNAs, piRNAs, snoRNAs, snRNAs, tRNAs) and those longer than ~200 nucleotides are known as long ncRNAs (lncRNAs, pseudogenes and circRNAs). [13][14][15] The regulatory networks consisting of miRNA, lncRNA and mRNA have received attention in the study of biological mechanisms involved in cancer occurrence and progression. 16 Now, the competing endogenous RNA (ceRNA) hypothesis, initially proposed by Salmena et al., is widely acknowledged by the scientific community. 17 According to this hypothesis, a complex post-transcriptional regulatory network mediated by miRNAs and sharing one or more miRNA response elements (MREs), protein-coding RNAs and ncRNAs competes for binding to miRNAs. This leads to the modulated expression of different molecules in the network. 18 There are mainly two cellular conditions needed for ceRNA to occur. Firstly, the relative concentration of ceRNAs and their miRNAs is important. Changes in the ceRNA expression levels need to be large enough to either overcome or relieve miRNA repression on competing ceRNAs. This is exemplified by RNA transcripts switched on or off at the transcriptional level in different developmental stages or physiological/pathological conditions. Secondly, the effectiveness of a ceRNA depends on the number of miRNAs it can sponge. This in turn depends on the accessibility of ceRNA to miRNA molecules, which is influenced by its subcellular localization as well as interaction with RNA-binding proteins. 17

| MicroRNAs
miRNAs are short RNA strands with 18-23 nucleotides that regulate critical cellular processes. They are transcribed by RNA polymerase II or III as short RNA hairpin structures which are subsequently processed by the nuclear and cytoplasmic RNase III-type enzymes. 19 They act post-transcriptionally via complementary base-pairing with 3′-untranslated region (3´-UTR) of the target gene, but may also interact with 5′-UTR and coding region. [20][21][22] At least, 60% of human genes harbour target sites for miRNAs. 23 miRNAs exert their effects through interaction between nucleotides 2-8 at their 5´-ends (seed region) and mRNA target sites, leading to translational repression, cleavage or mRNA degradation. 24 Regarding their function, miR-NAs are frequently found in oncogenesis-associated genomic regions. Therefore, they can be linked to relevant tumour properties, such as cell proliferation, apoptosis, differentiation and cell cycle regulation. [25][26][27][28][29] A growing range of evidence has demonstrated the contribution of miRNAs to HCC-related cellular processes and their potential use as prognostic and diagnostic markers. 10 For instance, miR-423 has been reported to play roles in HCC such as enhancing cellular invasiveness, 30 contributing to tumorigenesis, 31 cell cycle control, autophagy regulation, 32 promoting cell growth and regulating the G1/S transition by targeting p21Cip1/Waf1. 33 In another case, miR-10b has been reported to exert its oncogenic role in HCC by targeting the expression of CUB and sushi multiple domains 1 (CSMD1). 34 Also, miR-92a has been proposed to contribute to tumour growth in HCC by targeting FBXW7. 35 K E Y W O R D S circular RNA, competing endogenous RNA networks, hepatocellular carcinoma, long noncoding RNA, microRNA

| Long non-coding RNAs
LncRNAs are RNA molecules with more than 200 nucleotides and have been known to be involved in tumorigenesis in a variety of cancer types. [36][37][38] These molecules are transcribed by RNA polymerase ll from different regions of the target gene including enhancers (enhancer RNAs, eRNAs) and promoters (promoter upstream transcript, PROMPTS) and undergo post-transcriptional processing events involving 5′-end capping, 3′-end polyadenylation and splicing. 39,40 LncRNAs are mostly located in the cytosol, where they target mRNAs and down-regulate protein translation. 23 They are heterogeneous molecules that perform various functions via interacting with DNA, RNA, proteins, peptides, small weight molecules, miRNAs and mRNAs ( Figure 1). For instance, they regulate chromatin state and cell cycle, control mRNA stability, silence retrotransposons and competitively sponge miRNAs. [41][42][43][44] These regulatory RNAs play leading roles in the modulation of gene expression at epigenetic, transcriptional and post-transcriptional levels, thereby contributing to a variety of cellular phenomena such as RNA processing, chromatin modification, apoptosis and invasion. 45 Furthermore, some lncRNAs have been indicated to be abnormally expressed in human diseases, providing support for their involvement in pathogenesis. These functions underline the multi-faceted role of lncRNAs in the regulation of gene expression. Another noteworthy point about lncRNAs is that they perform their specific functions by interacting with multiple proteins and hence regulating numerous cellular processes. Studies have demonstrated that lncRNAs can activate post-transcriptional gene regulation, splicing and translation by binding to proteins. Therefore, determining possible lncRNA-protein interactions (LPIs) is essential for unravelling lncRNA-related activities.

