Comparison between the in vivo and in vitro expression of three adhesion-signaling proteins in embryonic cells

We have examined the relationship between the in vivo and in vitro expression of three adhesion-signaling proteins (FAK, PYK2 and Paxillin), using cells of the early chick embryo, where pure cell populations may be isolated and cultured, and in which epithelial-to-mesenchymal transformation is occurring. Focal Adhesion Kinase (FAK) and Proline-rich Tyrosine Kinase-2 (PYK2) are related in molecular structure, and may have some overlapping functions in signal transduction associated with cell-substratum adhesion. Paxillin, a cytoskeletal protein, is also localized to focal adhesions. We show that the immunocytochemical detection of these molecules in vivo does not reflect their in vitro localization. Focal Adhesion Kinase showed a developmentally regulated localization to the cytoplasm, but not to sites of adhesion, in epithelial cells in vivo, while Paxillin was associated with migrating mesoderm cells. Proline-rich Tyrosine Kinase-2 was undetectable in vivo. The level of expression of these molecules was compared under in vivo and in vitro conditions. While the expression of Focal Adhesion Kinase showed a tissue-specific regulation of expression with the change to in vitro conditions, Proline-rich Tyrosine Kinase-2 showed a more uniform and less tissue-specific up-regulation. Levels of Paxillin expression also showed an increase with this change in conditions. We conclude that despite the structural and functional relationships between these three molecules in the developing embryo, the expression and localization of each is independently regulated. We suggest that this provides these cells with the adaptability that they require in order to respond to the changing extracellular environment in the early embryo, and to undergo epithelial-to-mesenchymal transformation.     

Recognition of LD motifs by the focal adhesion targeting domains of focal adhesion kinase and proline‐rich tyrosine kinase 2‐beta: Insights from molecular dynamics simulations

The focal adhesion kinase (FAK) and the proline‐rich tyrosine kinase 2‐beta (PYK2) are implicated in cancer progression and metastasis and represent promising biomarkers and targets for cancer therapy. FAK and PYK2 are recruited to focal adhesions (FAs) via interactions between their FA targeting (FAT) domains and conserved segments (LD motifs) on the proteins Paxillin, Leupaxin, and Hic‐5. A promising new approach for the inhibition of FAK and PYK2 targets interactions of the FAK domains with proteins that promote localization at FAs. Advances toward this goal include the development of surface plasmon resonance, heteronuclear single quantum coherence nuclear magnetic resonance (HSQC‐NMR) and fluorescence polarization assays for the identification of fragments or compounds interfering with the FAK‐Paxillin interaction. We have recently validated this strategy, showing that Paxillin mimicking polypeptides with 2 to 3 LD motifs displace FAK from FAs and block kinase‐dependent and independent functions of FAK, including downstream integrin signaling and FA localization of the protein p130Cas. In the present work we study by all‐atom molecular dynamics simulations the recognition of peptides with the Paxillin and Leupaxin LD motifs by the FAK‐FAT and PYK2‐FAT domains. Our simulations and free‐energy analysis interpret experimental data on binding of Paxillin and Leupaxin LD motifs at FAK‐FAT and PYK2‐FAT binding sites, and assess the roles of consensus LD regions and flanking residues. Our results can assist in the design of effective inhibitory peptides of the FAK‐FAT: Paxillin and PYK2‐FAT:Leupaxin complexes and the construction of pharmacophore models for the discovery of potential small‐molecule inhibitors of the FAK‐FAT and PYK2‐FAT focal adhesion based functions.     

Serotonin Inhibition of the NMDA Receptor/Nitric Oxide/Cyclic GMP Pathway in Rat Cerebellum: Involvement of 5-Hydroxytryptamine2C Receptors

Abstract: Previous studies have shown that 5-hydroxytryptamine (5-HT) can potently inhibit glutamatergic transmission in rat cerebellum through the activation of multiple 5-HT receptors. The aim of this study was to subclassify the 5-HT₂ receptor mediating inhibition of the cyclic GMP response elicited by N -methyl- d -aspartate in adult rat cerebellar slices. Seven receptor antagonists, endowed with relative selectivities for the 5-HT_2A, 5-HT_2B, and 5-HT_2C subtypes, differentially affected the inhibition by (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane of the cyclic GMP response, suggesting that the receptor involved belongs to the 5-HT_2C subtype.     

