**5. miRNAs and cancer cell metabolism**

Along with changes on the genetic level, metabolic changes accompany cancer development in order to provide cells functioning in changing conditions, mainly hypoxia and glucose insufficiency due to intensive cell proliferation and clonal expansion and lagging in blood vessel formation. One of the main such metabolic reorganizations is Warburg effect, firstly reported in rat liver carcinoma in the 1920s and defined as an increase in the rate of glucose uptake and preferential production of lactate even in the presence of oxygen [56]. Main miRNAs, which are involved in Warburg effect realization in HCC cells, are miR-1, miR-122, and miR-338-3p [57]. The expression of the miR-1 targets G6PD and is mediated by NRF2, which, besides activating the transcription of genes encoding glycolytic enzymes, inhibits the conversion of pyruvate to acetyl-CoA by directly activating pyruvate dehydrogenase kinase 1 (PDK1) and leads to inhibition of tricarboxylic acid (TCA) cycle, promoting Warburg effect [58]. At the background of these interactions, it may appear to be regular that a higher expression of miR-1 showed a significant positive prognostic meaning for patients with HCC: Individuals with higher miR-1 serum levels showed longer OS than those with lower miR-1 serum concentrations (195 sera of HCC patients and 54 patients with liver cirrhosis; HR 0.451, 95% CI 0.228–0.856, P = 0.015). At the same time, serum miR-1 and miR-122 concentrations did not differ significantly between patients with HCC and liver cirrhosis [58].

miR-122 is another element of the processes that are assumed to restrain Warburg effect promotion, as far as among its main targets are Agpat 1 and Dgat 1 mRNAs, involved in triglyceride synthesis. One of the most important direct targets of miR-122 in HCC cells is PKM2, which is the most abundant pyruvate kinase iso-enzyme in liver tumors and a co-activator of several transcription factors, such as HIF-1α, β-catenin/c-Myc, NF-κB, and STAT3. Once in the nucleus, PKM2 can promote the transcription of target genes, such as HIF-1α targeted expression of GLUTs, PKM2, LDH-A, and VEGF-A, leading to the promotion of growth, positive feedback regulated glycolysis, and angiogenesis in cancer cells [59], and all these allow miR-122 to promote a decrease in lactate production and increase in oxygen consumption, thus reversing oxygen-independent glycolytic metabolism. Along with this, Yang G. et al. showed that miR-122 is downregulated in tissue samples from patients with HCC and also participates in ADAM17 regulation, which makes upregulation of miR-122 to inhibit proliferation of HCC cells *in vitro* [60]. In general, miR-122 is putatively one of the first examples of a tissue-specific miRNA and is highly expressed in the liver, where it constitutes 70% of the total miRNA pool [61]. Moreover, miR-122 has a prognostic role in HCC patients, and its downregulation is associated with poor prognosis: The overall survival time of the patients with low and high miR-122 expression in HCC was 30.3±8.0 and 83.7±10.3 months, respectively (P<0.001, tissue samples from 64 HCC patients and 28 matched nonneoplastic surrounding liver tissues) [62].

miR-338-3p has the same impact on Warburg effect in cancer liver cells such as miR-1 and miR-122, inhibiting it through decreasing expression of liver and red blood cell pyruvate kinase isoform (PKLR). miR-338-3p may be inactivated in HCC due to upregulation of circMAT2B, sequestering miR-338-3p due to its sponging activity, and disabling the regulation of its target gene PKM2 leading to increased proliferation, invasion, spheroid formation, and organoid dimensions, especially in hypoxic

conditions [59]. Expression of miR-338-3p in tissue samples from patients with HCC was also shown to be decreased [33].

miR-23 was shown to be involved in gluconeogenesis regulation in liver cancer developed in the mouse model. Reduction in serum glucose in tumor-bearing mice correlated with a reduction in the expressions of G6pc, Pepck, and Fbp1 encoding the key gluconeogenic enzymes glucose-6-phosphatase, phosphoenolpyruvate carboxykinase, fructose-1,6-phosphatase, respectively, and the transcription factor Pgc-1α along with upregulation of miR-23a expression. mRNA levels of these genes were reduced to ≈80% in the majority of primary human HCC tissue samples compared with matching peritumoral liver samples and miR-23a was also upregulated in human liver cancer samples. Moreover, PGC-1α and G6PC expression negatively correlated with miR-23a expression in human HCCs [63]. miR-23a has a significant diagnostic value as far as it may distinguish cirrhotic liver samples from cancer liver samples, being higher in the HCC group than cirrhotic. miR-23a was significantly higher in HCC patients with focal lesion size equal or more than 5 cm, patients with multiple focal lesions, and Okuda stage III. At cutoff value ≥ 210, miR-23a showed accuracy of 79.3% to diagnose HCC patients with sensitivity of 89.47% and specificity of about 64.91% ((57 patients with HCC, 57 patients with liver cirrhosis (LC), and 57 healthy subjects as control group) and serum alpha-fetoprotein at cut off level ≥ 200 ng/mL had 73.68% sensitivity and 52.63% specificity for diagnosis of HCC [64].

