Estrogen Receptor (ER)-α36 Is Involved in Estrogen- and Tamoxifen-Induced Neuroprotective Effects in Ischemic Stroke Models

The neuroprotection by estrogen (E2) and tamoxifen is well documented in experimental stroke models; however, the exact mechanism is unclear. A membrane-based estrogen receptor, ER-α36, has been identified. Postmenopausal-levels of E2 act through ER-α36 to induce osteoclast apoptosis due to a prolonged activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-related kinase (ERK) signaling. We hypothesized that ER-α36 may play a role in the neuroprotective activities of estrogen and tamoxifen. Here, we studied ER-α36 expression in the brain, as well as its neuroprotective effects against oxygen and glucose deprivation (OGD) in PC12 cells. We found that ER-α36 was expressed in both rat and human brain. In addition, OGD-induced cell death was prevented by l nmol/L 17β-estradiol (E2β). E2β activates the MAPK/ERK signaling pathway in PC12 cells under basal and OGD conditions by interacting with ER-α36 and also induces ER-α36 expression. Low-dose of tamoxifen up-regulated ER-α36 expression and enhanced neuronal survival in an ovariectomized ischemic stroke model. Furthermore, low-dose of tamoxifen enhanced neuroprotective effects by modulating activates or suppress ER-α36. Our results thus demonstrated that ER-α36 is involved in neuroprotective activities mediated by both estrogen and tamoxifen.     

ShRNA-Targeted COMMD7 Suppresses Hepatocellular Carcinoma Growth

Background

COMMD7 is a newly identified gene overexpressed in hepatocellular carcinoma (HCC) and associated with tumor invasion and poor prognosis. We aim to examine the biological function of COMMD7 in HCC by shRNA silencing.

Methods

COMMD7 expressions were examined in human HCC cell lines HepG2, Huh7, Hep3B, HLE, HLF, SK-Hep-1 and PLC/PRF/5 cells. Recombinant pGenesil-COMMD7-shRNA was transfected into COMMD7-abundant HepG2 cells to silence COMMD7 expression. The effects of COMMD7 silencing on HepG2 cell proliferation in vitro and xenograft tumor growth in vivo were evaluated. Flow cytometry profiling was used to detect the presence of apoptosis in COMMD7-silenced HepG2 cells and to differentiate cell cycle distribution. Electrophoretic mobility shift assay and luciferase reporter assays to examine the activities of nuclear factor-kappaB (NF-κB) signaling pathways in response to tumor necrosis factor (TNF)-α in COMMD7-silenced HepG2 cells.

Results

COMMD7 expression level was abundance in HepG2 and SK-Hep-1 cells. COMMD7 was aberrantly overexpressed in HepG2 cells, whilst pGenesil-COMMD7-shRNA exhibited a maximal inhibition rate of 75%. COMMD7 silencing significantly reduced HepG2 cell proliferation and colony formation. The knockdown of COMMD7 resulted in an increased apoptosis and cell cycle arrest at S-phase. COMMD7 knockdown also exhibited an antineoplastic effect in vivo, which manifested as tumor xenograft growth retardation. COMMD7 silencing also suppressed the responsiveness of NF-κB signaling pathway to the stimulation with TNF-α in vitro. Moreover, the similar suppressive effects of COMMD7 silence on SK-Hep-1 cells were also observed.

Conclusions

COMMD7 contributes to HCC progression by reducing cell apoptosis and overcoming cell cycle arrest. The proliferative and antiapoptotic effects of COMMD7 may be mediated by NF-κB signaling pathway.     

