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Dysregulated MicroRNA Fingerprints and Methylation Patterns in Hepatocellular Carcinoma, Cancer Stem Cells, and Mesenchymal Stem Cells

Hepatocellular carcinoma (HCC) is one of the top causes of cancer mortality worldwide. Although HCC has been researched extensively, there is still a need for novel and effective therapeutic interventions. There is substantial evidence that initiation of carcinogenesis in liver cirrhosis, a leading cause of HCC, is mediated by cancer stem cells (CSCs). CSCs were also shown to be responsible for relapse and chemoresistance in several cancers, including HCC. MicroRNAs (miRNAs) constitute important epigenetic markers that regulate carcinogenesis by acting post-transcriptionally on mRNAs, contributing to the progression of HCC. We have previously shown that co-culture of cancer cells with mesenchymal stem cells (MSCs) could induce the reprogramming of MSCs into CSC-like cells. In this review, we evaluate the available data concerning the epigenetic regulation of miRNAs through methylation and the possible role of this regulation in stem cell and somatic reprogramming in HCC.


HEPATOCELLULAR CARCINOMA
Hepatocellular carcinoma (HCC) is the most frequent primary malignancy of the liver. It is the third leading cause of mortality associated with cancer worldwide (Yang and Roberts, 2010;Dhanasekaran et al., 2012). HCC is a multifactorial disease that is influenced by several risk factors. It typically develops as a result of underlying liver disease and is commonly associated with cirrhosis . The major HCC-risk factors include viral infection with hepatitis B virus (HBV) and hepatitis C virus (HCV), which leads to liver cirrhosis and accounts for 75% of HCC cases (El-Serag, 2002). Other factors attributed to HCC include alcohol abuse, intake of food contaminated with aflatoxin and toxic chemical exposure, including dimethylformamide, dimethylacetamide, trichloroethylene, tetrachloroethylene, carbon tetrachloride and chloroform (Malaguarnera et al., 2012). In addition, Obesity is one of the highly recent factors that plays a significant role in developing non-alcoholic fatty liver disease (NAFLD) (Cholankeril et al., 2017). It can progress in many stages starting with lipid deposition in hepatocytes' cytoplasm and can lead to non-alcoholic steatohepatitis (NASH) (Marrero et al., 2002;Guzman et al., 2008;Reddy et al., 2012;White et al., 2012). NASH is the severe stage of NAFLD indicated by hepatocyte injury, uncontrolled inflammation, hepatocyte ballooning, cell death, infiltration of inflammatory cells, and collagen deposition (Guzman et al., 2008;Reddy et al., 2012). NASH has been determined to be one of the important events in promoting hepatic carcinogenesis (Ip and Wang, 2014).
Tissue damage and fibrosis result from chronic inflammation and oxidative stress, leading to cirrhosis and eventually tumor initiation, progression, and even metastasis (Lau and Lai, 2008;Shariff et al., 2009;Cabrera and Nelson, 2010). Although only ∼10-20% of HCC patients are eligible for surgical interference at the time of diagnosis, liver transplantation remains the first choice for treatment (Ji et al., 2009a). Furthermore, patients suffer a high frequency of relapse, and in patients who experience curative resection, the 5-year survival rate is 30-40% (Budhu et al., 2008). The low detection and high recurrence rates for the curable stages of HCC have increased interest in investigations of the molecular mechanisms underlying this disease .

CANCER STEM CELLS (CSCS)
The failure of conventional treatments to completely eliminate invasive tumor cells is thought to be due to the presence of a small subset of cancer cells, termed CSCs, which are accountable for cancer progression, metastasis, recurrence, and drug resistance. CSCs have been classified as immortal tumor-initiating cells which have pluripotent and self-renewal capacity . CSCs have been identified in numerous solid tumors, such as breast cancer, colon cancer, and HCC (Szotek et al., 2006;O'Brien et al., 2007;Kawai et al., 2015). CSCs were found to have a main contribution in tumor heterogeneity and to contribute to drug resistance (Beck and Blanpain, 2013;Bedard et al., 2013;Klein, 2013;Meacham and Morrison, 2013). While the origin of CSCs remains unclear, the proposed mechanisms for their generation include cell fusion, genetic mutations in stem cells, and regulatory factors within the tumor microenvironment (TME) (Bu and Cao, 2012). In addition, signaling pathways and genes that regulate stem cell differentiation, as Wnt/βcatenin, transforming growth factor β (TGF-β), and microRNAs (miRNAs), could contribute to the control and maintenance of CSC differentiation (Bedard et al., 2013;Meacham and Morrison, 2013). The Wnt/β-catenin signaling pathway seems to play major roles in the development of CSCs and in self-renewal, tumorigenesis, and cancer chemoresistance (Espada et al., 2009;Eaves and Humphries, 2010;Mohammed et al., 2016).