#### **6. Differential expression of miRNAs in chronic liver disease**

Pathological states in some cases underlying liver cancer development are also accompanied by changes in miRNA expression levels such as hepatitis B virus infection, hepatitis C virus infection, nonalcoholic fatty liver disease (NAFLD), and alcoholic liver disease. One of miRNAs, which is associated with hepatitis virus, is miR-23 [31]. Other miRNAs also may differentiate HCC samples at the background of viral hepatitis from others, such as miR-17-92. Together with miR-21, its expression level was increased in hepatitis B virus-positive human and woodchuck HCC samples. Possibly, hepatitis B virus X and miR-17-92 share common target gene, which is c-myc, which is activated by virus and is known to be the instrument of carcinogenesis promotion of miR-17-92 [65]. Unlike HCV infection, HBV is known to induce HCC development gapping cirrhosis stage, thus making molecular predictors of cancer in this case especially required. Besides miR-122 and miR-17-92, other miRNAs, involved in HBV pathogenesis following HCC development, are miR-184, miR-185, miR-196a, miR-199a-3p, miR-210, miR-217, and miR-34a, which are involved in HBV transcription process. The expression of the last is inhibited by HBV X protein (HBx) *via* p53 stimulation in hepatocytes, upregulating a macrophage-derived chemokine (CCL22), participating in regulatory T cells stimulation and effector T cells suppression, which results in increasing HBV genome transcription [66, 67]. Another miRNA, whose expression changes in HBV infection due to HBx, is miR-155. Upregulation of miR-155 leads to a reduction in the suppressor of cytokine signaling-1 (SOCS1) expression, increasing JAK/STAT signaling and suppressing HBV infection mediated by the induction of interferon (IFN) signaling [68]. Some of these miRNAs, which are involved in HBV transcription process, are also involved in the regulation of signaling pathways, disrupted in the cancer development. Among putative targets of miRNA-199a-3p are mTOR, c-Met, HIF-1α, CD44, ROCK1, and Axl, so this miRNA, being downregulated in

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

liver cancer samples, may inhibit cell proliferation, migration, and invasion [69]. miRNA-184 may function as an anti-apoptotic factor in liver cancer, and INPPL1 was identified as one of its targets. Through inhibition of the activities of caspases 3/7 and INPPL1 loss, it promotes cancer cell proliferation [70]. Similar function in HCC development through another target may play miRNA-196a. One of its direct targets is FOXO1, whose inhibition caused by miRNA-196a overexpression resulted in liver cancer cell migration and invasion *in vivo* [71]. miRNA-210 is known to be overexpressed in liver cancer samples and in HBV-associated liver cirrhosis; moreover, its expression was significantly higher in HepG2.2.15 cells than that in HepG2 cells. EGR3 was shown to be the target of miRNA-210, contrary to miRNA-210 the expression of EGR3 was downregulated in HBV-associated liver cirrhosis and liver cancer. In general, its role, like the role of miRNA-196a and miRNA-184, may be in proliferation promotion, and it is hard to assume whether the described mechanism with involvement in EGR3 regulation is specific for HBV-associated liver cancer, as far as EGR3 is known to be decreased in other cancers, like in head and neck cancers and gastric cancer [72]. miRNA-210 was also shown to be involved in bile acid-induced cholestatic liver injury through its direct target MLL4 (the histone methyltransferase mixed-lineage leukemia-4). miRNA-210 was the most highly elevated miR in mice with elevated hepatic BA levels and its expression was increased in patients with primary biliary cholangitis/cirrhosis (PBC) [73]. Opposite to the mentioned miRNAs, miRNA-217 inhibits liver cancer cell proliferation *via* targeting NAT2; moreover, its overexpression contributed to steatosis in hepatocytes as well as inflammation in mice, acting as a critical regulator in ethanol-induced hepatic inflammation [74]. miRNA-185 was shown to be involved in cholesterol metabolism in mouse model, and animals with knockout of this miRNA developed worsened hepatic steatosis upon high-fat high-cholesterol Western diet feeding with accumulation of triglyceride and cholesterol in the liver and developed hypercholesterolemia upon Western diet feeding. Treatment with miRNA-185 showed an improved accumulation of lipids in high-fat diet mouse model and insulin sensitivity *via* upregulation of the insulin-receptor substrate-2 [75, 76].