Estradiol and GPER Activation Differentially Affect Cell Proliferation but Not GPER Expression in the Hippocampus of Adult Female Rats

Estradiol increases cell proliferation in the dentate gyrus of the female rodent but it is not known whether the G protein-coupled estrogen receptor (GPER), a membrane receptor, is involved in this process, nor whether there are regional differences in estradiol’s effects on cell proliferation. Thus, we investigated whether estradiol exerts its effects on cell proliferation in the dorsal and ventral dentate gyrus through GPER, using the GPER agonist, G1, and antagonist, G15. Ovariectomized adult female rats received a single injection of either: 17β-estradiol (10 μg), G1 (0.1, 5, 10 μg), G15 (40 μg), G15 and estradiol, or vehicle (oil, DMSO, or oil+DMSO). After 30 min, animals received an injection of bromodeoxyuridine (BrdU) and were perfused 24 h later. Acute treatment with estradiol increased, while the GPER agonist G1 (5 μg) decreased, the number of BrdU+ cells in the dentate gyrus relative to controls. The GPER antagonist, G15 increased the number of BrdU+ cells relative to control in the dorsal region and decreased the number of BrdU+ cells in the ventral region. However, G15 treatment in conjunction with estradiol partially eliminated the estradiol-induced increase in cell proliferation in the dorsal dentate gyrus. Furthermore, G1 decreased the expression of GPER in the dentate gyrus but not the CA1 and CA3 regions of the hippocampus. In summary, we found that activation of GPER decreased cell proliferation and GPER expression in the dentate gyrus of young female rats, presenting a potential and novel estrogen-independent role for this receptor in the adult hippocampus.     

Articular cartilage chondrocytes express aromatase and use enzymes involved in estrogen metabolism

Introduction

Sex hormones, especially estrogens, have been implicated in articular cartilage metabolism and the pathogenesis of postmenopausal osteoarthritis. The conversion by aromatase (CYP19A1) of androstenedione into estrone (E1) and of testosterone into 17β-estradiol (E2) plays a key role in the endogenous synthesis of estrogens in tissue.

Methods

We analyzed the expression of aromatase (CYP19A1) in immortalized C-28/I2 and T/C-28a2 chondrocytes, as well as in cultured primary human articular chondrocytes and human articular cartilage tissue, by means of RT-PCR, Western blotting and immunohistochemistry. By means of quantitative RT-PCR and enzyme-linked immunosorbent assay, we also determined whether the aromatase inhibitor letrozole influences estrogen metabolism of cultured chondrocytes in immortalized C-28/I2 chondrocytes.

Results

Aromatase mRNA was detected in both immortalized chondrocyte cell lines, in cultured primary human chondrocytes, and in human articular cartilage tissue. By means of Western blot analysis, aromatase was detected at the protein level in articular cartilage taken from various patients of both sexes and different ages. Cultured primary human articular chondrocytes, C-28/I2 and T/C-28a2, and human articular cartilage tissue reacted with antibodies for aromatase. Incubation of C-28/I2 chondrocytes with 10⁻¹¹ M to 10⁻⁷ M letrozole as an aromatase inhibitor revealed significantly increased amounts of the mRNAs of the enzyme cytochrome P4501A1 (CYP1A1), which is involved in the catagen estrogen metabolism, and of the estrogen receptors ER-α and ER-β. Concomitantly, synthesis of estrone (E1) was significantly downregulated after incubation with letrozole.

Conclusions

We demonstrate that human articular cartilage expresses aromatase at the mRNA and protein levels. Blocking of estrone synthesis by the aromatase inhibitor letrozole is counteracted by an increase in ER-α and ER-β. In addition, CYP1A1, an enzyme involved in catabolic estrogen metabolism, is upregulated. This suggests that articular chondrocytes use ERs functionally. The role of endogenous synthesized estrogens in articular cartilage health remains to be elucidated.     

N-3 Poly-Unsaturated Fatty Acids Shift Estrogen Signaling to Inhibit Human Breast Cancer Cell Growth

