**7. Differential expression of miRNAs in HCC**

Concerning signaling pathways disrupted in HCC, almost all these pathways the regulation is double-sided: miRNA may regulate the expression of the genes and genes may regulate the expression of miRNAs. For example, TGF-β signaling may modulate miRNA expression level *via* canonical pathway, which is Smad-dependent requiring co-Smad Smad4, and non-canonical pathway, such as Smad4-independent. The Smad binding element found in the promoters of TGF-β/BMP-regulated genes contains a conserved sequence similar to the pre-miRNAs of TGF-β/BMP-regulated miRNAs, which is CAGAC [88]. miRNAs, containing this RNA-Smad binding element (R-SBE), are following: miR-105, miR-199a, miR-215, miR-421, and miR-529 [34]. The miRNAs upregulated by TGF-β signaling include miR-21, the miR-181 family, miR-10b, the miR-17/92 cluster, miR-155, miR-192, the miR-23/24/27 cluster, miR-216/217, miR-494, and miR-182. The miRNAs downregulated by TGF-β signaling include the miR-200 family, miR-203, let-7, miR-34a, and miR-584 [34]. Many miRNAs targeting different substrates of TGF-β pathway are frequently oncogenic, such as miR-200, having impact on the expression of TGF-β ligands and TGF-β receptors type I and II, miR-21, miR0211, miR-17/92, and miR-106b/206, mainly participating in regulation of expression TGF-β receptors type I and II and miR-182, involved in the regulation of R-Smad, co-Smad (I-Smad), and Smad7, and it modulates a negative feedback loop of TGF-β signaling as Smad7 is also induced by TGF-β. Besides TGF-β receptors type I and II, miR-17/92 and miR-106b/205 participate in the regulation of downstream targets of TGF-β pathway [34].

miR-141 and miR-200a expression levels were shown to be decreased and serum samples from patients with liver cancer (blood samples were taken from 30 patients with liver cancer and from 30 normal subjects, RNU6 or GAPDH as internal controls). The sensitivity and specificity of the investigated miRNAs for diagnosing tumor invasion in liver cancer and comparing metastasis of patients with liver cancer were higher in combination of miR-141 and miR-200a rather than alone, although the difference between AUC values in both cases—combination and one miR regimen—had no significant changes. The possible mechanism of cancer processes modulation was explained with E-cadherin and vimentin inhibition due to STAT4 inhibition, which was firstly reported as a target gene for these miRNAs [89, 90]. The study of Dhayat et al. also showed a significant negative correlation of miR-200a and miR-200b to the expression of the mesenchymal markers Vimentin and ZEB-1 and a significant positive correlation to the epithelial marker E-cadherin. Moreover, in this study, miR-200 family was significantly downregulated in HCC samples compared to liver cirrhosis and was shown to be able to distinguish between cirrhotic and HCC tissue [90].

miR-211-5p was significantly downregulated in patients with HCC (30 pairs of HCC tissues and matched adjacent tumor-free tissues, qRT-PCR, RNU6 (miRNA) as an endogenous control), although miR-211-5p expression in liver cancer samples was not significantly different from adjacent normal samples based on TCGA cohorts, it was considerably downregulated in 30 pairs of HCC tissues compared with matched adjacent tumor-free tissues from patients in clinics or real-world cohorts. It was also shown that miR-211-5p may have a prognostic role for HCC patients: Patients with a decreased expression of miR-211-5p had poor overall survival [91]. In another study, miR-211-5p was found to be decreased in 33 out of 40 HCC tissue samples compared with the corresponding non-tumor tissues; moreover, tissues from lymph node metastases also expressed lower levels of miR-211 compared with primary HCC tissues and the adjacent normal tissue (qRT-PCR, the

endogenous U6 snRNA or GAPDH as the internal control) [68]. Among miR-211-5p targets are STAB2, SPARC, ZEB2, and ACSL4, negatively regulating these genes, miR-211-5p participates in the suppression of cell proliferation, migration, and invasion in HCC tissues [92].

In contrast, miR-17/92 expression levels were shown to be highly expressed in HCC tissues compared to the non-tumor liver tissues (94 cases of HCC, 5 cases of cancer adjacent to normal hepatic tissue, and 5 cases of normal liver tissue; U6 small nuclear 2 (U6b) as an internal control, RT-PCR). In this study, it was shown that expression of miR-17-92 was negatively correlated with several target genes, including CREBL2, PRRG1, and NTN4, when analyzing the miRNA and mRNA sequencing data from the 312 hepatocellular cancer patients available from the TCGA database [68].

Expression level of miR-182-5p was also elevated in HCC tissues and its high expression level correlated with poor prognosis such as early recurrence in patients who underwent curative surgery (tissue samples from 119 patients; RT-PCR, U6 snRNA was probed as a loading control; the disease-free survival was calculated from the date of resection to the date of tumor recurrence). Promotion of HCC proliferation by miR-182-5p is partly possible due to the ability of the last to directly target 3′-UTR of FOXO3a and thus inhibits FOXO3a expression, activating AKT/FOXO3a pathway. MiR-182-5p interacts with 3`-UTR of FOXO3a by binding to the 72-79 site, but not the 914–921 site in the 3′-UTR of FOXO3a [93].