CHARACTERISTICS OF miRNAs
MiRNAs are small non-coding RNA molecules consisting of 21−23 nucleotides. They control gene expression by base pairing with the messenger RNAs (mRNAs) (Lu et al., 2005;Griffiths-Jones et al., 2006). Transcripts are regulated through either degradation or translational repression (Bartel, 2004). Full complementarity between a miRNA and an mRNA results in full degradation of the target mRNA. However, defects in perfect complementarity leads to less translation of the target gene without affecting the level of mRNA (Lewis et al., 2005;Cummins and Velculescu, 2006). MiRNAs target up to 90% of human genes (Miranda et al., 2006) and can be found in exons or introns of coding or non-coding genes, with their transcription dependent on genomic localization (Baskerville and Bartel, 2005;Lin et al., 2006). Although miRNAs have their own promoters and are self-sufficiently expressed some miRNAs that share the same transcriptional regulation are ordered in clusters. Hundreds of miRNAs have been known by molecular cloning and bioinformatics approaches in plants and animals (Bushati and Cohen, 2007;Liu et al., 2014). Interestingly, a subgroup of miRNAs, namely, epi-miRNAs, control the expression of epigenetic marks, such as DNA methyltransferases (DNMTs), histone deacetylases (HDACs), and polycomb genes, either directly or indirectly. DNA methylation has a key role in gene expression regulation via maintaining the stability of gene silencing. In mammals, DNA methylation takes place by covalent modification of cytosine residues through the addition of a methyl group to the fifth position of a cytosine ring, particularly in the CpG dinucleotides. This process is mediated by members of the DNMTs enzymes family (Chuang and Jones, 2007). Therefore, miRNAs function as both genetic and epigenetic regulators (Valeri et al., 2009). miRNAs control many cellular processes in eukaryotes, such as rate of growth, development, differentiation potential, cell cycle progression, and apoptosis, and their abnormal expression affects many human diseases (Valeri et al., 2009;Krol et al., 2010;Wahid et al., 2010;Pritchard et al., 2012). In addition to serving as essential players in tumor development, miRNAs have a role as possible biomarkers for cancer (Calin and Croce, 2006). Indeed, miRNA profiles reflect the stages of tumors and their developmental lineages (George and Mittal, 2010). MiRNAs have been found to modulate CSCs and metastasis. They can also act as oncogenes and tumor suppressors. Due to their functions as oncogenes and tumor suppressor genes, these miRNAs have been referred to as oncomirs (George and Mittal, 2010).

