Introduction
Hepatocellular carcinoma (HCC) is an aggressive cancer with poor long-term survival. Every year, up to 600,000 people die from the disease in the worldwide, and it has been a serious issue to human health [
1,
2]. High recurrence and metastasis rate of HCC after surgical resection is still a Gordian knot to further improve the patients’ survival. Therefore, the exploration of underlying mechanism of HCC recurrence and metastasis has been an important clinical significance for early diagnosis and intervention.
Normally, cell morphology and function are elaborately orchestrated by dynamic metabolisms of carbohydrates, lipids, and amino acids. Dysfunctions of metabolic reprogramming have been found to be an important characteristic of tumor cells during tumorigenesis and metastasis. Even in normoxia, tumor cells are inclined to produce ATP by glycolysis, which is called as aerobic glycolysis or Warburg effects [
3]. Although less efficient than oxidative phosphorylation, tumor cells usually turnover glucoses quickly in glycolytic pathway to meet their aggressive needs on energy and produce an intermediate, glutamine, the fastest-consuming amino acid in tumor microenvironment [
4]. The latter may provide carbon and nitrogen sources for de novo syntheses of amino acids, nucleotides, and lipids. Recently, many evidences suggest that the abnormities of lipid- and cholesterol-metabolisms are also involved in tumorigenesis and progression. Lipids are reported to be another important energy source to fuel tumor metastasis [
5]. Lipids, together with cholesterol, could form lipid rafts on cell membrane where a lot of receptors, ligands, and iron-channel proteins are aggregated as a functional unit. Destruction of lipid rafts is enough to prevent cell proliferation and tumor growth [
6]. In addition, invadopodia, another prominent membranous structure in migrated and invasive cancer cells, is closely regulated by lipid- and cholesterol-metabolisms. The cholesterol-biosynthesis pathway is under tight regulation by transcription factors, such as sterol regulatory element-binding protein 1 and 2 (SREBP1 and 2). The accumulation of SREBP2 in nucleus binds to sterol response elements (SREs) to activate the expression of cholesterol-biosynthesis enzymes, such as 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and squalene epoxidase (SQLE) [
7]. Whether this cascade could regulate cholesterol metabolism and invadopodia formation of HCC cells were still unknown. In virtue of invadopodia, cancer cells can degrade extracellular matrix, penetrate through blood vessels, and finally disseminate into distant target organs [
8]. Due to its important roles in tumor metastasis, a lot of components and regulators of invadopodia, such as integrins, actin-binding proteins, scaffolders [
9,
10], as well as signaling adaptors, are widely studied [
11]. However, the roles of lipid metabolisms on the formation and the function of invadopodia in HCC still need to be unveiled.
Non-coding RNAs (ncRNAs) have emerged as one important kind of molecules that can regulate altered genes contributing, to the establishment of metabolic reprogramming [
12]. For instance, SNHG16 could be regulated by the Wnt pathway in colorectal cancer and affect genes involved in lipid metabolism [
13]. HULC functions as an oncogene in hepatoma cells, acting mechanistically by deregulating lipid metabolism through a signaling pathway involving miR-9, PPARA, and ACSL1 [
14]. MicroRNAs (miRNAs), as a class of endogenous small noncoding RNAs, can regulate target protein expression at posttranscriptional level [
15,
16]. To date, multiple miRNAs have been discovered to involve in the regulation of lipid metabolism, such as miR-30c, miR-122, etc. [
17,
18]. In our previous study, miR-612 was found to have robustly suppressive effects on HCC proliferation and metastasis [
19]. And 167 genes were predicted as potential targets of miR-612 by miRanda, TargetScan, and miRTarget2. Two pivotal genes were drawn out from these predicted genes in gene interaction network based on function enrichment analysis (Additional file
1: Figure S1A). One is
akt2, which has been confirmed to mediate the suppressive effects of miR-612 in HCC invasive-metastatic cascade in our previously study. The other is
hadha, whose roles in HCC metastasis are unknown by far. As
hadha encodes the alpha subunit of mitochondrial trifunctional protein, hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase, the enzyme complex catalyze the three steps of beta-oxidation of fatty acids in mitochondria: long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD), long-chain enoyl-CoA hydratase, and long-chain thiolase activities [
20]. Therefore, we supposed that miR-612 might regulate tumor invasiveness and metastasis by HADHA-mediated lipid reprogramming and probably resulting in the abnormity of invadopodia in structure and function in HCC.
In this study, we mainly focused the roles of miR-612 on lipid reprogramming, invasiveness, and metastasis of HCC, which may lay a basis for its clinical application in precision diagnosis and therapy of metastatic HCC.