However, in almost all these pathways the regulation is double-sided: miRNA may regulate the expression of the genes, and genes may regulate the expression of miRNAs. TGF-β signaling may modulate miRNA expression level *via* the canonical pathway, which is Smad-dependent requiring co-Smad Smad4, and non-canonical pathway, such as Smad4-independent. The Smad binding element found in the promoters of TGF-β/BMP-regulated genes contains a conserved sequence similar to the pre-miRNAs of TGF-β/BMP-regulated miRNAs, which is CAGAC [34]. miRNAs, containing this RNA-Smad binding element (R-SBE), are following: miR-105, miR-199a, miR-215, miR-421, and miR-529 [34]. The miRNAs upregulated by TGF-β signaling include miR-21, the miR-181 family, miR-10b, the miR-17/92 cluster, miR-155, miR-192, the miR-23/24/27 cluster, miR-216/217, miR-494, and miR-182. The miRNAs downregulated by TGF-β signaling include the miR-200 family, miR-203, let-7, miR-34a, and miR-584 [34]. Many miRNAs targeting different substrates of TGF-β pathway are frequently oncogenic, such as miR-200, having an impact on the expression of TGF-β ligands and TGF-β receptors type I and II, miR-21, miR0211, miR-17/92, and miR-106b/206, mainly participating in regulation of expression TGF-β receptors type I and II and miR-182, involved in regulation of R-Smad and co-Smad (I-Smad) and Smad7, and it modulates a negative feedback loop of TGF-β signaling as Smad7 is also induced by TGF-β. Besides TGF-β receptors type I and II, miR-17/92 and miR-106b/205 participate in regulation of downstream targets of TGF-β pathway [34].

It may seem interesting that almost none of these miRNAs were included in the list of miRNAs, implying HCC signature based on TCGA database, which consists of 540 miRNA expression profiles from 348 HCC patients, of whom 248 had early-stage and 90 had advanced-stage HCC. SVM-HCC, based on an SVM29 incorporating the feature selection algorithm IBCGAa proposed method, used a feature selection algorithm (IBCGA) to select a significant miRNA signature associated with early and advanced stages of HCC. This signature contains 23 miRNAs: in order of decreasing MED (Main

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

Effect Difference) scores, miR-550a, miR-549, miR-518b, miR-512, miR-1179, miR-574, miR-424, miR-4286, let-7i, miR-320a, miR-17, miR-299, miR-3651, miR-2277, miR-621, miR-181c, miR-539, miR-106b, miR-1269, miR-139, miR-152, miR-2355, and miR-150. Moreover, the significance of 10 top-ranked miRNAs in distinguishing HCC tissue samples from normal tissue samples and their prognostic values were proved on different datasets [94]. For some of these miRNAs with the highest MED scores, there are known targets in the context of liver cancer development. Among direct targets of miRNA-320a, there are β-catenin, c-myc, cyclin D1, and dickkopf-1; functional studies have shown a significantly decreased capability of cell proliferation and G0/G1 growth arrest *in vitro* when miRNA-320 is overexpressed [95]. miRNA-424 demonstrated a strong prognostic value in liver cancer: Its expression level in HCC tissues was associated with a relapse after liver transplantation. The study involved samples from 121 patients, the median follow-up duration was 25.12 months, U6 snRNA was used as the endogenous control [96]. In liver cancer cell lines, it was shown that miRNA-574-3p has an ADAM28 as a direct target and binds 3′-untranslated region of the ADAM28 mRNA leading to reduced cell proliferation and migration and promoted cell apoptosis [97]. Like miRNA-200 and miRNA-211-5p, which are mentioned above in the context of HCC development and which are not listed in this miRNA list, miRNA-1179 directly interacts with zinc-finger E-box-binding homeobox 2 (ZEB2) and has antitumor function, leading to attenuated proliferation and migration of HCC cells. Its expression was decreased in HCC tissues compared with corresponding noncancerous tissues (40 paired HCC samples with matched normal tissues, U6 were used as internal reference) [98]. miRNA-550a plays a controversial role in HCC development, promoting HCC cell migration and invasion, and cytoplasmic polyadenylation element-binding protein 4 (CPEB4) is one of its potential targets, which is commonly decreased in liver cancer. The function of CPEB4, which is from the gene family CPEB involved in regulation of translation by controlling the polyadenylation of target genes, is yet to be elucidated. There are contradictional data of its function in cancer development: In pancreatic ductal cancer and neuroblastoma, its expression is upregulated, driving the growth and invasion of cancer cells. In HCC, it was shown that CPEB4 siRNA could promote the migration and invasion of HCC cells. This contradiction may be explained by different downstream targets regulated by CPEB4 in different cells because CPEB4 along with involvement in polyadenylation can control the translation by binding to the CPE sequence in 3′ UTR of the corresponding genes [99]. miRNA-512 may play one of the crucial roles in HCC progression, being significantly upregulated in human HCC samples and HCC cell lines. Along with miRNA-519, it targets tumor suppressors MAP3K2 and MAP2K4, and the integration of these two miRNAs into the AJCC staging system significantly improved the accuracy of the prediction of HCC recurrence [100]. Some of these miRNAs, such as miRNA-518b, miRNA-549, miRNA-2277, and miRNA-2355, are rarely mentioned in the context of liver cancer development, and their role in this process is yet to be elucidated.

This emphasizes the importance of working with customized algorithms and validated big datasets, when many of the aspects of the process you are studying like miRNA involvement in carcinogenesis—stay unclear, making identification of the most promising diagnostic and/or prognostic and/or therapeutic molecules *via* analyzing only mRNA-miRNA interactions or miRNAs in signaling or metabolic pathways not very effective.

In **Table 1**, there are listed miRNAs, whose expression levels are changed in the HCC development in order of mention in the text.



**Table 1.**

*miRNAs in liver cancer development.*
