Hepatocellular carcinoma is a primary liver cancer and one of the leading causes of mortality with wide geographic variation [1]. Any factor that leads to chronic liver injury and cirrhosis can be considered an oncogenic agent. Prevalent hepatocellular carcinoma (HCC) risk factors are Hepatitis viruses B and C infection, excessive alcohol intake, nonalcoholic steatohepatitis (NASH), and aflatoxin B1 exposure ^[2][3][2,3].

The major problem with the HCC treatment is the diagnosis is usually made in progressed disease stadiums when conventional systemic chemotherapy is ineffective [4]. To overcome this problem, researchers are developing molecularly targeted treatments that represent a more promising approach for advanced HCC. Several such drugs are clinically available, but their efficacy is limited [5]. Therefore, elucidating the molecular processes at the basis of hepatocarcinogenesis is critical for improving therapy outcomes and diagnosis.

The family of Runt-related (RUNX) genes (RUNX1, RUNX2, and RUNX3) is crucial for the different tumour types’ development and progression. RUNX proteins are transcription factors that behave in opposing ways, promoting or suppressing tumourigenesis ^[6][7][8][6,7,8]. However, the exact mechanisms of their deregulation in HCC, especially for RUNX1 and RUNX2, have not yet been sufficiently investigated.

Human RUNX genes participate in neuro-, blood, and bone development ^[7][8][7,8]. The role in developmental processes implies tight control of RUNX genes at transcriptional and posttranscriptional levels. RUNX genes’ chromosome location differs depending on the species, and human RUNX1, RUNX2, and RUNX3 genes’ locations are on chromosomes 21, 6, and 1, respectively. Two alternative promoters of RUNX genes, P1 and P2, generate two dominant isoforms with differing 5′-untranslated regions (5′-UTRs) (Figure 1A). These transcripts give two polypeptides with different N-terminal sequences, distal and proximal [9]. The diversity of RUNX transcripts is the result of alternative splicing. RUNX1 and RUNX2 have nine exons and twelve isoforms, whereas RUNX3 has six exons and two isoforms ^[7][10][7,10]. RUNX proteins’ molecular weight is 44, 50, and 57 KDa [7].

RUNX proteins received their name from the Runt homology domain (RHD) on the protein N-terminus, which has approximately 90% sequence homology in all three proteins [6]. The RHD domain binds to the target genes’ DNA through the consensus sequence of seven nucleotides, ‘PyGPyGGTPy’. Nuclear localisation and interaction with the other proteins are also functions of the RHD domain [10]. The RUNX proteins’ C-terminus encompasses the transactivation domain (TAD) and inhibitory domain (ID), with the consensus sequence of five amino acids, valine–tryptophan–arginine–proline–tyrosine (VWRPY). This sequence recruits Groucho/Transducin-like enhancer protein (TLE) corepressors ^[7][10][11][7,10,11]. TLE corepressors further control several target genes’ transcription. The TAD domain is the place of RUNX proteins’ interactions with the other regulatory proteins and control of their transcriptional activity [12]. In complex with the coregulators, RUNX proteins influence various cell processes [10] (Figure 1B).

1.2. The Role of the RUNX Genes in Normal Development

RUNX genes engage in normal cell development and differentiation processes, playing a tissue-specific role ^[13][14]. RUNX1 is crucial for cell growth and differentiation of immune cells, epithelial stem and epithelial cells, and neurodevelopment ^[11][14][11,15]. RUNX1 acts in the development of hematopoietic cells in vertebrates ^[15][16][13,16]. Consequently, the chromosomal rearrangements and gene mutations involving RUNX1 lead to various leukaemia types ^[15][13]. RUNX2 is a factor in bone generation [11]. RUNX2 knockout mice lack osteoblast differentiation, leading to osteoporosis [17]. RUNX3 is essential for embryogenesis, and RUNX3 knockout mice die quickly [18].