miRNAs AND HCC
Recent studies on liver miRNAs investigated the overexpression of specific miRNAs or the inhibition of other miRNAs both in vitro and in vivo. These studies showed the crucial biological roles of miRNAs for proper liver function (Takata et al., 2013). In HCC, Murakami et al. initially reported dysregulated miRNA expression, with four miRNAs, namely, miR-18, miR-92, miR-20, and precursor miR-18 being inversely associated with the extent of HCC development (Murakami et al., 2006). Later, several studies confirmed that miRNAs play an essential regulatory role in hepatic carcinogenesis progression and malignant transformation. Some miRNAs showed abnormal expression during the progression of liver cancer . The expression of some miRNAs was shown to influence HCC development via dysregulation of a number of cancerassociated molecular pathways, including TGF-β, p53, WNT/βcatenin, P13K/AKT/mTOR, RAS/MAPS, MET, and MYC (Negrini et al., 2011). Many oncogenic miRNAs have shown aberrant expression in HCC, including miR-1275 (Shaalan et al., 2018), miR-17-5p (Habashy et al., 2016), miR-96-5p, miR-182-5p (Assal et al., 2015), miR-155 (El Tayebi et al., 2015, and miR-181a (Lashine et al., 2011). Other tumor suppressor miRNAs involved in HCC include miR-34a (Yacoub et al., 2016), miR-486-5p (Youness et al., 2016), miR-615-5p (El Tayebi et al., 2012, and miR-Let7i . Genome-wide approaches have identified hundreds of miRNAs in HCC tumor tissues that were to be dysregulated compared to non-tumor tissues (Borel et al., 2012). MiR-122 is among many unique and well-studied dysregulated miRNAs that are highly expressed specifically in human liver. In HCC patients, a shorter recurrence time were attributed to lower levels of miR-122. While elevated expression of cyclin G1, a target of miR-122, was associated with a lower survival rate. Moreover, miR-122 acts as a tumor suppressor in HCC, and was subsequently reported to be downregulated in around 70% of cases (Callegari et al., 2013). MiR-221 is another critical oncogenic miRNA that is upregulated in 70-80% of HCC cases. Its overexpression leads to enhanced proliferation potential, migration, invasion, rate of growth, and decreased the rate of apoptosis in HCC patients (Fornari et al., 2008). Additionally, miR-221 modulates several gene targets involved in cancer-related pathways, including PTEN (P13K/AKT/mTOR), CDKN1B/p27, and CDKN1C/p57 (Fornari et al., 2008;Garofalo et al., 2009).
Due to their non-invasive detection, good specificity, and sensitivity, miRNAs are considered effective biomarkers for HCC (Shen et al., 2016). . MiR-155-5p and miR-21-5p which are reported as biomarkers for the prognosis of HCC in tissues, were found to have upregulated expression levels. On the other hand others were down-regulated Wang et al., 2014;Yunqiao et al., 2014;Tu et al., 2015). Circulating miR-122-5p and miR-16-5p could be used as presumed biomarkers for HCC. MiR-122-5p and miR-16-5p belong to this group which were particularly detected to be up and down-regulated, respectively (Cho et al., 2015;El-Abd et al., 2015).
As miRNAs expression levels can be used as biomarkers for HCC diagnosis and prognosis, miRNA specific methylation patterns are of importance for therapeutic applications as well. Acting as a tumor suppressor miRNA, decreased expression of miR-10a due to hypermethylation can be used as a biomarker for early HCC diagnosis and risk assessment (Shen et al., 2012). Furthermore, some miRNAs methylation patterns can be HCC cell-specific and therefore used as diagnostic biomarkers. Such miRNAs cell-specific diagnostic methylation patterns include the hypermethylation of miR-129-2, miR-34a, and miR-148a (Anwar et al., 2013;Lu et al., 2013). Also, hypermethylation of mir-9-1 has been shown to be a biomarker for poor diagnosis and aggressiveness (Anwar et al., 2013). In addition to their implications in diagnosis and prognosis, miRNA specific aberrant methylation patterns can be used for therapeutic applications. For example, administration of miR-124, which is hypermethylated in HCC, stopped HCC progression in animal models and was considered safe. Moreover, sorafenib (anticancer drug) increased the expression of miR-1274, which is hypermethylated in HCC, leading to an increased response to therapy (Zhou C. et al., 2011).

miRNAs AND CSCs IN HCC
miRNAs play essential roles in regulating CSCs (Garg, 2015), and in regulating apoptosis in CSCs by acting on mRNAs of apoptosis proteins or regulating mRNAs that are downstream targets in specific apoptotic pathways. These control mechanisms aid in the regulation of metastasis, drug resistance, tumor invasion, pluripotency, and self-renewal potential.