Effects of prostaglandin D2 on Na-dependent phosphate transport activity and its intracellular signaling mechanism in osteoblast-like cells

Inorganic phosphate (Pi) transport probably represents an important function of bone-forming cells in relation to extracellular matrix mineralization. In the present study, we investigated the effect of prostaglandin D₂ (PGD₂) on Pi transport activity and its intracellular signaling mechanism in MC3T3-E1 osteoblast-like cells. PGD₂ stimulated Na-dependent Pi uptake time- and dose-dependently in MC3T3-E1 cells during their proliferative phase. A protein kinase C (PKC) inhibitor calphostin C partially suppressed the stimulatory effect of PGD₂ on Pi uptake. The selective inhibitors of mitogen-activated protein (MAP) kinase pathways such as ERK, p38 and Jun kinases suppressed PGD₂-induced Pi uptake. The inhibitors of phosphatidylinositol (PI) 3-kinase and S₆ kinase reduced this effect of PGD₂, while Akt kinase inhibitor did not. These results suggest that PGD₂ stimulates Na-dependent Pi transport activity in the phase of proliferation of osteoblasts. The mechanisms responsible for this effect are activation of PKC, MAP kinases, PI 3-kinase and S₆ kinase.     

Serotonin 5-HT2C Receptor Stimulates Cyclic GMP Formation in Choroid Plexus

Abstract: The serotonin 5-HT_2C receptor (formerly designated the 5-HT_1C receptor) of the choroid plexus triggers phosphoinositide turnover. In the present study, we demonstrate that receptor activation also triggers the formation of cyclic GMP (cGMP). Application of 1 µM 5-HT to porcine choroid plexus tissue slices resulted in stimulation of cGMP formation to a maximum of five-fold basal level, with an EC₅₀ of 11 nM. This response was not inhibited by muscarinic or β-adrenergic receptor antagonists. Serotonin receptor antagonists inhibited cGMP formation with apparent K_i values of 1.3 (mianserin), 200 (ketanserin), and 5,500 (spiperone) nM, respectively. Neither serotonin-stimulated cGMP formation nor PI turnover was inhibited by pertussis toxin pretreatment. Preliminary biochemical studies suggested that serotonin-stimulated cGMP formation was calcium, phospholipase A₂, and lipoxygenase dependent, as incubation in low calcium buffers or inclusion of the phospholipase A₂ or lipoxygenase inhibitors p-bromophenacyl bromide or BW 755c resulted in significant reduction of cGMP formation. The present results suggest that in addition to triggering phosphoinositide turnover, choroid plexus serotonin 5-HT_2C receptors trigger cGMP formation in a calcium-sensitive manner.     

Regulation of the CYP27B1 5'-flanking region by transforming growth factor-beta in ROS 17/2.8 osteoblast-like cells

The biologically active form of vitamin D, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), regulates osteoblast proliferation and differentiation. Production of 1,25(OH)₂D₃ is catalysed by the enzyme 25-hydroxyvitamin D₃-1-hydroxylase (CYP27B1). Though highly expressed in the kidney, the CYP27B1 gene is also expressed in non-renal tissues including bone. It is hypothesised that local production of 1,25(OH)₂D₃ by osteoblasts plays an autocrine or paracrine role. The aim of this study was to investigate what factors regulate expression of the CYP27B1 gene in osteoblast cells. ROS 17/2.8 osteoblast cells were transiently transfected with plasmid constructs containing the 5′-flanking sequence of the human CYP27B1 gene fused to a luciferase reporter gene. Cells were treated with either parathyroid hormone (PTH), 1,25(OH)₂D₃, transforming growth factor-beta (TGF-β) or insulin-like growth factor-1 (IGF-1) and luciferase activity was measured 24 h later. The results showed that 1,25(OH)₂D₃ did not alter expression of the reporter construct, however treatment with PTH, IGF-1 and TGF-β decreased expression by 18, 53 and 58% respectively. The repressive action of TGF-β was isolated to the region between −531 and −305 bp. These data suggest that expression of the 5′-flanking region for the CYP27B1 gene in osteoblast cells may be regulated differently to that previously described in kidney cells.     

