**1. Introduction**

miRNAs are small noncoding RNAs ~20–25 nt long, which are involved in epigenetic regulation of gene expression. miRNAs were firstly discovered in 1993, when research groups led by Victor Ambros and Gary Ruvkun published two side-by-side papers in the journal Cell, describing the regulatory effects of tiny RNA discovered in Caenorhabditis elegans. Years later the term "microRNA" (miRNA, miR) was introduced [1]. Nowadays, more than 2600 miRNAs have been predicted to be encoded by the human genome, with the ability to modulate more than 15,000 genes [2]. Being small and noncoding RNAs, they have a huge and significant function in gene regulation and cancer development. Liver cancer, although not being at the top of most common cancers, remains among cancers with high mortality. Hepatocellular carcinoma (HCC) accounts for approximately 80% of all liver cancers and is a main cause of cancer mortality [3]. Hereinafter, when using the term liver cancer, it is meant to indicate hepatocellular carcinoma. The most obvious and significant reason for this high mortality rate in HCC patients is the late diagnosis of HCC. Against this background, every aspect of molecular pathogenesis becomes a valuable detail, which may aid in understanding HCC development. Some of the main miRNAs involved in the development of liver cancer will be discussed further. Changes in their expression levels were detected in comparable conditions: mostly in human liver tissue samples or human blood samples (plasma or serum) by qRT-PCR.

#### **2. miRNA biogenesis and regulation**

Despite the simple structure of a mature miRNA molecule—single-stranded RNA molecule of 20–25 nt—its biogenesis, like almost any process relating to nucleic acids, is multistage and multifactorial: It takes place both in the nucleus and in the cell cytoplasm, involves protein complexes for processing (miRNA maturation), may be performed *via* canonical and non-canonical pathways, and includes following transformations: primary miRNA (pri-miRNA), preliminary miRNA (pre-miRNA), miRNA duplex, and mature miRNA. Both canonical and non-canonical processing start in the nucleus.

miRNA processing may occur post- or co-transcriptionally [4]. miRNA biogenesis starts in the nucleus and requires RNA polymerase II/III; Drosha (an RNase III-like enzyme) with its cofactor, the RNA binding protein DGCR8 (DiGeorge Syndrome Critical Region 8), forms a microprocessor complex and functions in the nucleus, an exportin (frequently Exportin5 in the canonical pathway and Exportin1 in the non-canonical pathway) functioning as the transporter to the cytoplasm. Dicer, another RNase III-like endonuclease, RISC (RNA-induced silencing complex) functioning in the cytoplasm with Argonaute (AGO) as a core component. miRNA biogenesis starts with the processing of RNA polymerase II/III and forming primiRNAs, which are 5′ capped and 3′ polyadenylated, approximately several kilobases [5, 6]. In the canonical pathway, pri-miRNAs then are processed in the microprocessor complex of Drosha and DGCR8. The resulting ∼70 nucleotide RNAs with 2 nt 3′ overhang are known as precursor (pre-) miRNAs, which fold into mini-helical structures [7]. Pre-miRNAs are transported from the nucleus to the cytoplasm with Exportin 5/RanGTP complex, where they undergo processing with Dicer, which recognizes the pre-miRNA hairpin and cuts it at the loop end, resulting in the removal of the terminal loop and creating a ∼22 nt RNA duplex [8]. The final step of the miRNA biogenesis is processing the duplex miRNA into mature single-stranded miRNA by loading it onto an Argonaute (Ago) protein, which is the core protein in this final effector complex—RNA-induced silencing complex (RISC). The mature miRNA may be derived from both the 5' and 3' arms of the precursor duplex and are called the miRNA-5p and -3p, respectively [9].

As for regulation of miRNA expression with some miRNAs having their own promotors and some being regulated by other gene promotors, besides methylation, different endogenous factors, and hypoxia, different transcription factors (TF) also participate in miRNAs expression regulation and this is a double-edged process: TFs influence miRNA expression and miRNAs may repress TF expression [7, 10]. miRNA, TFs, and target genes form a complex relationship known as feedback loops (FBLs) and feed-forward loops (FFLs) [11]. Typically, FBLs occur when a TF activates or represses a miRNA, which in turn represses the TF; the miRNA and TF each regulate independent sets of TGs. FFLs are those where a regulator, such as a TF, controls the expression of a specific TG both directly, through promoting or enhancing its transcription, and indirectly, through another regulator, such as an miRNA that also regulates the TG [12].

mRNA and miRNA interaction implies binding of the last to the 3′ untranslated region (3′ UTR) of mRNA through base-pairing of the seed region of target mRNA, mainly at position 2–7 from the 5′ end of the miRNA; beyond the seed region, the binding between the whole mature miRNA sequence and the target mRNA is not perfectly complementary [13]. However, the interaction of miRNAs with other regions, including the 5′ UTR, coding sequence, and gene promoters, has also been reported.