Although evidence has shown the regulating effect of n-3 poly-unsaturated fatty acid (n-3 PUFA) on cell signaling transduction, it remains unknown whether n-3 PUFA treatment modulates estrogen signaling. The current study showed that docosahexaenoic acid (DHA, C22:6), eicosapentaenoic acid (EPA, C20:5) shifted the pro-survival and proliferative effect of estrogen to a pro-apoptotic effect in human breast cancer (BCa) MCF-7 and T47D cells. 17 β-estradiol (E2) enhanced the inhibitory effect of n-3 PUFAs on BCa cell growth. The IC50 of DHA or EPA in MCF-7 cells decreased when combined with E2 (10 nM) treatment (from 173 µM for DHA only to 113 µM for DHA+E2, and from 187 µm for EPA only to 130 µm for EPA+E2). E2 also augmented apoptosis in n-3 PUFA-treated BCa cells. In contrast, in cells treated with stearic acid (SA, C18:0) as well as cells not treated with fatty acid, E2 promoted breast cancer cell growth. Classical (nuclear) estrogen receptors may not be involved in the pro-apoptotic effects of E2 on the n-3 PUFA-treated BCa cells because ERα agonist failed to elicit, and ERα knockdown failed to block E2 pro-apoptotic effects. Subsequent studies reveal that G protein coupled estrogen receptor 1 (GPER1) may mediate the pro-apoptotic effect of estrogen. N-3 PUFA treatment initiated the pro-apoptotic signaling of estrogen by increasing GPER1-cAMP-PKA signaling response, and blunting EGFR, Erk 1/2, and AKT activity. These findings may not only provide the evidence to link n-3 PUFAs biologic effects and the pro-apoptotic signaling of estrogen in breast cancer cells, but also shed new insight into the potential application of n-3 PUFAs in BCa treatment.     

Estrogen Receptor-α36 Is Involved in Pterostilbene-Induced Apoptosis and Anti-Proliferation in In Vitro and In Vivo Breast Cancer

Pterostilbene (trans-3,5-dimethoxy-4′-hudroxystilbene) is an antioxidant primarily found in blueberries. It also inhibits breast cancer regardless of conventional estrogen receptor (ER-α66) status by inducing both caspase-dependent and caspase-independent apoptosis. However, the pterostilbene-induced apoptosis rate in ER-α66-negative breast cancer cells is much higher than that in ER-α66-positive breast cancer cells. ER-α36, a variant of ER-α66, is widely expressed in ER-α66-negative breast cancer, and its high expression mediates the resistance of ER-α66-positive breast cancer patients to tamoxifen therapy. The aim of the present study is to determine the relationship between the antiproliferation activity of pterostilbene and ER-α36 expression in breast cancer cells. Methyl-thiazolyl-tetrazolium (MTT) assay, apoptosis analysis, and an orthotropic xenograft mouse model were used to examine the effects of pterostilbene on breast cancer cells. The expressions of ER-α36 and caspase 3, the activation of ERK and Akt were also studied through RT-PCR, western blot analysis, and immunohistochemical (IHC) staining. ER-α36 knockdown was found to desensitize ER-α66-negative breast cancer cells to pterostilbene treatment both in vitro and in vivo, and high ER-α36 expression promotes pterostilbene-induced apoptosis in breast cancer cells. Western blot analysis data indicate that MAPK/ERK and PI3K/Akt signaling in breast cancer cells with high ER-α36 expression are mediated by ER-α36, and are inhibited by pterostilbene. These results suggest that ER-α36 is a therapeutic target in ER-α36-positive breast cancer, and pterostilbene is an inhibitor that targets ER-α36 in the personalized therapy against ER-α36-positive breast cancer.     

The aPKCι blocking agent ATM negatively regulates EMT and invasion of hepatocellular carcinoma

Epithelial-to-mesenchymal transition (EMT) has an important role in invasion and metastasis of hepatocellular carcinoma (HCC). To explore the regulatory mechanism of atypical protein kinase C ι (aPKCι) signaling pathways to HCC development, and find an agent for targeted therapy for HCC, immortalized murine hepatocytes were employed to establish an EMT cell model of HCC, MMH-RT cells. Our study showed that EMT took place in MMH-R cells under the effect of transforming growth factor-β1 (TGF-β1) overexpressing aPKCι. Furthermore, we showed that the aPKCι blocking agent aurothiomalate (ATM) inhibited EMT and decreased invasion of hepatocytes. Moreover, ATM selectively inhibited proliferation of mesenchymal cells and HepG2 cells and induced apoptosis. However, ATM increased proliferation of epithelial cells and had little effect on apoptosis and invasion of epithelial cells. In conclusion, our result suggested that aPKCι could be an important bio-marker of tumor EMT, and used as an indicator of invasion and malignancy. ATM might be a promising agent for targeted treatment of HCC.     