Effect of TNF-alpha on human osteosarcoma cell line Saos2--TNF-alpha regulation of bone sialoprotein gene expression in Saos2 osteoblast-like cells

Tumor necrosis factor-alpha (TNF-alpha) is a major mediator of inflammatory response in many diseases. It inhibits bone formation and stimulates bone resorption. To determine the molecular mechanisms involved in the regulation of gene expression of osteoblast-like cells, we analyzed the effects of TNF-alpha on the human osteosarcoma cell line Saos2. We used RT-PCR to examine the effects of TNF-alpha on bone sialoprotein (BSP), core binding factor a1 (Cbfa1), osterix, alpha 1 (I) collagen, cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), cathepsin B, cathepsin L and tissue inhibitors of metalloproteinase-1 (TIMP-1). TNF-alpha (10ng/ml) increased BSP, IL-6 and COX-2 mRNA levels after 3h, reaching maximal levels at 12 h. Cbfa1 mRNA levels increased after 3 h, but decreased by 24 h. Osterix, cathepsin B, cathepsin L and TIMP-1 mRNA levels did not change after stimulation with TNF-alpha. On the other hand, alpha 1 (I) collagen mRNA expression was suppressed by TNF-alpha at 24 h. Transient transfection analyses were performed using chimeric constructs of the rat BSP gene promoter linked to a luciferase reporter gene. TNF-alpha (10 ng/ml) had no effect on the promoter activities of BSP transfected into Saos2 cells. The results of gel mobility shift assays using radiolabeled double-stranded cAMP response element (CRE) and FGF2 response element (FRE) oligonucleotides in the proximal promoter of the rat BSP gene showed increased binding of nuclear proteins at 6 h. Gel mobility shift assays with radiolabelled COX-2-CRE and COX-2-NF kappa B oligonucleotides revealed an increase in the binding of nuclear proteins from TNF-alpha-stimulated Saos2 cells. These studies, therefore, showed that TNF-alpha indirectly increased BSP expression, and that it could be mediated through COX-2 and Cbfa1 expression in Saos2 osteoblast-like cells.     

5-Methoxytryptamine Inhibits Cyclic AMP Accumulation in Cultured Retinal Neurons Through Activation of a Pertussis Toxin-Sensitive Site Distinct from the 2-[125I]Iodomelatonin Binding Site

Abstract: Melatonin and 5-methoxytryptamine inhibited forskolin-stimulated cyclic AMP formation in cultured neural cells prepared from embryonic chick retina. Both methoxyindoles exhibited similar potency and efficacy, with EC₅₀ values of 0.8 n M for melatonin and 7.2 n M for 5-methoxytryptamine. Inhibition of cyclic AMP formation by 5-methoxytryptamine or melatonin was prevented by pretreatment with pertussis toxin. Pretreatment of cultures with 5-methoxytryptamine for 24 h reduced the subsequent inhibitory cyclic AMP response to 5-methoxytryptamine but not that to 2-iodomelatonin. Putative melatonin receptors on cultured retinal cells were labeled with 2-[¹²⁵I]iodomelatonin. Melatonin displaced specific 2-[¹²⁵I]iodomelatonin with a K _i value (0.8 n M ) similar to the EC₅₀ for inhibition of cyclic AMP formation. In contrast, 5-methoxytryptamine only inhibited 2-[¹²⁵I]iodomelatonin binding at very high concentrations ( K _i = 650 n M ). Pretreating cultured cells for 24 h with 2-iodomelatonin or melatonin, but not with 5-methoxytryptamine, reduced subsequent 2-[¹²⁵I]iodomelatonin binding. Thus, 5-methoxytryptamine appears to inhibit forskolin-stimulated cyclic AMP formation at a site distinct from the 2-iodomelatonin binding site.     