Discussion
Invadopodia is a kind of pseudopodia, which is mainly responsible for extracellular matrix degradation, cell migration and invasion in local, extravasation of blood vessels, and dissemination into a distant organ in a spatial and temporal manner [
8,
41]. Therefore, a variety of components, such as Cortactin, Caveolin-1, Tks4/5, and protein tyrosine kinase Src of invadopodia, are usually used as indicators to gauge the mobility and invasion potentials of cancer cells in vitro and in vivo system [
42,
43]. For example, Cortactin, a monomeric cytoplasmic protein, is able to be polymerized and rearranged in actin cytoskeleton of cell cortex after activated by external stimuli, thus promoting the formation of invadopodia, cell migration, and metastasis [
44]. Caveolin-1, a scaffolding protein, can interact with Src tyrosine kinases, cholesterol, and TGF-β receptor, etc., during the formation of invadopodia [
34,
35]. By now, invadopodia is regarded as a kind of microdomain on cell membranes which is rich of cholesterol. If the cholesterol was depleted, invadopodia formation and persistence could be impaired. On these microdomains, a lot of proteins such as Cortactin, Caveolin-1, Receptors, signaling adaptors, and trafficking proteins are assembled. And thus, depletion of cholesterol will impair the membrane fluidity and protein trafficking of invadopodia [
35]. Therefore, Cortactin, Caveolin-1, and cholesterol were chosen as the surrogated parameters for evaluating the formation and the function of invadopodia in the study. As to the subtle relationship among the cholesterol, invadopodia, and EMT, our results should be original. MiR-612/HADHA/cholesterol alteration will directly affect the lipid rafts construction, thereby influence the formation of invadopodia and invadopodia-mediated cellular matrix degradation, blunting EMT process of HCC. Furthermore, we discovered cholesterol metabolite, 27-hydroxycholesterol level change was shocking in HADHA-treated HCC cells. As reported, it could not only regulate the target genes controlling cholesterol, glucose, and fatty acid metabolism of liver but also increase the proliferation of various cancer cell [
45,
46].
It is well known that biological processes of cells are largely dependent on energy, saying ATP, which is usually derived from the metabolisms of carbohydrates, lipids, and amino acids. In normal, ATP is mainly derived from acetyl-CoA of aerobic oxidation of glucoses through tricarboxylic acid cycle. However, in tumor cells, glucoses are often catalyzed into pyruvate through hexokinase (HK) and phosphofructokinase (PFK) pathways for de novo biosyntheses of lipids and amino acids to meet their uncontrolled growth and metastasis, and only a small portion are reduced into lactic acids for ATP synthesis in mitochondria [
3]. As a result, lipid metabolism reprogramming, such as β-oxidation of fatty acids in mitochondria, is initiated rapidly as a compensatory pathway in these hypermetabolic cancerous cells to fuel cell proliferation, invasion, and metastasis [
47] and protect tumor cells from anoikis as well [
48]. Meanwhile, acetyl-CoA, a vital intermediate of β-oxidation of fatty acids, can either be used to synthesize ATP, or cholesterol, or so on, in mammal cells.
Although the dynamics of protein components, interactions, modifications, signaling, and functions during cytoskeletal remodeling in the processes of invadopodia have been extensively studied for more than 30 years [
49], the roles of lipid metabolism reprogramming on invadopodia are still in early stage. In virtue of living cell imaging, invadopodia was recently confirmed as a special microdomain on membrane enriched with phospholipid PI(4,5)P2 and cholesterol. Our results also revealed that upregulation of cholesterol significantly increase membrane fluidity accompanied with Cortactin aggregation, suggesting that lipid metabolism is involved in the formation of invadopodia, and thus promote tumor cell metastasis [
50,
51]. These results might open up a new perspective of lipid metabolism on tumor metastasis.
MiRNAs have been confirmed as important players in HCC metastasis using post-transcriptional mechanism [
19]. In our previous studies, we revealed that miR-612 had pleiotropic inhibitory effects on cell proliferation, EMT, stemness, and metastasis of HCC via directly suppressing
akt2 expression [
19]. In the present study, we found that
hadha is another pivotal gene in miR-612 target network by RNA immunoprecipitation, RNA-seq, and luciferase report assay. A large amount of acetyl-CoA and ATP are produced by β-oxidation of long-chain fatty acids [
52], resulting in many cancerous functions, such as autophagy [
53], apoptosis [
54], and so on. Here, we report a novel function of miR-612 on HCC invadopodia. Upregulation of miR-612 can significantly reduce cholesterol level and membrane fluidity of HCC, meanwhile, remarkably reduced the expressed level and function of Cortactin and Caveolin-1 in HCC invadopodia. Similar results were observed in HCC cells treated with β-oxidation inhibitor. Moreover, Wnt signaling pathway is able to lead to an increase in synaptic Cortactin and associated with cytoskeleton organization. Our finding also indicates that Cortactin is downstream of Wnt signaling, and associated to other cancer type s[
47]. Our findings confirmed that miR-612 suppressed migration and invasion of HCC partially by HADHA-mediating lipid reprogramming and inhibited the formation of invadopodia and Wnt/β-catenin-regulated EMT progression.
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