Comparison of the Regulation of β-Catenin Signaling by Type I,Type II and Type III Interferons in Hepatocellular Carcinoma Cells

Background/Objective

IFNs are a group of cytokines that possess potent antiviral and antitumor activities, while β-catenin pathway is a proliferative pathway involved in carcinogenesis. Interaction between these two pathways has not been well elaborated in hepatocellular carcinoma (HCC).

Methods

HCC cell lines, HepG2 and Huh7, were used in this study. β-catenin protein levels and corresponding signaling activities were observed by flow cytometry and luciferase assay, respectively. Cell proliferation was quantified by counting viable cells under microscope, and apoptosis by TUNEL assay. DKK1 and GSK3β levels were determined by flow cytometry. Secreted DKK1 was tested by ELISA. FLUD, S3I and aDKK1 were used to inhibit STAT1, STAT3 and DKK1 activities, respectively.

Results

Our findings show that all three types of IFNs, IFNα, IFNγ and IFNλ, are capable of inhibiting β-catenin signaling activity in HepG2 and Huh7 cells, where IFNγ was the strongest (p<0.05). They expressed suppression of cellular proliferation and induced apoptosis. IFNγ expressed greater induction ability when compared to IFNα and IFNλ (p<0.05). All tested IFNs could induce DKK1 activation but not GSK3β in HepG2 and Huh7 cells. IFNs induced STAT1 and STAT3 activation but by using specific inhibitors, we found that only STAT3 is vital for IFN-induced DKK1 activation and apoptosis. In addition, DKK1 inhibitor blocked IFN-induced apoptosis. The pattern of STAT3 activation by different IFNs is found consistent with the levels of apoptosis with the corresponding IFNs (p<0.05).

Conclusions

In hepatocellular carcinoma, all three types of IFNs are found to induce apoptosis by inhibiting β-catenin signaling pathway via a STAT3- and DKK1-dependent pathway. This finding points to a cross-talk between different IFN types and β-catenin signaling pathways which might be carrying a biological effect not only on HCC, but also on processes where the two pathways bridge.     

Salinomycin Inhibits Proliferation and Induces Apoptosis of Human Hepatocellular Carcinoma Cells In Vitro and In Vivo

The anti-tumor antibiotic salinomycin (Sal) was recently identified as a selective inhibitor of breast cancer stem cells; however, the effect of Sal on hepatocellular carcinoma (HCC) is not clear. This study aimed to determine the anti-tumor efficacy and mechanism of Sal on HCC. HCC cell lines (HepG2, SMMC-7721, and BEL-7402) were treated with Sal. Cell doubling time was determinated by drawing growth curve, cell viability was evaluated using the Cell Counting Kit 8. The fraction of CD133⁺ cell subpopulations was assessed by flow cytometry. We found that Sal inhibits proliferation and decreases PCNA levels as well as the proportion of HCC CD133⁺cell subpopulations in HCC cells. Cell cycle was analyzed using flow cytometry and showed that Sal caused cell cycle arrest of the various HCC cell lines in different phases. Cell apoptosis was evaluated using flow cytometry and Hoechst 33342 staining. Sal induced apoptosis as characterized by an increase in the Bax/Bcl-2 ratio. Several signaling pathways were selected for further mechanistic analyses using real time-PCR and Western blot assays. Compared to control, β-catenin expression is significantly down-regulated upon Sal addition. The Ca²⁺ concentration in HCC cells was examined by flow cytometry and higher Ca²⁺ concentrations were observed in Sal treatment groups. The anti-tumor effect of Sal was further verified in vivo using the hepatoma orthotopic tumor model and the data obtained showed that the size of liver tumors in Sal-treated groups decreased compared to controls. Immunohistochemistry and TUNEL staining also demonstrated that Sal inhibits proliferation and induces apoptosis in vivo. Finally, the role of Sal on in vivo Wnt/β-catenin signaling was evaluated by Western blot and immunohistochemistry. This study demonstrates Sal inhibits proliferation and induces apoptosis of HCC cells in vitro and in vivo and one potential mechanism is inhibition of Wnt/β-catenin signaling via increased intracellular Ca²⁺ levels.     