Regulation of the CYP27B1 5′-flanking region by transforming growth factor-beta in ROS 17/2.8 osteoblast-like cells

The biologically active form of vitamin D, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), regulates osteoblast proliferation and differentiation. Production of 1,25(OH)₂D₃ is catalysed by the enzyme 25-hydroxyvitamin D₃-1α-hydroxylase (CYP27B1). Though highly expressed in the kidney, the CYP27B1 gene is also expressed in non-renal tissues including bone. It is hypothesised that local production of 1,25(OH)₂D₃ by osteoblasts plays an autocrine or paracrine role. The aim of this study was to investigate what factors regulate expression of the CYP27B1 gene in osteoblast cells. ROS 17/2.8 osteoblast cells were transiently transfected with plasmid constructs containing the 5′-flanking sequence of the human CYP27B1 gene fused to a luciferase reporter gene. Cells were treated with either parathyroid hormone (PTH), 1,25(OH)₂D₃, transforming growth factor-beta (TGF-β) or insulin-like growth factor-1 (IGF-1) and luciferase activity was measured 24 h later. The results showed that 1,25(OH)₂D₃ did not alter expression of the reporter construct, however treatment with PTH, IGF-1 and TGF-β decreased expression by 18, 53 and 58% respectively. The repressive action of TGF-β was isolated to the region between −531 and −305 bp. These data suggest that expression of the 5′-flanking region for the CYP27B1 gene in osteoblast cells may be regulated differently to that previously described in kidney cells.     

Mechanical stretch increases CCN2/CTGF expression in anterior cruciate ligament-derived cells

Anterior cruciate ligament (ACL)-to-bone interface serves to minimize the stress concentrations that would arise between two different tissues. Mechanical stretch plays an important role in maintaining cell-specific features by inducing CCN family 2/connective tissue growth factor (CCN2/CTGF). We previously reported that cyclic tensile strain (CTS) stimulates α1(I) collagen (COL1A1) expression in human ACL-derived cells. However, the biological function and stress-related response of CCN2/CTGF were still unclear in ACL fibroblasts. In the present study, CCN2/CTGF was observed in ACL-to-bone interface, but was not in the midsubstance region by immunohistochemical analyses. CTS treatments induced higher increase of CCN2/CTGF expression and secretion in interface cells compared with midsubstance cells. COL1A1 expression was not influenced by CCN2/CTGF treatment in interface cells despite CCN2/CTGF stimulated COL1A1 expression in midsubstance cells. However, CCN2/CTGF stimulated the proliferation of interface cells. Our results suggest that distinct biological function of stretch-induced CCN2/CTGF might regulate region-specific phenotypes of ACL-derived cells.     

Caspase-2/NEDD-2 protease mediates execution of apoptosis in AK-5 tumor cells

AK-5 tumour cells have been shown to undergo apoptosis in vitro and in vivo. The efficient killing of tumour cells by necrosis and apoptosis leads to spontaneous regression of the tumour. To investigate a possible involvement of caspase-2/Nedd-2 protease in AK-5 apoptosis, we introduced Nedd-2 gene in antisense orientation and showed inhibition of tumour cell apoptosis. Similarly introduction of the bcl-2 gene in tumour cells also inhibited the apoptotic programme. NK cells which have previously been shown to be the effector cells also fail to induce apoptosis in Nedd-2 antisense and bcl-2 transfected clones whereas NK mediated cytotoxic activity is not altered in the transfectants. These results suggest participation of Nedd-2 protease in the induction of apoptosis in AK-5 cells leading to tumour regression. This revised version was published online in June 2006 with corrections to the Cover Date.     

Nuclear import of N-terminal FAK by activation of the FcepsilonRI receptor in RBL-2H3 cells

As FAK integrates membrane receptor signalling, yet is also found in the nucleus, we investigated whether nuclear FAK is regulated by membrane receptor activation. Activation of the mast cell FcepsilonRI receptor leads to the release and synthesis of inflammatory mediators as well as increased proliferation and survival. Using RBL-2H3 basophilic leukaemia cells, FAK and the FcepsilonRI receptor were co-localised following cross-linking of IgE with antigen. This also resulted in a significant increase in the nucleus of several N-terminal FAK fragments, the largest of which included the kinase domain but not the focal adhesion targeting domain. This was confirmed using cells that stably expressed recombinant EGFP-FAK. Furthermore, treatment of EGFP-FAK expressing cells with Leptomycin B, an inhibitor of nuclear export, resulted in increased nuclear localisation of EGFP-FAK. Therefore, FAK can shuttle between the nuclear and cytoplasmic compartments and the cellular distribution of N-terminal FAK is regulated by membrane receptor activation.     

