Abstract
BackgroundMicroRNA-612 (miR-612) has been proven to suppress EMT, stemness, and tumor metastasis of hepatocellular carcinoma (HCC) via PI3K/AKT2 and Sp1/Nanog signaling. However, its biological roles on HCC progression are far from elucidated.MethodsWe found direct downstream target of miR-612, hadha by RNA immunoprecipitation and sequencing. To explore its biological characteristic, potential molecular mechanism, and clinical relevance in HCC patients, we performed several in-vitro and in-vivo models, as well as human tissue chip.ResultsEctopic expression of miR-612 could partially reverse the level of HADHA, then suppress function of pseudopods, and diminish metastatic and invasive potential of HCC by lipid reprogramming. In detail, miR-612 might reduce invadopodia formation via HADHA-mediated cell membrane cholesterol alteration and accompanied with the inhibition of Wnt/β-catenin regulated EMT occurrence. Our results showed that the maximum oxygen consumption rates (OCR) of HCCLM3miR-612-OE and HCCLM3hadha-KD cells were decreased nearly by 40% and 60% of their counterparts (p < 0.05). The levels of acetyl CoA were significantly decreased, about 1/3 (p > 0.05) or 1/2 (p < 0.05) of their controls, in exogenous miR-612 or hadha-shRNA transfected HCCLM3 cell lines. Besides, overexpression of hadha cell lines had a high expression level of total cholesterol, especially 27-hydroxycholesterol (p < 0.005). SREBP2 protein expression level as well as its downstream targets, HMGCS1, HMGCR, MVD, SQLE were all deregulated by HADHA. Meanwhile, the ATP levels were reduced to 1/2 and 1/4 in HCCLM3miR-612-OE (p < 0.05) and HCCLM3hadha-KD (p < 0.01) respectively. Moreover, patients with low miR-612 levels and high HADHA levels had a poor prognosis with shorter overall survival.ConclusionmiR-612 can suppress the formation of invadopodia, EMT, and HCC metastasis and by HADHA-mediated lipid programming, which may provide a new insight of miR-612 on tumor metastasis and progression.
Highlights
Hepatocellular carcinoma (HCC) is an aggressive cancer with poor long-term survival
Low endogenous miR-612 level correlates with poor outcome of patients In our previous study, we have noticed that the level of miR-612 had a significant inversed correlation with tumor size and stage, intrahepatic metastasis, and microvascular invasion in one cohort with 37 primary hepatocellular carcinoma (HCC) tissues
(See figure on previous page.) Fig. 7 Clinical significance of Hydroxyacyl-CoAdehydrogenase/3-ketoacyl-CoAthiolase/enoyl-CoA hydratase (HADHA) in HCC patients. a The cholesterol and 27-hydroxycholesterol of HepG2 and HCCLM3 cells relative quantification based on liquid chromatography mass spectrometry (LC-MS)/MS system. (NS: no significance; **p < 0.01; ***p < 0.001). b Immunoblotting of proteins involved in cholesterol-biosynthesis pathway in HepG2hadha-OE, HCCLM3hadha-KD cell, and their negative control cells. (Cells treated with negative control lentivirus). c Negative correlation between miR-612 and hadha mRNA in 15 HCC and their adjacent normal tissues. d Representative images of negative and positive HADHA staining in tumor and peritumor tissue from one HCC patient
Summary
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]. 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. The accumulation of SREBP2 in nucleus binds to sterol response elements (SREs) to activate the expression of cholesterolbiosynthesis 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. Its biological roles on HCC progression are far from elucidated
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