Reproductive Senescence Blunts Response of Estrogen Receptor-α Expression to Estrogen Treatment in Rat Post-Ischemic Cerebral Microvessels

Background

Several studies demonstrate that estrogen treatment improves cerebral blood flow in ischemic brain regions of young ovariectomized (OVX) rats. Estrogen receptor-α (ER-α) may mediate estrogen’s beneficial actions via its effects on the cerebral microvasculature. However, estrogen-derived benefit may be attenuated in aged, reproductively senescent (RS) rats. Our goal was to determine the effects of aging, estrogen deprivation and estrogen repletion with oral conjugated estrogens (CE) on postischemic cerebral microvascular protein expression of ER-α and ER-β.

Methods

Fisher-344 (n = 37) female rats were randomly divided into the following groups: OVX, OVX CE-treated, RS untreated, and RS CE-treated. After 30 days pretreatment with CE (0.01 mg/kg) rats were subjected to15 min. transient global cerebral ischemia. Non-ischemic naïve, OVX and RS rats were used as controls. Expression of ER-α and ER-β in isolated cortical cerebral microvessels (20 to 100 µm in diameter) was assessed using Western blot and immunohistochemistry techniques.

Results

Age and reproductive status blunted nonischemic ER-α expression in microvessels of OVX rats (0.31±0.05) and RS rats (0.33±0.06) compared to naïve rats (0.45±0.02). Postischemic microvascular expression of ER-α in OVX rats (0.01±0.0) was increased by CE treatment (0.04±0.01). Expression of ER-α in microvessels of RS rats (0.03±0.02) was unaffected by CE treatment (0.01±0.02). Western blot data are presented as a ratio of ER-α or ER-β proteins to β-actin and. Oral CE treatment had no effect on ER-β expression in postischemic microvessels of OVX and RS rats. Statistical analysis was performed by One-Way ANOVA and a Newman-Keuls or Student’s post-hoc test.

Conclusion

Chronic treatment with CE increases ER-α but not ER-β expression in cerebral microvessels of OVX rats. Aging appears to reduce the normal ability of estrogen to increase ER-α expression in postischemic cerebral microvessels.     

Negative Regulation of Leptin-induced Reactive Oxygen Species (ROS) Formation by Cannabinoid CB1 Receptor Activation in Hypothalamic Neurons

The adipocyte-derived, anorectic hormone leptin was recently shown to owe part of its regulatory effects on appetite-regulating hypothalamic neuropeptides to the elevation of reactive oxygen species (ROS) levels in arcuate nucleus (ARC) neurons. Leptin is also known to exert a negative regulation on hypothalamic endocannabinoid levels and hence on cannabinoid CB₁ receptor activity. Here we investigated the possibility of a negative regulation by CB₁ receptors of leptin-mediated ROS formation in the ARC. Through pharmacological and molecular biology experiments we report data showing that leptin-induced ROS accumulation is 1) blunted by arachidonyl-2′-chloroethylamide (ACEA) in a CB₁-dependent manner in both the mouse hypothalamic cell line mHypoE-N41 and ARC neuron primary cultures, 2) likewise blocked by a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, troglitazone, in a manner inhibited by T0070907, a PPAR-γ antagonist that also inhibited the ACEA effect on leptin, 3) blunted under conditions of increased endocannabinoid tone due to either pharmacological or genetic inhibition of endocannabinoid degradation in mHypoE-N41 and primary ARC neuronal cultures from MAGL^−/− mice, respectively, and 4) associated with reduction of both PPAR-γ and catalase activity, which are reversed by both ACEA and troglitazone. We conclude that CB₁ activation reverses leptin-induced ROS formation and hence possibly some of the ROS-mediated effects of the hormone by preventing PPAR-γ inhibition by leptin, with subsequent increase of catalase activity. This mechanism might underlie in part CB1 orexigenic actions under physiopathological conditions accompanied by elevated hypothalamic endocannabinoid levels.     