In general, there is no direct correlation between miRNA and target mRNA expression levels. Multiple miRNAs can regulate a single gene/mRNA, and some miRNAs can target many mRNAs (up to more than 100 mRNAs) and from 1 to 2% of human transcripts interact with nine or more miRNAs, thus displaying sponge-like activity [14]. Furthermore, miRNAs have been shown to activate gene expression under certain conditions [5]. Along with mRNAs differing in their miRNA-binding capacity, binding activity of some highly expressed miRNAs may be weakened by either a high target-to-miRNA ratio or the relocation of this miRNA to the nucleus. Some miRNAs might be expressed at relatively low levels and interact with many mRNAs and, oppositely, some miRNAs might be expressed at a very high level and interact with only a few mRNAs [15].

Considering all of the above, it makes identifying the specific miRNA-target gene or transcription factor-target gene interactions difficult, and possibly unwarranted [12].

### **3. miRNA in liver functions**

Apparently, miRNAs are involved in all processes underlying normal liver functioning, so main pathologic processes, such as nonalcoholic fatty liver disease (NAFLD), fibrosis at the background of various diseases, and cancer are associated with significant changes in miRNA expression profiles, although these changes are not always associated with target mRNA expression changes and the mechanism of miRNA participation in these processes is not clear. A significant role of some miR-NAs was shown for main liver functions such as lipid metabolism and all the steps of glucose metabolism, including lipogenesis.

#### **3.1 miRNAs and the regulation of lipid metabolism**

Pivotal role of miR-34a in PPARα (the peroxisome proliferator-activated receptor alpha) pathway, which is a direct target of miR-34a and a master regulator of lipid metabolism, was shown in cultured cells transfected with miR-34a inhibitor and simultaneously consequences of miR-34a inhibition were shown in C57BL/6 mice injected with the miR-34a inhibitor [16]. The upregulation of miR-34a resulted in the downregulation of hepatic PPARα and SIRT1 (silent mating type information regulation 2 homolog 1), silencing miR-34a led to an initially increased expression of PPARα, SIRT1, and PPARα's downstream genes, and activation of the central metabolic sensor AMPK was also increased. In the mouse model, the miR-34a inhibitor suppressed lipid accumulation and improved the degree of steatosis, which is assumed to be regular as far as its level was significantly upregulated in liver tissues of high-fat diet-fed mice [17].

miR-122 is among those playing a crucial role in lipid metabolism in the liver: miR-122 expression in mice liver was increased by free fatty acids (FFAs) *via* activating the retinoic acid-related orphan receptor-alpha, inducing secretion of miR-122 to blood, entering muscle and adipose tissues of mice, reducing mRNA levels of genes involved in triglyceride synthesis, mainly, Agpat1 and Dgat1. It also led to the attenuated triglyceride synthesis and elevated β-oxidation pathway [18]. Before it was shown that cholesterol biosynthesis genes would be affected by miR-122, plasma cholesterol levels were reduced in antagomir-122-treated mice, thus illustrating attenuation of the cholesterol biosynthesis when silencing hepatic miR-122 [19]. There are another data,

also proving the meaning of miR-122 in lipid metabolism, its inhibition in normal mice resulted in reduced plasma cholesterol levels, increased hepatic fatty-acid oxidation, and a decrease in hepatic fatty-acid and cholesterol synthesis rates. Simultaneously, miR-122 inhibition in a diet-induced obesity mouse model also resulted in decreased plasma cholesterol levels and a significant improvement in liver steatosis, accompanied by reductions in several lipogenic genes [20]. An increase in miR-122 expression in obese subjects may appear to be the compensated mechanism to maintain lipid metabolism. Besides miRNAs, hepatic lipid accumulation recruits inflammatory response, impairing some signaling pathways involved in lipid metabolism, including the AMPK signaling pathway, the role of miR-122 in energy metabolism in the liver, skeletal muscle, and adipose tissues requires more evidence to evaluate [21].