| Circular RNA
Circular RNAs (circRNA) are transcript isoforms generated from precursor mRNAs of protein-coding genes in which back-splicing between 3′ and 5′ splice sites forms a covalent circular structure.
Compared with linear RNAs, circRNAs have a higher thermal stability and tissue specificity. Next-generation RNA sequencing and bioinformatic analyses have revealed that circRNAs constitute a substantial fraction of eukaryotic transcriptome. [46][47][48][49][50] CircRNAs perform various biological functions including miRNA sponging, interaction with RNA-binding proteins (RBPs), translation of proteins and acting as mRNA translation brake. Due to the high structural stability of circRNAs, they have an inherent sponging capacity through which bind to miRNAs and prevent them from sequestering or suppressing their target mRNAs. [51][52][53] For instance, SMARCA5 and circ-MTO1 sponge miR-17-3p and miR-181b-5p, respectively, thereby inhibiting the growth and metastasis of HCC cells. 54

| Exosomes
Exosomes are small endosomes extracellular vesicles with a size range of ~40 to 160 nm. 55 Exosomes have gained attention over the past decade owing to their role as carriers of a wide range of biomolecules, including lipids, proteins, DNAs, mRNAs, lncRNA and miRNAs in both physiological and pathological processes. Hence, they illustrate a novel mode of intercellular communication and play a principal role in many cellular processes, such as immune response, signal transduction and antigen presentation. They represent potential use as biomarkers in a variety of cancers including HCC. 56,57 It has been demonstrated that exosomes can promote the occurrence and development of tumours and make a significant contribution to tumour invasion and metastasis. 58 Exosomes can be divided into tumour and stromal cell secretions based on their origin in the tumour microenvironment. 59 In a recent report, a urinary exosomal miRNA, miR-532-5p, has been suggested as a predictive biomarker for biochemical recurrence after radical prostatectomy (RP) in intermediate-risk prostate cancer patients. 60 Han et al 61

| COMPE TING ENDOG ENOUS RNA NE T WORK (CERNA )
Here, we review the roles played by some of the important ceRNA regulatory networks in different steps of HCC development including cell growth and proliferation, metastasis, invasion, angiogenesis, apoptosis and chemoresistance. Different ceRNA networks and their mechanisms of action in HCC are summarized in Table 1. can also regulate cell cycle by binding directly to retinoblastoma (Rb) protein through its N-terminal 24 highly fidelity amino acids (N24). 64 Long intergenic non-protein-coding RNA (LINC00160) has been reported to correlate with chemoresistance of breast cancer cells by regulating TFF3 through the activity of the transcription factor C/EBPβ. 65 miR-132, a member of miR-212/132 cluster, has been demonstrated to be dysregulated in several malignancies. The function of this miRNA is complicated. It can act as an oncogene in squamous cell carcinoma of the tongue or as a tumour suppressor in osteosarcoma, prostate, ovarian and non-small-cell lung cancers. 66 Moreover, miR-132 has been recognized as a biomarker in colorectal cancer (CRC) and it can inhibit the invasion and metastasis of CRC by targeting ZEB2. 67 miR-132 has also been found to play some roles in pancreatic cancer. Down-regulation of this miRNA, through promoter methylation, can promote the progression and metastasis of pancreatic and prostate cancers. 68,69 There are multiple lines of reports supporting the role of miR-132 in HCC. miR-132 functions as a tumour suppressor in HCC by directly targeting PIK3R3 and regulating the AKT/mTOR pathway. It can also suppress cell proliferation, colony formation, migration and invasion, as well as induce apoptosis in HCC cells. 66 There is some evidence showing the relationship between LINC00160 and miR-132 in HCC. It has been found that miR-132 is down-regulated in HCC compared with normal adjacent tissues and its overexpression directly targets PIK3R3,     However, further investigation is required to pave the way for using these findings in the clinic.

ACK N OWLED G EM ENTS
We would like to thank all authors responsible for the insights that we attempted to summarize. This work was supported by Tarbiat Modares University, Tehran, Iran.

CO N FLI C T O F I NTE R E S T
The authors report no competing interest.