Activation of GPER Induces Differentiation and Inhibition of Coronary Artery Smooth Muscle Cell Proliferation

Background

Vascular pathology and dysfunction are direct life-threatening outcomes resulting from atherosclerosis or vascular injury, which are primarily attributed to contractile smooth muscle cells (SMCs) dedifferentiation and proliferation by re-entering cell cycle. Increasing evidence suggests potent protective effects of G-protein coupled estrogen receptor 1 (GPER) activation against cardiovascular diseases. However, the mechanism underlying GPER function remains poorly understood, especially if it plays a potential role in modulating coronary artery smooth muscle cells (CASMCs).

Methodology/Principal Findings

The objective of our study was to understand the functional role of GPER in CASMC proliferation and differentiation in coronary arteries using from humans and swine models. We found that the GPER agonist, G-1, inhibited both human and porcine CASMC proliferation in a concentration- (10⁻⁸ to 10⁻⁵ M) and time-dependent manner. Flow cytometry revealed that treatment with G-1 significantly decreased the proportion of S-phase and G2/M cells in the growing cell population, suggesting that G-1 inhibits cell proliferation by slowing progression of the cell cycle. Further, G-1-induced cell cycle retardation was associated with decreased expression of cyclin B, up-regulation of cyclin D1, and concomitant induction of p21, and partially mediated by suppressed ERK1/2 and Akt pathways. In addition, G-1 induces SMC differentiation evidenced by increased α-smooth muscle actin (α-actin) and smooth muscle protein 22α (SM22α) protein expressions and inhibits CASMC migration induced by growth medium.

Conclusion

GPER activation inhibits CASMC proliferation by suppressing cell cycle progression via inhibition of ERK1/2 and Akt phosphorylation. GPER may constitute a novel mechanism to suppress intimal migration and/or synthetic phenotype of VSMC.     

Selective GPER activation decreases proliferation and activates apoptosis in tumor Leydig cells

We have previously shown that estrogens binding to estrogen receptor (ER) α increase proliferation of Leydig tumor cells. Estrogens can also bind to G protein-coupled ER (GPER) and activation of this receptor can either increase or decrease cell proliferation of several tumor types. The aim of this study was to investigate GPER expression in R2C rat tumor Leydig cells, evaluate effects of its activation on Leydig tumor cell proliferation and define the molecular mechanisms triggered in response to its activation. R2C cells express GPER and its activation, using the specific ligand G-1, is associated with decreased cell proliferation and initiation of apoptosis. Apoptosis after G-1 treatment was asserted by appearance of DNA condensation and fragmentation, decrease in Bcl-2 and increase in Bax expression, cytochrome c release, caspase and poly (ADP-ribose) polymerase-1 (PARP-1) activation. These effects were dependent on GPER activation because after silencing of the gene, using a specific small interfering RNA, cyt c release, PARP-1 activation and decrease in cell proliferation were abrogated. These events required a rapid, however, sustained extracellular regulated kinase 1/2 activation. G-1 was able to decrease the growth of R2C xenograft tumors in CD1 nude mice while increasing the number of apoptotic cells. In addition, in vivo administration of G-1 to male CD1 mice did not cause any alteration in testicular morphology, while cisplatin, the cytotoxic drug currently used for the therapy of Leydig tumors, severely damaged testicular structure, an event associated with infertility in cisplatin-treated patients. These observations indicate that GPER targeting for the therapy of Leydig cell tumor may represent a good alternative to cisplatin to preserve fertility in Leydig tumor patients.     

Estrogen Receptor-β-selective Ligands Alleviate High-fat Diet- and Ovariectomy-induced Obesity in Mice