Among other miRNAs, possibly acting as modulators of lipid and cholesterol levels in the maintenance of cholesterol and fatty acid metabolism, are miR-33, miR-103, miR-104, and miR-307 [22, 23]. Obviously, some miRNAs may be involved in both lipid and glucose metabolisms, as far as one miRNA may have multiple mRNA targets. One of these miRNAs is miR-33a and miR-33b, intronic miRNAs located within the sterol regulatory element-binding protein (SREBP) genes, working in concert with its host gene to ensure a fine-tuned regulation of lipid and glucose homeostasis. miR33b also cooperates with SREBP1, having an impact on key regulatory enzymes of hepatic gluconeogenesis glucose metabolism—phosphoenolpyruvate carboxykinase (PCK1) and glucose-6-phosphatase (G6PC). Overexpression of miR-33b in human hepatic cells leads to a significant reduction of glucose production *via* inhibition of PCK1 and G6PC expression [24].

miR-206 was shown as a potent lipid and glucose production inhibitor by simultaneously facilitating insulin signaling and impairing hepatic lipogenesis due to promoting phosphorylation of INSR (insulin receptor) and impaired hepatic lipogenesis by inhibiting Srebp1 (sterol regulatory element-binding transcription factor 1) transcription and inhibition of PTPN1 (protein tyrosine phosphatase, non-receptor type 1) *via* interaction with its 3′ untranslated region and following degradation. miR-206 reduced lipid and glucose production in human hepatocytes and livers of dietary obese mice [25].

#### **3.2 miRNAs and insulin signaling**

miR-103 and miR-107 were the first two miRNAs shown to regulate insulin sensitivity in liver and adipose tissue in mice: Their overexpression in these mouse models led to downregulation of caveolin-1 expression, a component of caveolae lipid raft required for insulin receptor signaling. miR-802 was also shown to be involved in the regulation of insulin sensitivity and glucose transport: elevated miR-802 decreased expression of HNF1β while increasing expression of the insulin suppressors, SOCS1 and SOCS3. Increased expression of both SOCS1 and SOCS2, in turn, desensitizes insulin signaling, resulting in increased hepatic glucose production in these mouse models [26]. It is worth noting that miR-23a was first reported as a regulator of gluconeogenesis through direct binding at the 3'-UTRs of both G6Pase and PGC-1α mRNAs, and later its expression was found to be elevated in hepatocytes of hepatocellular carcinoma mice where gluconeogenesis is attenuated [27].

#### **3.3 Circular RNAs**

In general, when discussing miRNA functions and interactions, it should be noted, besides its elusive relations with mRNA, that they are not limited to mRNA

#### *miRNAs in Liver Cancer DOI: http://dx.doi.org/10.5772/intechopen.106171*

and involve other RNAs as well, that may influence its activity and function, for example, circRNAs have been validated as microRNA (miRNA) sponges, which have complementary sequences binding to their target miRNAs, thereby inhibiting the function of those miRNAs and abolishing the inhibition of target gene expression [27]. Circ-0000092 with such miRNA sponging activity and miR-338-3p as a target miRNA was shown to be elevated in HCC tissue (40 patients, RT-qPCR, GAPDH) and cell lines, while miR-338-3p was shown to be decreased (40 patients, RT-qPCR, U6 as an internal control) [28]. Simultaneously, miRNA-338-3p target, HN1, shown to be overexpressed by Liu et al. in liver cancer and known to be involved in metastasis and invasion development in breast and prostate cancer partly due to negative impact on the b-catenin/E-cadherin interaction, was shown to be elevated along with circ-0000092 [29]. These data along with the effects of delivery of a series of mimic, inhibitor, or siRNA plasmids into HCC cells on cell proliferation, migration, invasion, and angiogenesis *in vitro* may allow to assume that circ-0000092, absorbing miRNA-338-3p and positively influencing HN1 expression, and promote cancer cell proliferation and invasion. A possible mechanism for maintaining CSC (cancer stem cell) self-renewal with HN1 involvement is enhancing of oncogenic factor MYC, and the LEPR–STAT3 pathway [29]. Another circRNA with specific sponging activity for miRNA-338-3p is circMAT2Bm, which also negatively influences miRNA-338-3p and, as a result, positively one of its targets—PKM2, which encodes one of the key enzymes in the process of glycolysis [30]. CircASAP1 in liver cancer acts as a competing endogenous RNA for miRNA-326 and miRNA-532-5p, which play a tumor suppressor role in liver cancer, regulating MAPK1 and CSF1. CircUHRF1, which is predominantly secreted into plasma in exosomes by HCC cells, inhibits the activity of miRNA-449, upregulating the expression of TIM3 and inhibiting NK cell function. The expression of circUHRF1 is higher in HCC tissues than in corresponding adjacent nontumor tissues [31]. There are other circRNAs, whose expression level is decreased in HCC tissues compared with noncancerous tissues, and that were shown to have miRNAs among their targets, such as circTRIM33-12 and miRNA-191, circHIAT1 and miRNA-3171, circLARP4 and miRNA-761, and circMTO1 and miRNA9. Decreased expression of these circRNAs in HCC resulted in elevated expression level of the corresponding miRNAs and sustaining of proliferation, invasion, and metastasis of cancer cells [31].