The tumorigenicity of liver CSCs was found to be significantly suppressed by inhibition of miR-181. This miRNA regulates the differentiation potential of liver CSCs through activating transcription factors, including caudal homeobox gene 2 (CDX2) and GATA6, and negatively regulating the Wnt/β-catenin pathway via nemo-like kinase (NLK) (Ji et al., 2009b;Leal and Lleonart, 2013;Bessède et al., 2014). MiR-Let-7 and miR-Lin28 have been reported to be related to the rate of growth and metastasis of HCC. Lin28 is highly expressed in normal embryonic stem cells (ESCs). It maintains the selfrenewal of liver CSCs by inhibiting the interaction of Let-7 with the mature miRNA. Let-7 degradation, which is caused by excessively active Lin28 and c-MYC, dis-equilibrates liver CSCs, leading to accelerated growth and metastasis of HCC (Heo et al., 2008). MiRNAs positively regulate liver CSCs via high expression of EpCAM, which is a prominent marker of liver CSCs. This upregulation is mediated by inhibition of TGF-β by downstream transcription factors of miR-18, such as CDX2, GATA6, and NLK. The EpCAM intracellular domain (EpICD) enters the nucleus and induces overexpression of cyclin D1, c-MYC, and miR-181 after binding to LIM domain protein 2 (FHL2), β-catenin and lymphoid enhancer factor 1 (Lef-1) (Ji et al., 2009b). Another group showed that TGF-β1 downregulate TP53INP1 by targeting miR-155 and promote epithelial-mesenchymal transition (EMT) and liver CSC phenotypes (Liu F. et al., 2015). The Wnt/βcatenin signaling pathway that regulates tumor heterogeneity is mainly related to miRNA, but the mechanism by which this balance between liver CSCs and cancer cells is maintained has not been elucidated.
Based on previous studies, some miRNAs expression was reported to be dysregulated in both HCC and CSCs. In Table 1, we compiled the mutually dysregulated miRNAs to establish the links between these miRNAs and the initiation and progression of HCC.
We classified the previously reported miRNAs as mutually upregulated, mutually downregulated, and mutually dysregulated but not mutually upregulated or mutually downregulated, as illustrated in Figure 1.
A schematic presentation of the role of dysregulated miRNAs in HCC initiation, progression, and aggressiveness is presented in Figure 2.
As reported previously, MSCs aid in cancer development by enhancing the metastatic capability of tumor cells (Hill et al., 2017). This has been also reasoned by the fact that MSCs can home to tumor microenvironment mainly due to the action of stromal cell-derived factor 1 (SDF-1) (Gao et al., 2009). After homing, MSCs start to trans-differentiate into cancer associated fibroblasts mainly due to the action of TGF-beta1 (Ghaderi and Abtahi, 2018). Afterwards, cancer associated fibroblasts (CAFs) start to induce metastasis in neighboring tumor cells by inducing EMT . Some of the significant genes involved in such pro-metastatic signature of the tumor MSCs have been identified in lung cancer and they include GREM1, LOXL2, ADAMTS12, and ITGA11 (Fregni et al., 2018). Also, as investigated by our research group, soluble factors secreted from cancer cells when cocultured with MSCs have shown to induce cancer stem cell-like characteristics in the cocultured MSCs (El-Badawy et al., 2017). Relating the previous information, we are trying to highlight the mutual dysregulated miRNA in HCC, CSCs, and MSCs to investigate whether miRNAs play a vital role in the acquirement of MSCs to pro-metastatic characteristics or development into Cancer stem cells-like cells or even CAFs. So, in Table 2, we summarize the roles of miRNAs that are mutually dysregulated in HCC, CSC, and in MSCs. The functions of these miRNAs may provide insight into their regulatory roles in the development of cancer (Schraivogel et al., 2011). Based on these proposed functions ( Table 2), we classified these miRNAs according to their roles in MSC differentiation. Figure 3 shows the potential pathways that may be involved in the reprogramming of MSCs and their acquisition of CSC-like characteristics after co-culture with cancer cells.
The relationship between the expression of miRNAs expression, their target genes' expression, and the fate of HCC is detailed in Table 3.