Obesity is an epidemic problem affecting millions of people in the Western hemisphere and costs the United States economy more than $200 billion annually. Currently, there are no effective treatments to combat obesity. Recent studies have implicated the constitutive activity of estrogen receptor (ER) β as an important regulator of metabolic diseases. However, the potential of ER-β-selective ligands to offset obesity is not clear. We evaluated the pharmacological effect of ER-β-selective ligands (β-LGNDs) in animal models of high-fat diet- and ovariectomy-induced obesity. Ligand binding, transactivation, and uterotrophic studies with β-LGNDs demonstrated selectivity for ER-β over ER-α. Animals fed a high-fat diet showed a significant increase in body weight, and this weight gain was attenuated by β-LGNDs. High-fat diet-mediated increases in serum cholesterol, leptin, glucose, and fat accumulation in organs were also reduced by β-LGNDs. In addition, MRI scanning indicated that β-LGNDs altered body composition by reducing fat mass and increasing lean body mass. Organ weights and gene expression analyses demonstrated that adipose tissue is the center of action for β-LGNDs, and the reduction in body weight is likely due to increased energy expenditure. In vitro and in vivo mechanistic studies indicated that the anti-obesity effects of β-LGNDs were due to indirect peroxisome proliferator-activated receptor γ antagonistic actions requiring the ligand binding domain of ER-β and through abrogation of the ability of PGC-1 to coactivate peroxisome proliferator-activated receptor γ. In conclusion, these studies indicate that ligand-activated ER-β is a potential therapeutic target to combat obesity and obesity-related metabolic diseases.     

CircRNA hsa_circRNA_104348 promotes hepatocellular carcinoma progression through modulating miR-187-3p/RTKN2 axis and activating Wnt/β-catenin pathway

Circular RNAs (circRNAs) have confirmed to participate in diverse biological functions in cancer. However, the expression patterns of circRNAs on hepatocellular carcinoma (HCC) remains unclear. In the present study, we clarified that hsa_circRNA_104348 was dramatically upregulated in HCC tissues and cells. Patients with HCC displaying high hsa_circRNA_104348 level possessed poor prognosis. Has_circ_104348 facilitated proliferation, migration, and invasion, meanwhile suppressed apoptosis of HCC cell. Furthermore, hsa_circRNA_104348 directly targeted miR-187–3p, could regulate miR-187-3p to affect proliferation, migration, invasion, and apoptosis of HCC cells, and may have effect on Wnt/β-catenin signaling pathway. Moreover, RTKN2 could be a direct target of miR-187-3p. In addition, knockdown of hsa_circRNA_104348 attenuated HCC tumorigenesis and lung metastasis in vivo. Taken together, these findings indicated that circular RNA hsa_circRNA_104348 might function as a competing endogenous RNA (ceRNA) to promotes HCC progression by targeting miR-187–3p/RTKN2 axis and activating Wnt/β-catenin pathway.Subject terms: Biotechnology, Cancer     

Neuronal Differentiation Dictates Estrogen-Dependent Survival and ERK1/2 Kinetic by Means of Caveolin-1

Estrogens promote a plethora of effects in the CNS that profoundly affect both its development and mature functions and are able to influence proliferation, differentiation, survival and neurotransmission. The biological effects of estrogens are cell-context specific and also depend on differentiation and/or proliferation status in a given cell type. Furthermore, estrogens activate ERK1/2 in a variety of cellular types. Here, we investigated whether ERK1/2 activation might be influenced by estrogens stimulation according to the differentiation status and the molecular mechanisms underling this phenomenon. ERK1/2 exert an opposing role on survival and death, as well as on proliferation and differentiation depending on different kinetics of phosphorylation. Hence we report that mesencephalic primary cultures and the immortalized cell line mes-c-myc A1 express estrogen receptor α and activate ERK1/2 upon E₂ stimulation. Interestingly, following the arrest of proliferation and the onset of differentiation, we observe a change in the kinetic of ERKs phosphorylation induced by estrogens stimulation. Moreover, caveolin-1, a main constituent of caveolae, endogenously expressed and co-localized with ER-α on plasma membrane, is consistently up-regulated following differentiation and cell growth arrest. In addition, we demonstrate that siRNA-induced caveolin-1 down-regulation or disruption by means of ß-cyclodextrin treatment changes ERK1/2 phosphorylation in response to estrogens stimulation. Finally, caveolin-1 down-regulation abolishes estrogens-dependent survival of neurons. Thus, caveolin-1 appears to be an important player in mediating, at least, some of the non-genomic action of estrogens in neurons, in particular ERK1/2 kinetics of activation and survival.