### **4. miRNA expression patterns in HCC**

It is evident that most of the processes taking place in the liver in normal condition require different miRNAs and assumably healthy hepatocyte should have its "normal" miRNA profile with wide ranges, which make it possible to suggest—there are strong pieces of evidence that will be discussed further—those different pathologic processes, forming the diseases, are accompanied with different changes in miRNA expression levels. With regard to hepatocellular carcinoma, it could be speculated that in the very initial stages, particularly at preclinical stages when it is favorable for cancer background but still no clinical manifestation of cancer, liver cancer may have different miRNA expression profiles within the same cancer type. Further will be discussed the liver cancer development and partly the background of viral hepatitis B, viral hepatitis C, nonalcoholic fatty liver disease (NAFLD), and alcohol-related liver disease (ARLD).

miRNAs along with other nucleic acids have a significant impact on cancer development, where they may have both the role of cancer promotion and cancer suppression; therefore, miRNAs with increased expression in tumors are thought to function as oncogenes and are termed as oncomirs. On the contrary, miRNAs with decreased expression in cancer cells are considered tumor suppressor genes, presumably preventing tumor development by negatively inhibiting oncogenes and/or genes that control cell differentiation or apoptosis [32]. miRNAs are known to be involved in most signaling pathways, and in the liver cancer development, the same signaling pathways are involved, like in most other cancer types, such as TGF-β, Wnt/B-catenin, Hh, Notch, EGF, HGF, VEFG, JAK/STAT, Hippo, and HIF, which lead to uncontrolled cell division and metastasis [32].

Main miRNAs, involved in TGF-β regulation, are miR-200, miR-21, miR-211, miR-17/92, miR-106b/25, and miR-182 [33]. miR-200 and miR-21 are one of the main players among noncoding RNAs in interaction with TGF-β signaling in the process of EMT. miR-200 forming a double-negative feedback loop with ZEB factors (zinc finger E-box-binding homeobox) plays a significant role in EMT (epithelial-mesenchymal transition): miR-200 is downregulated because of reversible DNA methylation of the miR-200 loci as a result of prolonged autocrine TGF-β signaling, driving a sustained ZEB expression, and thus maintaining a stable mesenchymal phenotype. miR-200 is known to interact with both ZEB factors—(ZEB1; also known as deltaEF1) and SIP1 (also known as ZEB2) [34]. miR-200a is responsible for significant inhibition of cell proliferation and colony formation rate in HCCLM3 and HepG2 cell lines, while knocking out miR-200a restores the rate of proliferation and colony formation of cancer cells [35]. miRNA expression levels of miR-200 family tend to be decreased in individuals with liver cancer (plasma and tissue), compared with healthy individuals, and have a prognostic value for patients with HCC: microRNA-200a and miR-200c were independent prognostic factors for hepatocellular carcinoma and induced cell cycle arrest by targeting CDK6 or MAD2L1, respectively [36]. Opposite to miR-200 family, expression of miR-21 is induced in response to TGF-β signaling and is associated with tumor invasion and chemoresistance *in vitro*. Besides this, Wang Z et al. mention, indicating this is unpublished data, that Notch-1 could be one of miR-200b targets because overexpression of miR-200b significantly inhibited Notch-1 expression [37]. Moreover, miR-21 is able to directly interact with TGF-beta receptors: Mishra S. et al. revealed that miR-21 suppresses a tumor-suppressor gene TGFBR2 (transforming growth factor-beta receptor II) levels by binding to its 3 0-UTR, hence inhibiting the tumor-suppressive activity of TGFβ pathway [38]. Reported target genes for miR-21 in HCC are the following: FASLG, PTEN, HBP1, IL-12, RECK, and TIMP-3; some of these genes were shown simultaneously to be miR-21 targets in other liver diseases, such as ALD (FASLG), NAFLD (HBP1), and liver fibrosis (TIMP3) [39]. There are plenty of data showing an increase in miR-21 expression level in the background of liver cancer development or the chronic liver diseases, which are the risk factors for liver cancer development.