DYSREGULATED miRNA METHYLATION IN HCC
Genome-wide abnormal DNA methylation of miRNA host genes in HCC was recently reported. One study analyzed tumor and neighboring normal non-tumor tissues in 62 HCC patients. This analysis was performed using Infinium Human Methylation Analysis Bead Chips. One hundred ten miRNAs from 64 different host genes were covered in this analysis through assessing the methylation of 254 CpG sites. Methylation levels were found to be significantly different at 54 CpG sites from 27 host genes between tumor and neighboring normal non-tumor tissues (Shen et al., 2012). In addition, the expression of three identified miRNAs were measured. MiR-10a was downregulated in tumor tissues and therefore its action on its oncogenic target genes as a tumor suppressor miRNA diminished. This decline appeared to be related to hypermethylation of the host genes. Accordingly, aberrant methylation and expression of miRNAs were considered valuable molecular biomarkers for HCC early diagnosis (Shen et al., 2012). In another study, miRNA genes, from HCC cells and normal liver hepatocytes, showed significantly different profiles of global DNA methylation. In the same study, in HCC cells, miRNAs CpG-poor regions were more commonly hypomethylated rather than being hypermethylation . Investigations using miRNA expression microarray data identified 10 dysregulated miRNAs in HCC that are regulated by DNA methylation. Of the 10 studied miRNAs, miR-23a/27a and miR-25/93/106b constituted two miRNA clusters in which five miRNAs were upregulated. On the other hand, the other five miRNAs including miR-375, miR-195, miR-497, miR-378, and miR-148a were downregulated . The cluster containing miR-25/93/106b, with upregulated expression, was required for cell proliferation including the anchorageindependent growth. It was also shown to target the E2F1 transcription factor in HCC, which inhibits apoptosis . Additionally, miR-331-3p was shown to target PHLPP, a protein that plays a central role in inducing apoptosis and reducing metastasis (Ma et al., 2010;Liu J. et al., 2011;Chang et al., 2014;Peuget et al., 2014). These data provide further evidence for the potential role of miR-331-3p in HCC metastasis. The miR-23a/27a cluster, with upregulated expression, enhanced anti-apoptotic pathways in addition to promoting cells proliferation in HCC (Huang et al., 2008). In another study, miR-429 functioned by manipulating liver 1 | miRNAs whose expression was reported to be dysregulated in HCC and CSCs.
Due to several reports of miRNA hypomethylation in HCC, DNA hypomethylation was shown to play a significant role in miRNA regulation. Moreover, DNA methylation might be responsible for the abnormal expression of these miRNAs.
Data in Table 4 shows some consistent pattern between the role of different miRNAs and their methylation patterns. To illustrate, miRNAs that act as tumor suppressors are hypermethylated while oncomiR are hypomethylated, which finally leads to HCC progression. On the other hand, in HCC, some miRNAs showed an opposite pattern. MiR-10a, miR-10b, miR-9, and miR-196b have been shown to be hypermethylated and their hypermethylation state would lead to reduced tumorigenesis. Therefore, further studies are needed to investigate other roles for these specific miRNAs, and especially the function of miR-596 in HCC progression.

CONCLUSION
Several studies showed how tumor microenvironment enhances cancer development and progression (Whiteside, 2008;Wang et al., 2017;Klymenko and Nephew, 2018). Studies from our laboratory have shown that soluble HCC factors play a vital role in the induction of chemoresistance properties in human bone marrow (hBM)-MSCs and trigger their transformation into CSC-like cells (El-Badawy et al., 2017). However, the mechanism of this transformation remains unclear. Although the initiation of HCC is known to be preceded by cirrhosis, the initiation mechanism itself is thought to involve CSCs. CSCs were proposed to be responsible for chemoresistance and relapse in most cancers. Previous data reported by our research group highlighted the role of CSCs in HCC initiation and progression (El-Badawy et al., 2017). Studies also demonstrated that liver CSCs are associated with liver cancer metastasis and relapse and that CSCs play a substantial role in the resistance of liver cancer to conventional treatment . These data indicate that targeting CSCs as a potential therapeutic approach for liver cancer holds huge promise for improving the treatment outcomes (Lou et al., 2018). Since reprogramming events responsible for the transformation and initiation processes are mainly controlled by epigenetic modifications, determining the role of such epigenetic fingerprints, including miRNAs and their methylation, in initiation, relapse, and chemoresistance in HCC and CSCs is central to understanding tumor biology and developing effective therapies.