In case of HCV infection, there are miRNAs, which not just regulate expression of the genes, involved in virus replication, but are able to directly target the viral genome. Among these miRNAs are miR-196, miR-448, and miR-122, which stabilize the 5′ and 3′ UTRs of the HCV genome, so inhibition of this miRNA dramatically reduces the replication of HCV RNA [77, 78]. miRNAs, involved in viral replication *via* reducing tumor suppressor deleted in liver cancer 1 (DLC-1) and cell entry *via* the PI3K/AKT signaling pathway, are miR-141 and miR-491, respectively [76].

When it concerns alcoholic liver disease, miRNA expression profile changes especially due to increase in expression of inflammation-related miRNAs, such as miR-132, miR-155, miR-146, and miR-21, which influence alcohol/lipopolysaccharide (LPS)/TLR4 pathways, transmitting proinflammatory stimuli *via* a mitogen-activated protein kinases (MAPKs) or TIR domain-containing adaptor-inducing IFN-β (TRIF) [79, 80]. The imbalance between expression of let-7 and LIN28/28B has the crucial consequences, as far as regular alcohol overdose consumption diminishes let-7 expression, and loss of let-7 induces transformation of hepatic stellate cells (HSC) to mesenchymal phenotype, enhancing liver injury *via* inhibiting LIN28B, and thus promoting oncogenesis [81]. One of mechanisms underlying fibrosis formation in response to alcohol consumption is miR-34a, and its upregulation causes deregulation of its direct targets such as caspase-2, SIRT1, and matrix metallopeptidase (MMP) 1 and MMP2 [82].

Another risk factor for HCC development is NAFLD. It was revealed that miR-34a and miR-122 identified in blood serum are potential markers for discriminating NAFLD patients from healthy controls with an area under the curve (AUC) values of 0.781 and 0.858, respectively, along with miR-21, miR-125b, and miR-375 did not show significant difference in level expression between NAFLD patients and healthy controls. Serum levels of miR-34a and miR-122 were found to be significantly higher among NAFLD patients and were positively correlated with VLDL-C and triglyceride levels [83]. The other study showed the same tendency for miRNA-34a and miRNA-122, and also found a significant difference in level expression of following miRNA: miR-21and miR-451. Moreover, the serum level of miR-122 was correlated with the severity of liver steatosis; however, in the previous study, the expression levels of miR-34a and miR-122 did not correlate with the histological features of NAFLD [83]. There is at least one common signaling pathway, involving both miRNA-34a and miRNA-122, which is AMPK [21, 82]. It should be admitted that along with being possibly involved in the regulation of the same targets, miRNA-122 and miRNA-34 definitely should have similar effects as far as miRNA-122 expression is decreased in cancers including liver cancer, and miRNA-34 expression is decreased in liver cancer while being elevated in chronic hepatitis C patients. Targets and mechanisms of miRNA-34 involvement in the liver cancer development are known in less details than miRNA-122. It is supposed, that through involvement in the Sirt1/p53 pathway regulation, miRNA-34 promotes liver fibrosis in patients with HCV [84]. It is possible to speculate that with loss of the benign liver cells phenotype, malignant liver cells lose possibility of normal miRNA-34 expression as far as miRNA-34 expression is significantly decreased in liver cancer cells. One of the possible mechanisms of miRNA-34 involvement in the liver cancer development is glucose metabolism, in which LDHA, which is the target gene of miRNA-34, participates in glucose metabolism reprograming with its switch to the increased glycolysis [85]. In its turn, miRNA-122 is also indirectly involved in glucose metabolism, having Igf1R as one of its targets [63]. The other putative intersection of these two miRNAs is p53 pathway, as far as both participate in its regulation. miRNA-34 in the context of HCV fibrosis via Sirt1 regulation and miRNA-122 through cyclin G1, which results in increased p53 protein stability activity and reduction in invasion capabilities of HCC cells with elevated miRNA-122 expression [86]. Another miRNA-132, which possibly shares with miRNA-34 SIRT1 as a common target, is miRNA-132. This miRNA is elevated in the response to alcohol consumption, while *in vivo* and *in vitro* studies suggest miR-132 targets SIRT1, being increased in HCC cells and associated with unfavorable survival in HCC patients [87].

With the cancer development, differences in miRNA expression profiles smooth out as far as cancer cell phenotype and functions despite its different background development including such common features as promoted proliferation, disrupted apoptosis, and increased migratory and invasion capabilities. miRNA expression during the process of malignization changes in conformity with these demands, so far miRNA-34, being elevated in HCV liver cirrhosis, is decreased in the liver cancer cells. Obviously, all changes in miRNA expression during cancer development tend to upregulation of oncogenic miRNAs and downregulation of tumor suppressor miRNAs, and with evolution of the stage of the tumor differences in miRNA expression associated with different background liver disease level out. However, different miRNAs may be involved in the same signaling pathways or share common target genes, which is allowed by the sequence and molecular nature of miRNA—mRNA interaction—and indirect influence of miRNA on the expression levels of the genes, which are not its direct targets.