Members of the miR-17-92 and the miR-106b-25 clusters have been implicated in the progression of liver fibrosis through the influence on the expression of TGF-β receptor II (TGF-βRII), having opposite effects on this expression. miR-19b has been shown to play an inhibitory role in hepatic stem cell-mediated fibrogenesis and to be decreased in fibrotic rats and human livers. Overexpression of miR-19b inhibited the expression of TGF-βRII, which in turn inhibited SMAD3 expression and, as a result, reduced type-1 collagen production. Unlike miR-19b, miR-93 and miR-106b were observed to be consistently upregulated during the development of cirrhosis, and miR-106b along with miR-181was shown to have a diagnostic value for liver cirrhosis irrespective of the etiology [40].

#### *miRNAs in Liver Cancer DOI: http://dx.doi.org/10.5772/intechopen.106171*

miR-211, which is known to be involved in TGF-β interaction in prostate cancer cells, was shown to be involved in WNT-β signaling regulation via SATB2. In prostate cancer cells, increased expression of miR-211 inhibited expression of TGF-β1, TGF-β2, smad2, smad3, phosphorylated smad2, and smad3, and stem cell markers and *in vitro* resulted in reductions in the proliferation, invasion, colony-forming ability, sphere-forming ability, and stemness of prostate cancer stem cells, *in vivo* in decreased tumor growth, and cell apoptosis [41]. In HCC cells, miR-211 is supposed to suppress cancer cell proliferation *via* WNT-β and SATB2 downregulation. 3′-UTR of SATB2 was shown to be the direct target of miR-211, it contains a conserved target site for miR-211, and *in vitro* miR-211 mimics repressed the luciferase activity of the luciferase gene with inserted 3′-UTR of SATB2 in the pGL3-control vector. miR-211 expression in HCC tissues and cells is inversely correlated with SATB2, when in HCC tissues miR-211 expression was decreased, SATB2 expression was upregulated.

MiR-125b is known to interact with the Hh pathway, which is a well-known factor regulating liver reconstitution. miR-125b, produced by CP-MSCs (chorionic platelet-derived mesenchymal stem cells), attenuates Hh activation partly due to Smo expression inhibition and the consequence of this regulation is the promotion of the regression of fibrosis, contributing to liver regeneration [42]. It was demonstrated that another target of miR-125b in HCC cells is LIN28B, and simultaneously miR-125b may increase p21Cip1/Waf1 expression and arrest cell cycle at G1 to S transition, which may contribute to suppression of HCC cell migration, invasion, and growth *in vitro* and *in vivo* [43]. With regard to this data, Liu W. et al. demonstrated that the expression level of serum exosomal miR-125b in patients with HCC (158 samples, qRT-PCR, normalization normalized to caenorhabditis elegans miRNA (CelmiR-39)) was decreased in comparison with the expression level of serum exosomal miR-125b in patients with chronic hepatitis B (n=30) and liver cirrhosis (n=30). Moreover, the exosomal serum miR-125b level was shown to have a prognostic value for HCC patients: It predicted the recurrence and survival of HCC patients with an area under the ROC curve of 0.739 (83.0% sensitivity and 67.9% specificity) and 0.702 (82.5% sensitivity and 53.4% specificity) [44]. Moreover, inhibition of miR-125b suppressed the expression of profibrogenic genes in culture-activated primary HSCs and reduced the basal and transforming growth factor β (TGF-β)-induced alpha-smooth muscle actin (α-SMA) expression and cell contraction of the immortalized HSC cell line [45].

miRNA-199a-3p, being one of the putative therapeutic tools in liver cancer, may perform its anticancer effect through involvement in NOTCH signaling. miRNA-199a-3p is downregulated in liver cancer tissues and most liver cancer cell lines; in liver cancer cell lines (MHCC97H, Hep3B, SMMC-7721, Huh7, and HepG2), its expression was significantly lower than in normal liver cell lines; simultaneously, mRNA YAP1 expression was significantly higher than in normal liver cell lines. It was shown that miRNA-199a-3p targets YAP1, downregulates Jagged1, and suppresses the Notch signaling, which results in HCC cell proliferation inhibition and apoptosis promotion [46]. In a mouse model with induced HCC treatment with miRNA-199a-3p showed regression of hepatocellular carcinoma with the restoration of normal architecture on histopathological examination of liver specimens [47].

