We identified in HBL transcriptomes a central dysregulation of noncoding RNAs, associated with changes in the expression of genes linked to metabolism and oxidative reactions of lipids and carbohydrates. The assessment of these findings together with our previous data reinforced that the disturbance of metabolic pathways in a major event in HBLs, besides disclosing an aberrant ncRNA network, which can be highlighted as a new epigenetic player in the control of gene expression of this tumor type, along with the already reported abnormal DNA methylation [9, 11, 45]
The analysis of the PCA and the heatmap showed two clusters of samples, one exclusively tumoral and the other composed of control liver samples but one tumor (79T). It is noteworthy that this particular tumor did not carry somatic coding mutations or copy number alterations, maybe explaining its similarity of expression with control livers. Four clusters with similar expression patterns were observed in HBLs, three of them consisting of upregulated genes affecting biological pathways associated with development, and morphogenesis, which is linked to the disruption of the differentiation proposed for HBL genesis [46–48]. Most of these increased processes are linked to developmental events, and associated with gene changes such as DKK1 , BMP4 , RUNX2 , LEF1 , as well as homeobox genes such as DLX1, DLX6, MSX1 , among others. HMGA2 and CDH3 upregulation are associated with embryonic development, cell differentiation, and communication. HMGA2 polymorphisms were recently linked to HBL progression ; and CDH3 encodes a classical cadherin component of glycoproteins, and alterations were not described in HBL before. The overexpression of DKK1 was reported in HBL as a novel biomarker related to uncontrolled wingless/WNT signaling . We also detected upregulation of DKK4, previously reported with increased expression in HBLs  and other cancers, such as colorectal, gastric, pancreatic, and renal; however, in hepatocellular carcinoma was observed a reduced DKK4 expression, suggesting that effects of DKK4 in tumorigenesis and progression can vary in the different tumors [57, 58].
On the other hand, the identified cluster 4, composed of downregulated genes, was linked to metabolic processes, revealing a crucial role of metabolism interference in HBLs. Changes in HBL metabolism are still poorly described and understood [59, 60]. THRSP, for example, a gene associated with lipid metabolism, was found to be downregulated in our data; it is expressed in liver and adipocytes, and we previously showed THRSP promoter hypermethylation in HBLs , suggesting that expression control by an epigenetic mechanism can interfere in the tumoral lipid metabolism. In addition, the observed downregulation of TTC36 can be associated with alterations in the metabolism of amine and small molecules, development process, and cell differentiation. This gene is associated with tumor suppressor activities, and its inactivation in gastric cancer might promote cell proliferation, at least in part through activating the Wnt/β-catenin signaling pathway . Another interesting gene for future studies is GCK, which contributes to the reprogramming of energy metabolism in cancer cells . Recently, bioinformatic analyses of microarray expression in HBLs also highlighted changes in biological processes associated with small-molecule catabolism, organic acid metabolism, lipid metabolism, and oxidation-reduction reactions .
Differentially expressed genes in HBLs were found to be widely distributed on chromosomes, with only the 14q32 region showing an enriched cluster of upregulated genes. The overexpression of a 300 kb region located at the 14q32 DLK1/DIO3 locus, previously reported in association with DNA hypomethylation, indicates a direct role of this epigenetic mechanism in gene expression control . In a previous study, this locus was associated with a major increase in the expression of a cluster of miRNAs and snoRNA of the C/D box family (namely SNORD113 and SNORD114), especially in metastatic tumors . Our data corroborated these recent findings regarding 14q32 locus overexpression on HBLs; we detected upregulation of the genes at this locus (DLK1, MEG3, SNORD113-3, SNORD114-22) that Carillo et. al proposed to classify HBLs according to the level of 14q32 gene expression.
Analysis of the protein network revealed which proteins encoded by DEGs were decisive in the pathways and mechanisms highlighted throughout this work. Increased HDAC2 expression was detected, and this protein presents a hub-bottleneck centrality associated with methylation-dependent chromatin silence, DNA methylation, and negative regulation of metabolism. It is interesting to note that recent work showed epigenetic silencing by histone deacetylases (HDAC) as a critical step for the development of pediatric liver cancer . Particularly, the elevation of HDAC1 and HDAC2 proteins were found in a large group of patients with HBLs . Also classified as a hub-bottleneck centrality protein, CCND1 presented downregulation in tumor samples; as a major regulator of the cell cycle transition, this protein reduction can directly interfere in cell proliferation. CCND1 germline polymorphisms have been proposed as contributors of HBLs development . CCND1 protein was associated in the network with methylation-dependent chromatin silence, DNA methylation, negative regulation of metabolism, leukocyte mediated cytotoxicity, and cytokine-mediated signaling pathway. It is important to note that our data suggested a CCND1 regulation by mir-494 in the miRNA-mRNA analysis. GCK and CAMK2B, downregulated in HBLs, were also classified with a centrality degree; the first one was associated with metabolism and cytokine pathways, and the second with epigenetic processes (DNA methylation and gene silencing) as well as cytokine-mediated signaling. The mRNAs GCK and CAMK2B were two of the ten genes emphasized in the GOcircle, and CAMK2B was predicted to be regulated by miR-186 in the miRNA-mRNA analysis. Therefore, CCND1, GCK and CAMK2B are strong candidates for future analyses.
From the 1,492 DEGs detected in HBLs, 85 were exclusively expressed in tumors, 47 of them being lncRNAs or miRNAs. Altered ncRNA expression has been observed in several tumor types, including colorectal , hepatocellular carcinoma , gastric , and renal cell carcinoma , indicating that aberrant expression of ncRNAs contributes to carcinogenesis. In HBLs, a previous study has shown that the microRNA profile can distinguish tumors from control liver tissues . In our work, miRNAs found to be upregulated were associated with possible oncogenic activities through the putative regulation of downregulated mRNAs. Based on the miRNA-mRNA analysis, we propose that 18 of the differentially expressed miRNA precursors here detected in HBLs were linked with regulation of mRNA expression of 82 genes. It is important to clarify that this analysis is limited because not related to mature/processed miRNAs, and functional validation was not performed. Yet, the upregulation of four miRNA precursors (miR-186, miR-214, miR-377, and miR-494) detected in HBLs were central and could possibly result in reduced expression of their target protein-coding genes. In our HBL transcriptome data, the overexpression of miR-186 was predicted to be linked to 26 down-regulated mRNAs; recently, Cui et al. described miR-186 reduced expression targeting an N6-methyladenosine gene (MTTL3) and affecting HBL progression. The upregulate miR-214 precursor was associated with the possible downregulation of 12 mRNAs; however, the literature reported opposite miR-214 downregulation[62, 70] a discrepancy that should be validated by other technique. The overexpression of miR-377 was predicted to be related to 22 down-regulated mRNA in HBLs, and recent publications supported this upregulation pattern [62, 71]. Lastly, the miR-494 precursor was potentially linked to the downregulation of 11 mRNA genes. The miRNA-494 is overexpressed in HCC (hepatocellular carcinoma), being considered an epigenetic regulator associated with tumor invasiveness, proliferation, and migration [72, 73], and linked to TET1 activity and tumor progression . We previously indicated an increased expression of the TET family genes (TET1, TET2, and TET3) in HBLs, connected with an active demethylation activity and increased 5hmC. Therefore, upregulation of miR-494 is a novelty in the HBL biology, and future studies can provide further links among its expression and TET1 regulation.