1.3. The Role of RUNX Genes in Cancer

RUNX genes’ mutations and abnormal expression lead to various cancer types. RUNX genes can hinder or activate tumourigenesis [12]. A recent study by Pan and colleagues ^[19][22] suggests that RUNX genes’ aberrant expression causes disparate cancer types and influences disease prognosis. RUNX1 plays a cardinal role in hematopoiesis and, consequently, in haematological tumours. The researchers documented mutations of the RUNX1 gene in acute myeloid leukaemia (AML) ^[20][23], acute lymphoid leukaemia (ALL), and familial platelet disorder with a predisposition to acute myeloid leukaemia (FPD/AML). These mutations either influence or do not influence the binding ability of RUNX1 to the other target genes, with the more or less changed function, depending on the mutated region (DBD, TAD, or nuclear localisation domain). RUNX1 expression changes are also found in solid tumours, like glioblastoma, ovarian, colon, breast, and hepatocellular carcinoma, where in tumour cells and available patient samples, it predominantly acts as a tumour suppressive. However, its oncogenic function is also documented ^{[21][22][23][24][25][26]}[31,32,33,34,35,36]. RUNX2 is involved in the progression of various human tumours by regulating cell proliferation, angiogenesis, cancer stemness, and metastasis ^[27][37]. Its expression could be deregulated by several mechanisms. Recent research has uncovered numerous somatic mutations in the RUNX2 gene in various cancers, including missense, nonsense, and nonstop mutations and frameshift insertions and deletions ^[27][37]. RUNX3 is involved in carcinogenesis by interacting with several oncogenic signalling pathways, acting as a tumour suppressor in some tumours ^{[28][29][30][31][32]}[40,41,42,43,44] and as an oncogene in others ^[33][34][35][45,46,47]. RUNX3 is frequently inactivated in human cancers by promoter DNA hypermethylation ^{[28][29][30][31][32][36][37][38][39][40][41]}[40,41,42,43,44,48,49,50,51,52,53], histone modification ^[42][43][54,55], hemizygous deletion ^[38][39][50,51], and protein mislocalisation ^[6][31][6,43]. 

1.4. The Mechanisms of Action of RUNX Proteins

Also, the co-expression of RUNX genes with epigenetic regulators could affect the onset of some cancer types. Researchers should pay attention to epigenetic mechanisms of RUNX genes’ regulation, including DNA methylation and miRNAs ^[19][22]. As epigenetic regulators, RUNX proteins cooperate with diverse coregulators and involve many signal transduction processes. In addition, they participate in chromatin landscape remodelling [10]. There is evidence that RUNX proteins can function as pioneer transcription factors that recruit various chromatin remodelling enzymes and other transcription factors to open the condensed chromatin structure and thus activate the transcription of target genes ^[10][19][44][10,22,65]. This epigenetic role of RUNX genes appears to play a pivotal role in both physiological and pathological conditions, including cancer ^[10][19][10,22].

2. RUNX1 in HCC

2.1. RUNX1 Role in HCC

The role of RUNX1 in solid tumours is controversial, acting in two opposite ways. It can impede or promote carcinogenesis, as reviewed in ^[7][45][7,76]. Databases that collect transcriptomic studies and link data on the genomic and clinical parameters with various cancer groups show controversial results on the RUNX1 expression. According to the Gent2 and TIMER2.0 databases, there was a significantly higher expression of RUNX1 transcript in hepatocellular carcinoma compared to normal tissue ^[46][47][77,78]. However, the UCSC database shows the opposite results ^[48][79]. It has an upgraded version ^[49][80]. The RUNX1 expression was decreased in hepatocellular carcinoma. That was consistent with the work of Miyagawa and colleagues, who noticed that RUNX1 mRNA was 76% and 47% lower in HCC and cirrhotic tissue than in normal tissue. Also, there was a significant decrease in RUNX1 mRNA in HCC compared to cirrhotic liver samples ^[50][81]. One of the crucial processes in cancer progression is angiogenesis. In hematopoietic cell differentiation from hemogenic endothelium cells, RUNX1 has a vital role. The RUNX1-deficient mice lack hematopoiesis and angiogenesis ^[51][83]. Added exogenously, IGFBP-3 inhibited RUNX1-promoted angiogenesis dose-dependently ^[52][84]. Thus, it can conclude that RUNX1 has a cardinal role in angiogenic differentiation and vascularisation. Vascular endothelial growth factor (VEGF) is an angiogenesis modulator in a cancer cell environment and the negative prognostic factor for acute myeloid leukaemia. In the HCC cell culture, Elst and colleagues found that RUNX1 inhibited VEGF expression ^[53][85]. 