In liver cancer, HGF, ERBB3, and NF-κB form a positive feedback loop: higher expression of ERBB3 makes liver cancer cells more sensitive to HGF stimulation; moreover, HGF enhances ERBB3 expression by NF-κB transcriptional activity. miR-17-5p and miR-20a-5p in liver cancer cell lines and mice xenograft models were shown to suppress liver cancer cell proliferation after hepatectomy *via* blocking

HGF, ERBB3, and NF-κB positive feedback loop. HCC patients with lower levels of miR-17-5p and miR-20a-5p or higher levels of ERBB3 had significantly shorter OS and PFS survivals after surgical resection [48]. Simultaneous deregulation of VEFG and miRNA expression was shown in tissue samples of patients with liver cirrhosis, while VEGF did not show a significant difference in expression level in HCC samples compared to control (non-cancer and non-cirrhotic) samples. Expression level of VEGF was 12.97-fold higher in cirrhotic patients compared to liver cancer samples; concurrently, miR-206 and miR-637 (RT-qPCR, U6, RNU44, and RNU48 were used as reference genes) were down-expressed in LC samples. miR-637 was downregulated in HCC samples too [49]. Before it was shown that in HCC cells, miR-637 is responsible for suppressing autocrine leukemia inhibitory factor (LIF) expression and exogenous LIF-triggered activation of the transcription factor Stat3, which regulates several growth factors, including the VEGFA gene [50]. miR-146a indirectly influences VEGF expression in HCC cells through upregulating APC, which inhibits β-catenin accumulation in nucleus, and downregulating NF-κB p65 by targeting HAb18G [51]. An increase in expression of mRNA Jak2 and Stat3 along with reduced expression of miRNA-409 and reduction of Jak2 and Stat3 protein in response to miRNA-409 overexpression in liver cancer cells may allow to assume miRNA-409 in liver cancer playing an antitumor function through interaction with Jak2 and Stat3. Increased expression of miRNA-409 in liver cancer cells led to a decrease in cell viability and increased apoptosis. This miRNA expression level was significantly decreased in liver cancer tissues compared with paracancerous and normal liver tissues and was negatively correlated with tumor stage, tumor size, and overall survival time of patients with liver cancer [52]. Another miRNA, which is putatively involved in interaction with Jak2 and Stat3 in liver cancer, is miRNA-543, whose expression level was also shown to be decreased in liver cancer tissues. Like miRNA-409, it has a protective role in liver cancer and OS in patients with liver cancer and increased miRNA-543 is longer than in patients with decreased miRNA-543. Inhibition of miRNA-543 expression resulted in liver cancer cells with exactly the same consequences like inhibition of miRNA-409: increased cancer cell proliferation and decreased apoptosis. It also activated the protein expression of phosphorylated JAK2, phosphorylated STAT3, c-Myc, and B-cell lymphoma 2 (Bcl-2) in liver cancer cells [53]. miRNA-3662, which downregulated in HCC tissues and cell lines, may be involved in reprogramming cancer cells' glucose metabolism and forming of Warburg effect while having hypoxia-inducible factor-1α (HIF-1α) as one of the direct targets. Gain-of-function and loss-of-function assays showed that miR-3662 dampened glycolysis by reducing lactate production, glucose consumption, cellular glucose-6-phosphate level, ATP generation, and extracellular acidification rate, and increasing oxygen consumption rate in HCC cells [54]. Another putative target of miRNA-3662 in HCC cells, which allows its regulation of glucose metabolism, is hexokinase 2 (HK2). miR-3662 expression was decreased in liver cancer tissues and cells, while overexpression of miR-3662 or knockdown of HK2 inhibited cell proliferation, invasion, and glucose metabolism in cancer liver cells, which could be reversed by upregulating HK2 [55].

Taking into account the ambiguous relation between miRNA and mRNA expression levels and other factors, including circRNAs and proteins, associated with miRNA biogenesis and those involved in miRNA and mRNA interactions, prediction of changes in miRNA expression levels in any cancer, including liver cancer, becomes not that obvious task.