Serotonin 5-HT1D and 5-HT1A Receptors Respectively Mediate Inhibition of Glutamate Release and Inhibition of Cyclic GMP Production in Rat Cerebellum In Vitro

Abstract: The K⁺-evoked overflow of endogenous glutamate from cerebellar synaptosomes was inhibited by serotonin [5-hydroxytryptamine (5-HT); pD₂ = 8.95], 8-hydroxy-2-(di- n -propylamino)tetralin (8-OH-DPAT; pD₂ = 7.35), and sumatriptan (pD₂ = 8.43). These inhibitions were prevented by the selective 5-HT_1D receptor antagonist N -[4-methoxy-3-(4-methyl-1-piperazinyl)phenyl]-2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)(1,1-biphenyl)-4-carboxamide (GR-127935). The three agonists tested also inhibited the cyclic GMP (cGMP) response provoked in slices by K⁺ depolarization; pD₂ values were 9.37 (5-HT), 9.00 (8-OH-DPAT), and 8.39 (sumatriptan). When cGMP formation was elevated by directly activating glutamate receptors with NMDA or α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), the inhibition of the cGMP responses displayed the following pattern: 5-HT (pD₂ values of 8.68 and 8.72 against NMDA and AMPA, respectively); 8-OH-DPAT (respective pD₂ values of 9.15 and 9.00); sumatriptan (0.1 µ M ) was ineffective. The 5-HT_1A receptor antagonist ( S )-(+) N-tert -butyl-3-[4-(2-methoxyphenyl)piperazin-1-yl]-2-phenylpropionamide dihydrochloride [(+)-WAY 100135] did not prevent the inhibition of glutamate release by 5-HT but blocked the inhibition by 8-OH-DPAT of the NMDA/AMPA-evoked cGMP responses. It is suggested that presynaptic 5-HT_1D receptors mediate inhibition directly of glutamate release and indirectly of the cGMP responses to the released glutamate; on the other hand, activation of (postsynaptic) 5-HT_1A receptors causes inhibition of the cGMP responses linked to stimulation of NMDA/AMPA receptors.     

Evidence for [D-Ala2,D-Leu5]Enkephalin-Induced Supersensitivity to 5-Hydroxytryptamine in a Neurotumor × Brain Hybrid Cell Line (NCB-20)

A neuroblastoma X Chinese hamster embryonic brain explant hybrid cell line (NCB-20) expressed 5-hydroxytryptamine (5-HT1) receptors, linked to adenylate cyclase, which closely resembled 5-HT1 receptors previously characterized in central nervous tissue. However, the affinity of the receptors for 5-HT was only 150 nM compared to 5 nM in membranes prepared from cerebral cortex. The elevation of cyclic AMP levels in NCB-20 cells produced by 5-HT was found additive to that produced by cholera toxin but synergistic with that produced by either prostaglandin E1 (PGE1) or forskolin, suggesting that these latter two agents elevate cyclic AMP levels by a different mechanism than 5-HT. The elevation of cyclic AMP levels by either 5-HT or PGE1 was reversed by [D-Ala2,D-Leu5]enkephalin (DADLE), morphine, clonidine, and 3,4-dihydroxyphenylethylamine (dopamine) on a short (30 min) time scale. However, continued exposure to DADLE resulted in loss of the initial inhibitory effects of DADLE after 6 h and return of cyclic AMP levels to that seen with either 5-HT or PGE1 alone. When the DADLE exposure time was increased to 48 h, 5-HT produced a further twofold increase in cyclic AMP levels, but there was no increase in the responsiveness of the cells to PGE1 unless naloxone was added 1 h prior to treatment with PGE1. Scatchard analysis showed that the increased potency of 5-HT resulted from an increase in receptor affinity for 5-HT (from a KD of 150 +/- 20 nM to one of 20 +/- 7 nM), with a reduction in the number of apparent binding sites. The 5-HT supersensitivity observed in NCB-20 cells may be a good model for neurotransmitter interactions that produce desensitization or facilitation in the intact nervous system.     