2.2. RUNX1 and miRNAs

There are not many studies of epigenetic processes involving the RUNX1 gene. Tuo and colleagues noticed the hypomethylation of the RUNX1 promoter in hepatocellular carcinoma ^[54][86]. Some studies show the association between several miRNAs and RUNX1 in HCC. Transcript 1 of RUNX1 (RUNX1-IT1) is a long non-coding sequence of an RNA transcript of the RUNX1 gene ^[55][87]. Yan and colleagues noticed the RUNX1 expression decrease in the HCC patients’ samples, and the knocking-down of RUNX1-IT1 increased the proliferation and reduced apoptosis in HCC cells ^[56][88]. Sun and colleagues observed the association of decreased RUNX1-IT1 expression with shorter DFS and OS. RUNX1-IT1 binds mir-632, competing with the other RNAs in HCC cells for target gene GSK-3β binding and modulating the WNT/β-catenin signalling cascade. Added hypoxia-prompted histone deacetylase 3 (HDAC3) in HCC cells reduced the RUNX1-IT1 expression. They concluded that the goal of HCC therapy should be to activate RUNX1-IT1 ^[57][89]. On the other hand, Vivacqua and colleagues noticed that oestrogen receptor agonists, such as the G protein-coupled oestrogen receptor agonist (G-1) and 17β-oestradiol (E2), increased miR-144 expression in HepG2 hepatocarcinoma cells, via the G protein-coupled oestrogen receptor 1 (GPER) and the PI3K/ERK1/2/Elk1 pathway. miR-144 then downregulates RUNX1, promoting the cell cycle ^[58][90].

In conclusion, RUNX1 binds target genes (VEGF and COL4A1) and involves signalling pathways of cancer proliferation, metastasis, and angiogenesis. RUNX1 also interacts with several miRNAs, for example, mir-632 and mir-144. Given the importance of RUNX1 in hepatocellular carcinoma, its potential suitability as a treatment target requires additional studies.

3. RUNX2 in HCC

3.1. General Role of RUNX2 in HCC

According to literature data, the expression of RUNX2 on mRNA and/or protein level is elevated in HCC cell lines, as well as in liver tumour tissue ^[59][60][61][95,103,104], suggesting that this transcription factor has a role in hepatocarcinogenesis. Previous findings confirmed higher RUNX2 expression in HCC patients than expression detected in non-tumour tissues or healthy controls ^[59][60][62][95,103,105]. Wang and colleagues noticed that increased expression of RUNX2 significantly correlates with unfavourable clinicopathological features in HCC. These adverse features included the onset of multiple tumour nodes, higher histological grades and TNM stages, and venous invasion presence ^[60][103].

3.2. RUNX2 Tumour Invasion Activity in HCC

The general mechanisms underlying the role of RUNX2 in various tumour types provide directions for detailed studies on the impact of RUNX2 on the pathogenesis of HCC. Many reports showed that increased RUNX2 expression enhances tumour cell migration and invasive properties ^{[63][64][65][66][67][68][69]}[106,107,108,109,110,111,112]. Previous studies revealed the crucial role of the RUNX2 in the regulation of the epithelial-to-mesenchymal transition (EMT) process in many tumours ^[61][70][104,113], which is the first step toward tumour invasion and metastatic potential (Figure 23).