Accumulating data on the expression profiles, roles, and regulation of miRNAs are essential for designing effective stem In the context of clinical applications, miRNAs could represent an opportunity to develop safe strategies for achieving early diagnosis, monitoring disease status, and improving the effectiveness of non-invasive HCC treatment (Valeri et al., 2009). Several studies have shown the effect of miRNAs on enhancing  Zhu et al., 2016) Hypermethylation of this miRNA is expected to reduce levels of migration, proliferation, and invasion potential Hypermethylation (Shen et al., 2012) Downregulation miR-196b Targets FOXP2 (Yu et al., 2018) Decreased metastasis, proliferation, and migration potential Hypermethylation (Shen et al., 2012) Downregulation miR-1 It acts as an oncomiR (Hu et al., 2015) Decreased proliferation and migration potential Hypermethylation (Datta et al., 2008) Silenced miR-124 Acts as a tumor suppressor by targeting Baculoviral IAP repeat containing 3 (BIRC3) gene (Cao et al., 2018) Promotion of proliferation and migration potential Hypermethylation (Furuta et al., 2010) Silenced miR-125b Acts as a tumor suppressor by targeting (TAZ) transcriptional co-activator  Increased migration and invasion Hypermethylation (Alpini et al., 2011) Silenced miR-203 Acts as a tumor suppressor by targeting survivin (Wei W. et al., 2013) Increased proliferation potential Hypermethylation (Furuta et al., 2010) Silenced miR-1247 Acts as a tumor suppressor with Wnt3 being its target (Chu et al., 2017) Increased proliferation and invasion potential Hypermethylation (Anwar et al., 2013) Downregulation miR-132 Acts as a tumor suppressor by targeting PIK3R3 (Hu et al., 2015) Increased proliferation, invasion, and migration potential Hypermethylation (Wei X. et al., 2013) Downregulation miR-320 Acts as a tumor suppressor where c-Myc is its target  Increased proliferation and invasion Hypermethylation (Shen et al., 2012) Downregulation miR-596 No data available No data available Hypermethylation (Anwar et al., 2013) Downregulation miR-663 Inhibits proliferation by targeting the HMGA2 gene (Huang et al., 2016) Increased proliferation and invasion Hypermethylation (Potapova et al., 2011) Downregulation miR-9 Different data are reported: It may act as a tumor suppressor role by targeting TAZ (WWTR1) (Higashi et al., 2015). Moreover, it targets KLF17 and increases the migration and invasion properties (Sun et al., 2013) Data reported that hypermethylation could lead to HCC progression (Higashi et al., 2015), while other reports suggested that hypermethylation can lead to reduced migration and invasion properties (Sun et al., 2013) Hypermethylation (Anwar et al., 2013) Downregulation the sensitivity of liver CSCs to treatment. Many dysregulated miRNAs in liver CSCs exert their roles by binding to specific target genes that are key molecules in many pathways. Targeting these miRNAs, their targeted genes, or respective pathways may thus effectively target CSCs, and disturb their role in metastasis, recurrence, and resistance to therapy (Lou et al., 2018). Together with conventional treatment, targeting specific miRNAs involved in tumor progression in combination therapy represent an attractive approach to multifactorial effective therapy to liver cancer (Tao et al., 2018). Although no miRNAs-based drugs are available in current clinical practice (Lou et al., 2018), the antitumor efficiency of modern anticancer drugs like sorafenib on HCC was significantly increased in vivo upon delivery of miR-122-exosome to the tumor (Blechacz and Gores, 2008;Lou et al., 2015). Such enhancement is promising to patients of unresectable HCC whose treatment with sorafenib was of limited efficacy, by prolonging survival for only 3 months (Blechacz and Gores, 2008;Kane et al., 2009). Investigating the expression signature of those candidate miRNAs in HCC diagnosis, prognosis, metastasis, and recurrence and determining how these miRNAs genetically and epigenetically regulate the transformation of somatic stem cells to a more chemoresistant phenotype is needed for translation to clinical practice (Valeri et al., 2009;El-Badawy et al., 2017).

AUTHOR CONTRIBUTIONS
Each author has substantially contributed to conducting this study and drafting this manuscript. MN and RS wrote the manuscript. MN, ME, and SE analyzed data and moderate figures and tables. While, NE-B contributed in writing and editing of the manuscript.

FUNDING
This study was funded by the Egyptian Science and Technology Development Fund (STDF Grant ID: 5300).