Differential Coupling of Serotonin 5-HT1A and 5-HT1B Receptors to Activation of ERK2 and Inhibition of Adenylyl Cyclase in Transfected CHO Cells

Although the subtypes of serotonin 5-HT1 receptors have distinct structure and pharmacology, it has not been clear if they also exhibit differences in coupling to cellular signals. We have sought to compare directly the coupling of 5-HT1A and 5-HT1B receptors to adenylyl cyclase and to the mitogen-activated protein kinase ERK2 (extracellular signal-regulated kinase-2). We found that 5-HT1B receptors couple better to activation of ERK2 and inhibition of adenylyl cyclase than do 5-HT1A receptors. 5-HT stimulated a maximal fourfold increase in ERK2 activity in nontransfected cells that express endogenous 5-HT1B receptors at a very low density and a maximal 13-fold increase in transfected cells expressing 230 fmol of 5-HT1B receptor/mg of membrane protein. In contrast, activation of 5-HT1A receptors stimulated only a 2.8-fold maximal activation of ERK2 in transfected cells expressing receptors at 300 fmol/mg of membrane protein but did stimulate a 12-fold increase in activity in cells expressing receptors at 3,000 fmol/mg of membrane protein. Similarly, 5-HT1A, but not 5-HT1B, receptors were found to cause significant inhibition of forskolin-stimulated cyclic AMP accumulation only when expressed at high densities. These findings demonstrate that although both 5-HT1A and 5-HT1B receptors have been shown to couple to G proteins of the Gi class, they exhibit differences in coupling to ERK2 and adenylyl cyclase.     

Mianserin-Induced Down-Regulation of Human 5-Hydroxytryptamine2A and 5-Hydroxytryptamine2C Receptors Stably Expressed in the Human Neuroblastoma Cell Line SH-SY5Y

Abstract: We have assessed the ability of the serotonergic antagonist mianserin to modulate the number and functional activity of human 5-hydroxytryptamine_2A (5-HT_2A) and 5-HT_2C receptors stably expressed in the human neuroblastoma cell line SH-SY5Y. Incubation of cells expressing the 5-HT_2A receptor with mianserin (100 n M ) for 24 h caused a significant decrease (48%) in the binding capacity of [³H]ketanserin. This receptor down-regulation was associated with a corresponding decrease in the maximal production of inositol phosphates induced by 5-HT but not by carbachol. Exposure of cells expressing the 5-HT_2C receptor to mianserin (100 n M ) for 72 h but not for 24 h similarly resulted in a significant reduction (44%) in [³H]mesulergine binding. Corresponding analysis of inositol phosphate production by 5-HT at the 5-HT_2C receptor after incubation with mianserin showed no change in maximal response after 24 h. No change in the binding capacity of either radioligand was seen after incubation with mianserin for 1 h. A decrease in the binding affinity of both radioligands was also observed after mianserin treatment, but this decrease was similar after 1 h of incubation to that seen after 24 or 72 h, and was probably due to the retention of mianserin within the tissue. We conclude that antagonist down-regulation is evident at human 5-HT_2A and 5-HT_2C receptors stably expressed in a human neuroblastoma cell line and is probably mediated by a direct action of mianserin at the receptor.     

lncRNA RBM5-AS1 promotes cell proliferation and invasion by epigenetically silencing miR-132/212 in hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is regarded as one of the most common malignancies worldwide leading to cancer-related death. Long noncoding RNAs (lncRNAs) are a critical modulator affecting HCC progression. Whereas, the pathogenesis of lncRNA RBM5-AS1 in the development of HCC remains unclear. Quantitative RT-PCR or western blot assays were applied to detect the expression of genes and proteins, respectively. The proliferation and metastasis abilities were assessed using Cell counting kit-8 (CCK-8), EdU and transwell assays. RNA immunoprecipitation (RIP) experiment was employed to validate the molecular interactions. RBM5-AS1 is highly expressed in HCC tissues and cell lines, especially in Hep3B and HepG2 cells. RBM5-AS1 knockdown dramatically restrains cell proliferation, invasion and migration of HCC cells. Importantly, RBM5-AS1 acts as an epigenetic regulator to elevate the H3K27me3 level of miR-132/212 promoter regions via recruiting PRC2 (EZH2, SUZ12, EED), and eventually reducing miR-132/212 expressions. The recovery experiments demonstrated that downregulation of miR-132/212 markedly eliminate the antitumor effects mediated by RBM5-AS1 silencing in HCC cells. The data of this work illustrate that RBM5-AS1 acts as an epigenetic regulator to promote the HCC progression by repressing miR-132/212 expressions, which would provide a new insight for understanding the action mechanism of RBM5-AS1 in HCC development.